2010 APS Annual Meeting
Abstracts of Presentations
Abstracts submitted for presentation at the APS 2010 Annual Meeting August 7–11, 2010. The abstracts are arranged alphabetically by the first
author’s name.
Interception, identification and molecular characterization of three
Potato virus S isolates infecting potato germplasm introduced from South
America
J. A. ABAD (1), Y. Lin (2), C. J. Maroon-Lango (1), C. Loschinkohl (1), M.
R. Smither (1), H. R. Pappu (2)
(1) USDA APHIS PPQ PHP Plant Germplasm Quarantine Program, Beltsville, MD, U.S.A.; (2) Plant Pathology Dept., Washington State University,
Pullman, WA, U.S.A.
Phytopathology 100:S1
In the last three years, the USDA-APHIS-PPQ Plant Germplasm Quarantine
Program intercepted several potentially highly infectious unknown and
unusual virus isolates in potato germplam imported from South America.
Recently, three PVS isolates, Q1, Q3 and Q5, were intercepted and
characterized. The infected potato accessions were symptomless, and PVS
was detected by bioassay, ELISA and molecular procedures. Chenopodium
quinoa and Nicotiana debneyii showed symptoms following mechanical
inoculations with Q1. However, Q3 and Q5 failed to produce symptoms in
these plants. ELISA using PVS-specific antiserum was strongly positive for
Q1 and inconclusive for Q3 and Q5. RT-PCR using Carlavirus generic
primers was positive for all three PVS isolates. PVS-O-specific primer pair
were positive for Q1 but negative for Q3 and Q5. Primers for PVS-A were
negative for all isolates. The coat protein gene of each isolate was amplified,
cloned, sequenced, and compared with those of known PVS isolates.
Phylogenetic tree constructed using the CP amino acid sequences indicated
that Q3 and Q5 clustered with the PVS-Andean group. The Q1 isolate was
more closely related to the isolate Ha6-2 from Syria as well as U.S. isolates
and clustered with other known PVS-Ordinary strains. Sequence alignments
also suggested that isolates Q3 and Q5 have insertions in three regions of the
CP gene when compared with the Vltava isolate of the Andean group of PVS.
Aflatoxin persistence in corn residues under no-till and conventional
tillage management
H. K. ABBAS (1), R. M. Zablotowicz (2), C. Accinelli (3), C. A. Abel (4), N.
A. Phillips (2), B. J. Johnson (2)
(1) USDA ARS CG&PRU, Stoneville, MS, U.S.A.; (2) Stoneville, MS,
U.S.A.; (3) Bologna, ITALY; (4) Aimes, IA, U.S.A.
Phytopathology 100:S1
Little is known about the occurrence of aflatoxin in corn plant debris during
the intercropping period. Corn was planted in two randomized complete block
design experiments under no till (NT) and conventional till (CT) practices
using Bt and Non-Bt corn in Elizabeth, MS in 2007 and 2008. Aflatoxin levels
were determined at harvest and over-wintering corn within corn stover, cobs,
and cobs with kernels. These plant components were collected from the soil
surface of no-till (NT) plots or from the upper 5 cm of soil in conventional-till
(CT) plots. At time of harvest, the aflatoxin in corn kernels from Bt and non-
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
Bt hybrids were similar with greater aflatoxin found in grain from CT
compared to NT plots. Likewise, a similar level of aflatoxin was found in
various plant components of Bt and non-Bt residues. Regardless of genotype
or tillage, the highest levels were found in cobs containing grain and the
lowest content in stover. Aflatoxin concentrations in these tissues dissipated
more rapidly in CT compared to NT plots. The levels found in these studies
were much less than previously reported (Abbas et al., 2008), however,
relatively high aflatoxin levels (100 to > 3000 ppb) persist in grain remaining
on cobs under NT conditions one to seven months post-harvest.
Improving scab suppression and tuber yield of potatoes with multiple
repeated applications of low rates of fish emulsion to a commercial field
P. A. ABBASI (1)
(1) Agriculture & Agri-Food Canada, London, ON, CANADA
Phytopathology 100:S1
Fish emulsion (FE), a liquid co-product of the processing of fish into fish
meal, is an excellent organic product to enrich soil microbes and generate
disease suppressive conditions against soil-borne diseases such as seedling
damping-off, potato scab, and Verticillium wilt. Our studies suggest that
biological control and organic acids play a role in FE-mediated disease
suppression depending on substrates and soils. However, the broadcast rates
(20,000 L/ha) of FE that provided effective control of potato scab may be too
costly for commercial use. This long-term study was initiated to improve
disease suppression and tuber yield of potatoes with multiple repeated
applications of much lower broadcast rates (100 and 200 L/ha) of FE after
harvesting and before planting potatoes starting in fall of 2007 for 3 years.
Both rates (100 and 200 L/ha) of FE reduced scab severity by 34 and 42% in
2008 and 18 and 57% in 2009, reduced the percentage of tubers with deeppitted scab by 23 and 30% in 2008 and 18 and 51% in 2009, and increased
tuber yield by 16 and 19% in 2008 and 14 and 20% in 2009, respectively. FE
soil amendment enhanced the numbers of soil bacteria including those of
Pseudomonads and Bacilli. Lower rates applied more frequently may provide
longer lasting disease suppression and may be economically feasible. FE is an
excellent model system for development of an organic amendment to enhance
biological control potential of natural soils.
Management of the root-knot nematode, Meloidogyne incognita on
tomato in Egypt
M. M. ABD-ELGAWAD (1), S. S. Kabeil (2)
(1) National Research Center, Dokki, EGYPT; (2) Mubarak City for Scientific
Research and Technology Applications, Alexandria, EGYPT
Phytopathology 100:S1
The efficacy of carbofuran at 1 mg a.i./kg soil, Serratia marcescens (1 × 109
bacterium cells/ml water) at 2 ml of the suspension/kg soil, and three
different Trichoderma harzianum isolates each separately added at 50 ml./kg
soil against the root-knot nematode Meloidogyne incognita on two tomato
cultivars Super Strain B and Alisa was assessed in the glasshouse. Fresh
and dry weight of shoots were higher (P ≤ 0.05) in nematode-free plants of
the two cultivars than both M. incognita-infested plants and the
above-mentioned treatments. Carbofuran followed by S. marcescens and T.
Vol. 100, No. 6 (Supplement), 2010
S1
harzianum generally decreased nematode development and reproduction
parameters compared to the untreated control. Other effects of M. incognita
infestation on protein content and enzyme activities are presented and
discussed.
Impact of mycorrhizal infection on sensitivity of wheat to sorghum
allelopathy
M. M. ABDELKARIM (1), R. J. Gualandi (1), K. D. Gwinn (1), B. H.
Ownley (1)
(1) University of Tennessee, Knoxville, TN, U.S.A.
Phytopathology 100:S2
Sorgoleone, an allelochemical exuded from the roots of sorghum, decreases
growth of subsequent crops. The aim of this experiment was to determine
impact of two species of mycorrhizae (Glomus intraradices or Gigaspora
margarita) on allelopathic effects of sorghum (Sorghum bicolor Dekalb
‘DK39Y’) on wheat (Triticum aestivum Pioneer ‘26R22’). Sorghum plants
were planted either in the presence or absence of mycorrhizae-infected
sorghum roots. Vegetative growth of sorghum was removed after 12 weeks,
and wheat (20 seeds/pot) was grown for one month in medium containing
sorghum roots. The experiment had eight replicates with four treatments: no
sorghum (control), non-mycorrhizal sorghum, Glomus-infected sorghum, and
Gigaspora-infected sorghum. All growth parameters (shoot height, fresh shoot
weight, fresh and dry root weights, and stem diameter) were greater for
control than treatments that followed sorghum [P < 0.0001 for all parameters
except stem diameter (P = 0.009)]. Plant height of mycorrhizal treatments was
significantly higher than the non-mycorrhizal treatment, but there were no
differences between mycorrhizal species (P < 0.0001). Root weight (fresh and
dry) was greater in treatment with Gigaspora than in treatment with Glomus;
neither was different from non-mycorrhizal treatment (P < 0.0001). In a
natural infestation, the bird cherry oat aphid, Rhopalosiphum padi, was
preferentially attracted to the non-mycorrhizal sorghum treatment (33.5 ± 8.1
aphids/plant); other treatments had fewer than one aphid/plant.
Two undescribed viral species isolated from native grapevines
N. Abou Ghanem-Sabanadzovic (1), S. SABANADZOVIC (1)
(1) Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, MS, U.S.A.
Phytopathology 100:S2
More than sixty viruses have been reported from Vitis vinifera and related
rootstock species/hybrids while very little is known about viruses infecting
grape germplasm native to the American continent. A survey aimed at the
identification of viruses present in native Vitis germplasm in the Southeastern
U.S.A. was carried out in 2007 and 2008 exclusively targeting wild
specimens. Shotgun sequencing of reverse-transcribed dsRNAs extracted from
a specimen of Vitis aestivalis collected from Great Smoky Mountains National
Park revealed co-infection by two unrelated viruses. Complete sequences were
generated for both viruses: i) a multipartite dsRNA virus related to Rice
ragged stunt virus (gen. Oryzavirus; fam. Reoviridae) and ii) a luteovirid with
similarities with Pea enation mosaic virus 1 (PEMV-1; gen. Enamovirus; fam.
Luteoviridae). While the oryza-like virus was detected in only one additional
specimen, enamo-like virus seems to be rather widespread in the native
grapes. No members of these taxa were previously reported from grapevines.
Susceptibility of cultivated species/hybrids to these viruses is yet to be
examined.
Detection of toxin and non-toxin forms of Aspergillus flavus and A.
parasiticus by RT-PCR in Georgia peanuts
P. ACHAR (1)
(1) Kennesaw State University, Kennesaw, GA, U.S.A.
Phytopathology 100:S2
Aspergillus flavus and Aspergillus parasiticus are common contaminant in
peanuts and are responsible for the production of carcinogenic aflatoxin. PCR
are routinely used to detect Aspergillus spp. in peanuts. In the present study
Real-Time PCR (RT-PCR), was used to detect aflatoxigenic from nonaflatoxigenic strains. Universal primers (ITS) 1 and (ITS) 4 were used to
amplify a conserved region of the internal transcribed space for the non-toxin
producing sequences. Specific primers, Nor 1 & 2 and Ver 1 & 2, were used to
identify the toxin producing sequences. The Roche Light Cycler 480 and
SYBR Green were used for RT-PCR. The melting points gave insight into the
amplicon length and G-C content of both spp. In all samples tested, the ITS
gene concentration was a factor of 181 and 1,024 times more than the Nor and
Ver genes amplified, respectively. PCR products with ITS primers ranged
from 550-600 bp while Nor at 350 bp and Ver at 475 bp using 2.0% agarose
gel. Our results showed the ability of RT-PCR to detect the presence of toxin
and non-toxin producing genes in Aspergillus spp. Relative quantification
supported the prediction that a greater concentration of housekeeping genes
would be present in all the samples compared to the genes for aflatoxin
biosynthesis. Our results indicated that RT-PCR is faster and more accurate
S2
PHYTOPATHOLOGY
than traditional methods and hence recommended for early detection of
aflatoxin strains in peanuts since aflatoxin is a potential carcinogen.
Tracing the emergence of resistance breaking variants of Beet necrotic
yellow vein virus in nature
R. ACOSTA-LEAL (1), B. K. Brian (2), C. M. Rush (2)
(1) AgriLife Research (Texas A&M), Amarillo, TX, U.S.A.; (2) Texas
AgriLife Research, Amarillo, TX, U.S.A.
Phytopathology 100:S2
The capability of BNYVV to overcome the sugar beet Rz1-mediated
resistance is frequently associated with the presence of a specific amino acid
in the hypervariable region of the viral p25 (RNA-3). The underlying C to U
mutation has independently occurred in Minnesota and California where
resistance breaking (RB) variants cause characteristic yellow spots of Rz1plants in the field. Multiple yellow spots often develop in a single field. Given
the nature of the RB mutation, the objective of this work was to determine if
RB variants can emerge in parallel multiple times from sympatric avirulent
haplotypes during the same crop season. A fragment of the viral RNA-3 from
single-plant isolates was cloned and sequenced from several Rz1-plants inside
and outside yellow spots to analyze the genetic composition of these
populations. Co-infections of avirulent and RB haplotypes were commonly
found in diseased plants and occasionally in asymptomatic plants. Plants close
together were sometimes infected by the same predominant haplotype. Several
avirulent progenitors have been identified and two evolutionary pathways to
overcome Rz1 apparently have taken place in the Imperial Valley.
Selection of isolates of Penicillium expansum with reduced sensitivity to
fludioxonil and pyrimethanil from sensitive, single-spored isolates
J. Adaskaveg (1), H. FORSTER (2)
(1) University of California, Riverside, CA, U.S.A.; (2) University of
California, Davis, CA, U.S.A.
Phytopathology 100:S2
Isolates of Penicillium expansum with reduced sensitivity against the two new
postharvest fungicides fludioxonil and pyrimethanil could be readily obtained
when large numbers of conidia (108/plate) of single-spored sensitive isolates
were plated on selection plates. Agar plates contained a continuous
concentration gradient for each fungicide. There was no correlation between
the number of resistant isolates obtained and the degree of heterogeneity of
the sensitive population (1, 4, or 60 isolates) that was used in the selection
assays. Resistance frequencies ranged from 1 × 10–8 to 3.6 × 10–5 for
fludioxonil and from 1.2 × 10–8 to 1.8 × 10–6 for pyrimethanil. For fludioxonil,
isolates were either moderately resistant (EC50 0.77 to 3.5 mg/L; sensitive
isolates: <0.02 mg/L) or highly resistant (EC50 >40 mg/L), whereas for
pyrimethanil a range of sensitivities (EC50 1.8 to >75 mg/L; sensitive isolates:
<0.70 mg/L) was observed. Isolates insensitive to both fungicides were
recovered at very low frequency in some tests and always displayed a lower
level of resistance. Most resistant isolates were stable in culture and were
pathogenic in apple fruit inoculations. In these experiments, no isolates with
reduced sensitivity to difenoconazole, another fungicide planned for
registration, were obtained. Our data indicate that the risk of resistance
development against new postharvest fungicides for pome fruit is high and
that resistance management is crucial.
Competitiveness of Penicillium expansum isolates with reduced sensitivity
to fludioxonil and pyrimethanil during infection of apple fruit
J. Adaskaveg (1), H. FORSTER (2)
(1) University of California, Riverside, CA, U.S.A.; (2) University of
California, Davis, CA, U.S.A.
Phytopathology 100:S2
Isolates of Penicillium expansum resistant to the new postharvest fungicides
fludioxonil and/or pyrimethanil and pathogenic to pome fruit were selected in
the laboratory from natural populations of the pathogen. The competitiveness
of these isolates was evaluated in co-inoculations of apple fruit with a
sensitive wild-type isolate of P. expansum. Competiveness was based on the
proportion of resistant to sensitive progeny that were grown from conidia
collected from decaying fruit. Either of two isolates highly resistant to
fludioxonil (EC50 >40 mg/L as compared to <0.02 mg/L for sensitive isolates)
were not recovered after co-inoculation with the sensitive isolate, whereas
when using an isolate highly resistant to pyrimethanil (EC50 >75 mg/L as
compared to <0.70 mg/L for sensitive isolates) 27.1 to 33.3% of conidia from
decaying fruit displayed the resistant phenotype. In co-inoculations with either
of two isolates of P. expansum with an intermediate level of resistance to both
fungicides (EC50 0.12 or 2.42 mg/l for fludioxonil, 1.74 or 2.08 mg/L for
pyrimethanil), 22.9 to 35.4% of the collected conidia displayed the doubleresistant phenotype. These data indicate that differences in competitiveness
exist among resistant isolates, that with repeated fungicide applications some
isolates may become predominant, and that proper anti-resistance strategies
have to be followed in the use of these new fungicides.
Evaluation of winter wheat accessions for resistance to Xanthomonas
translucens pv. undulosa
T. B. ADHIKARI (1), J. Hansen (1), S. Gurung (1), J. M. Bonman (2)
(1) North Dakota State University, Fargo, ND, U.S.A.; (2) USDA-ARS, Small
Grains and Potato Germplasm Research Unit, Aberdeen, ID, U.S.A.
Phytopathology 100:S3
Bacterial leaf streak (BLS) caused by Xanthomonas translucens pv. undulosa
(Xtu), has re-emerged and become an important disease of wheat in the
northern Great Plains of the United States. Breeding for disease resistance
and planting resistant varieties offers the best approach to control BLS
in the absence of effective bactericides. Most of the currently grown wheat
varieties in the United States appear to be susceptible to BLS. Over 400 winter
wheat accessions from a core subset of the USDA National Small Grain
Collection, representing landraces, cultivars, and breeding lines of diverse
origin, were assessed for resistance to BLS at the flag leaf stage during
2009 and 2010 in a greenhouse at NDSU. Results showed that the wheat
accessions have a wide range of susceptibility to BLS but 41 accessions
exhibited resistance to the disease. Resistance was significantly more frequent
in accessions from North America, Australia-New Zealand, Western Asia,
and South-central Asia compared to accessions from Northern and
Eastern Europe. The United States had the most resistant accessions
followed by China and Germany. There was no association between kernel
color or accession improvement status (e.g., landrace, cultivar, breeding line)
and BLS resistance. Overall, this study identified potential new sources of
resistance to BLS, which could be utilized in winter wheat improvement
programs.
Potential viral threats to Miscanthus x giganteus and switchgrass
production for bioenergy in the United States
B. O. AGINDOTAN (1), M. O. Ahonsi (2), M. E. Gray (2), C. A. Bradley
(2)
(1) University of Illinois, Urbana, IL, U.S.A.; (2) Energy Biosciences
Institute, University of Illinois, Urbana-Champaign, Urbana, IL, U.S.A.
Phytopathology 100:S3
Miscanthus x giganteus and Panicum virgatum (switchgrass) are two potential
biomass crops being evaluated for cellulosic ethanol production. Because they
could be cultivated in large scale for biomass purposes, it is important to
identify viruses that are potential threats to these crops. Identification of
viruses infecting these crops is important for quarantine purposes, virus
resistance breeding, and production of virus-free planting materials. Using a
combination of methods, viruses were identified that infected these crops in
fields in Illinois, Iowa, Wisconsin, Kentucky, Tennessee and Georgia. The
most common viruses infecting both M. x giganteus and switchgrass were
Sugarcane mosaic virus (SCMV) and Wheat soil-borne mosaic virus
(WSBMV). Barley yellow dwarf-PAV virus (BYDV-PAV) was detected only
in switchgrass in Illinois. A new virus of switchgrass was identified, which
was closely related to, but significantly different from Maize rayado fino virus
(MRFV). Other virus-like symptoms were observed on collected samples of
M. x giganteus and switchgrass, and identification of causal agents is in
progress.
Isolation and characterization of Pectobacterium carotovorum mutants
host-signal responsive genes
P. A. AGYEMANG (1)
(1) Tennessee State University, Nashville, TN, U.S.A.
Phytopathology 100:S3
Pectobacterium carotovorum (Pc) is a phytopathogenic bacterium that causes
soft rot disease in economically important crops by producing extracellular
enzymes triggered by its host signals and other environmental cues. Enzyme
production and pathogenicity depends on a coordinated regulation of a
number of genes in the pathogen. Approximately 20% of the putative proteins
of Pc have unknown function. Pc strain AC5006N3 (lacZ-, NalR) was
mutagenized using mini-Tn5 Km containing a promoterless lacZ reporter.
About 31,122 colonies were screened in the presence and absence of celery
extract (CE) to obtain mutants in the host extract-regulated genes. After
repeated screening and evaluation of promoter activity of the mutants based
on betagalactosidase ( -gal) assays, 98 mutants were obtained of which 83
were induced (> 5-fold) and 15 were repressed (<0.5-fold) in the presence of
CE. Semiquantitative assays on selected (26 induced and 3 repressed) mutants
showed increased production of cellulase (Cel), pectate lyase (Pel) and
protease (Prt) in the presence of CE. Quantitative assays revealed that,
compared to the parent, 6 of these mutants overproduced (> 2-fold) Pel while
one was deficient (< 0.5-fold). Three mutants overproduced Prt while 5 were
deficient (< 0.5-fold). In a pathogenicity test using celery petioles and potato
tubers, 6 mutants macerated at least 2-fold more tissues compared to the
parent. Nine mutants had reduced ability to macerate the tissues. Identification
of genes of unique mutants is imperative.
Grapevine necrotic union, a newly recognized disease in grapevines on
110 Richter rootstock in California
M. Al Rwahnih (1), A. Rowhani (1), R. J. Smith (2), J. K. Uyemoto (3), M. R.
SUDARSHANA (3)
(1) University of California, Davis, CA, U.S.A.; (2) University of California
Cooperative Extension, Santa Rosa, CA, U.S.A.; (3) USDA ARS, Davis, CA,
U.S.A.
Phytopathology 100:S3
In early fall of Yr 2004, inspection of a 7-year old vineyard of Pinot noir (PN)
clone 02A (Vitis vinifera L.) grafted on rootstock 110 Richter (110R; V.
berlandieri x V. rupestris) in Sonoma County, CA, revealed ~ 2.1% of the
grapevines with symptoms of solid red leaf blades, weak shoot growth and
grape clusters with reduced set. Examination of trunk specimens indicated a
necrotic line at the scion-rootstock junction and hence named “grapevine
necrotic union” (GNU). Yearly surveys indicated that GNU incidence
gradually increased to 22% in Yr 2009. This disease was also observed in PN
clones 02A (PN02A), 04, 667, and 777, in Napa County, and Pinot gris 152
on 110R in Monterey County, and the incidence ranged from 2.0% to 45%.
RT-PCR assays did not indicate any known grapevine viruses that could be
considered associated with diseased vines. Repeated chip bud grafts of
diseased vines onto test plants of Cabernet Sauvignon 08 on 110R rootstock
failed to demonstrate a graft-transmissible agent. However, bench grafts of
several PN clones and Chardonnay-04 (Ch-04) produced GNU on 110R but
not on rootstock 3309 Couderc (V. riparia x V. rupestris). Ultradeep
sequencing analysis of cDNA made from dsRNA obtained from bark
scrapings indicated the association of Grapevine redglobe virus and Grapevine
rupestris vein-feathering virus in Ch-04 and PN02A vines. These two viruses
were also found associated with GNU affected vines in Napa County.
Characterization of pervasive latent viral infection of olive trees in the
National Clonal Germplasm Repository
M. AL RWAHNIH (1), Y. Guo (1), S. Daubert (1), D. Golino (1), A.
Rowhani (1)
(1) University of California-Davis, Davis, CA, U.S.A.
Phytopathology 100:S3
The olive industry in California is booming. Oil olive acreage is growing at
4000 acres per year, and acreage of table olives is also expanding. Propagation
material is being requested in ever-larger quantities from the U C DavisUSDA National Clonal Germplasm Repository, which is recognized as one of
the richest collections of olive material in California. The repository maintains
107 different olive varieties, imported from nineteen different countries.
However, the collection at the Germplasm Repository has never been
systematically characterized as to its viral infection status. We have now
completed the first comprehensive virus testing of the collection using
molecular diagnostic tools, and have compiled a list of the viruses that were
detected. A total of 54 trees from 36 different cultivars were sampled. Though
these trees were asymptomatic, the samples from 97.9% of them showed
dsRNA profiles indicating viral infection. 91.5% of those trees tested positive
for Olive leaf yellowing-associated virus (OLYaV) by RT-PCR analysis,
while 39.5% were positive for Cucumber mosaic virus. PCR amplicons of the
OLYaV HSP70h gene were cloned and sequenced to analyze the molecular
variability between isolates from trees originating from different geographical
regions. The sequence analysis showed a maximum of 27% divergence
between amplicons obtained from these selections.
Profiling host-specific and virus-derived small RNAs in a woody
perennial plant species infected with an ampelovirus
O. J. ALABI (1), Y. Zheng (2), G. Jagadeeswaran (3), R. Sunkar (3), R. A.
Naidu (4)
(1) Washington State University, Prosser, WA, U.S.A.; (2) Institute of
Developmental Biology and Molecular Medicine and School of Life Sciences,
Fudan University, Shanghai, China, 200433, Shanghai, PRC PEOPLES REP
OF CHINA; (3) Department of Biochemistry and Molecular Biology,
Oklahoma State University, Stillwater, OK, U.S.A.; (4) Department of Plant
Pathology, Irrigated Agriculture Research and Extension Center, Washington
State University, Prosser, WA, U.S.A.
Phytopathology 100:S3
Grapevine leafroll disease (GLRD) is an economically important viral disease
of wine grapes. In this study, we used Solexa deep sequencing technology to
compare small RNA (sRNA) profiles between GLRD-affected and unaffected
leaves. Total RNA extracted from symptomatic leaves tested positive for
Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus and
family Closteroviridae) and uninfected leaves were used for constructing
small RNA libraries and sequenced. Approximately 2.3 and 1.5 million small
RNA reads of 18 to 28 nt length were obtained from uninfected and infected
libraries, respectively. Sequence analysis indicated that the host microRNAs
(miRNAs) and other endogenous small interfering RNAs (siRNAs) were
differentially regulated in infected samples. In addition, sRNAs derived from
Vol. 100, No. 6 (Supplement), 2010
S3
GLRaV-3 were identified in the infected sample. Virus-derived sRNAs were
mapped to the genome of GLRaV-3 according to their polarities and the
results suggested the presence of potential hotspots that generate siRNAs from
the virus genome. To our knowledge, this is the first study to profile miRNAs
in closterovirus-infected perennial crop by deep sequencing approach.
Genetic variation of Sclerotinia sclerotiorum on four crops from the north
central United States
L. ALDRICH-WOLFE (1), S. E. Travers (2), B. D. Nelson (3)
(1) Plant Pathology, North Dakota State University, Fargo, ND, U.S.A.; (2)
Biological Sciences, North Dakota State University, Fargo, ND, U.S.A.; (3)
North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S4
Sclerotinia sclerotiorum is a pathogen of many commonly-grown crops in the
north central United States, yet little is known about genetic variability in the
region and across crops. In 2008 149 isolates were collected from four crops
(canola, dry bean, soybean, and sunflower) in twelve North Central states and
WY, MT and CO. Isolates were evaluated for mycelial compatibility group
(MCG) and microsatellite haplotype at twelve loci. Forty-six MCGs were
identified. The most common was MCG 9 found in nine states and on all four
crops. Six of the most common MCGs represented 58% of the isolates and
were found across crops. There was a strong association between MCGs and
microsatellites. For example, all isolates within MCG 9 shared a single
microsatellite haplotype, while in MCG 8 there were three microsatellite
haplotypes. To date, MCGs do not appear to be restricted to particular
geographic regions or host crops. Gene diversity was as high among samples
from eastern North Dakota and western Minnesota as among all samples,
suggesting the pathogen may be as diverse genetically in local areas as
across broad geographic regions. Microsatellite analysis found no evidence
for differences in genotype or genetic diversity of the pathogen among the
crops. Within eastern North Dakota and western Minnesota (where crops were
more equally represented in the samples), gene flow appeared to be high
between soybean and dry bean and lowest between sunflower and the two
legumes.
Selection indexes comparison to improve maize resistance to Aspergillus
flavus, Fusarium moniliforme and Rhizoctonia solani grain and plant
infection
J. M. ALEZONES (1), R. D. Perdomo (1), B. Borges (1), A. D. Gonzalez (1),
J. J. Salazar (1), F. R. Hernandez (1)
(1) Fundacion Danac, San Felipe, VENEZUELA
Phytopathology 100:S4
Preharvest contamination of maize by Aspergillus flavus (AF) and Fusarium
moniliforme (FM) mycotoxins is considered a serious health problem.
Rhizoctonia solani (RS) is also a harmful pathogen for this crop in several
countries. The identification of cultivars with adequate performance in
multiple traits is important in any breeding program, and for that, several
selection indexes (SI) have been developed. In order to identify which index is
more suitable for selection to these diseases, a two year trial was conducted
involving the evaluation of 740 inbreds. To evaluate AF and FM resistance,
kernel screen assay (KSA) technique was used, for RS a screening technique
involving inoculation of plants with rice grains colonized by the fungus was
performed. Genetic progress percent (GP), a “balance value” (BV), that is a
relation between the trait with less advance and the one with the biggest
progress, and the product of these two variables “PB”, were calculated for five
SI (Classic, Base, Free of weights, Multiplicative and Sum of Ranks) under a
selection fraction of 25%. For both years the SI with the highest GP was the
“Classic”, but it posses a great lack of balance, which means that some
diseases have a great advance when others keeps the same. On the other hand
the “Sum of ranks” index was the one with higher BV. Finally, when PB is
calculated, Sum of Ranks index posses the higher value, meaning that GP and
BI for this index were simultaneously high, making it suitable for selection to
these diseases.
Pustule density and latent period of Puccinia hordei on Iraqi barley
genotypes
M. AL-HAMDANY (1), M. M. Salih (1)
(1) Ministry of Science and Technology, Baghdad, IRAQ
Phytopathology 100:S4
Disease severity of Puccinia hordei on barley genotypes and latent period
were thoroughly investigated under field and growth room conditions
respectively in Baghdad region. Barley genotypes included 9 cultivars, 13
induced mutants, 8 selected germplasims, and 300 M2 variants following
gamma irradiation on Numar cultivar were used. Data of pustule density on
these genotypes under artificially heavy epidemic form, indicated that disease
responses of all tested genotypes could be classified to four groups: Highly
susceptible such as cultivars Golden melon, Aimer, Beacher, Weah and
Arivat, mutants D/21, D/30 and D/32; Susceptible such as cultivar Prior,
S4
PHYTOPATHOLOGY
mutants D/24, C/50, NA/20, and C/63, selected germplasms 480, 552, and
557; Moderate susceptible as in cultivar Gazera 2, the mutants D/34, TB/5
and VB/7, and selected germplasms 102, 576, 577 and 657 and mildew
resistance source H-421; Moderate resistance included both cultivars Gazera 1
and Numar, the mutants OA/15, VB/6, and SA/12, all M3 Numar
variants regardless the dose used of gamma rays. The latent period of P.
hordei on detached leaves was 132 hrs. in the first two groups, 156 hrs. in the
third and 168 hrs. on barley genotypes might have a partial resistance to P.
hordei in Iraq. However, promising new source leaf rust resistance was
identified.
Frequency of 3ADON and 15ADON isolates of Fusarium graminearum
from field plots of wheat inoculated with mixed pathogen populations in
North Dakota
S. ALI (1), K. Puri (1), M. McMullen (1), S. Zhong (1)
(1) North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S4
Isolates of Fusarium graminearum can be identified as one of the three
chemotypes, 3-acetyl-Deoxynivalenol (3ADON), 15-acetyl-Deoxynivalenol
(15ADON) and nivalenol (NIV) based on their trichothecene profile.
Recently, 3ADON isolates have increased in the fungal population in the
Northern Great Plains of the U.S. and Canada; the reason for the population
change is still not known. We inoculated FHB susceptible (Briggs) and
moderately resistant (Alsen) spring wheats with individual populations of
3ADON and 15ADON chemotypes (ten isolates each) and a mixed population
(ten isolates of each chemotype), and added a flowering-time fungicide
treatment, under field conditions. F. graminearum isolates (575) were
randomly recovered from the inoculated plots and analyzed for chemotype
using polymerase chain reaction (PCR). Results showed a significantly higher
recovery frequency of 3ADON isolates than of 15ADON isolates in the mixed
population inoculated on Briggs, with or without fungicide treatment, and on
Alsen without fungicide treatment. No significance was observed in recovery
frequency of the two chemotypes from mixed-inoculation on Alsen with
fungicide treatment, but the number of isolates recovered from this treatment
was low (34). The results suggest that the 3ADON isolates may be more
competitive than the prevalent 15ADON isolates under North Dakota field
conditions, regardless of cultivar resistance or use of fungicide.
Viruses infecting cucurbit crops in Oklahoma
A. ALI (1), A. Khattab (2)
(1) University of Tulsa, Tulsa, OK, U.S.A.; (2) Department of Biological
Science, The University of Tulsa, Tulsa, OK, U.S.A.
Phytopathology 100:S4
During the growing season of 2008, surveys were conducted to detect and
determine the incidence of viruses in the major cucurbit growing areas of
Oklahoma. A total of 588 symptomatic leaf samples from 36 fields in 3
counties (Atoka, Blaine and Tulsa) were collected from five cucurbit crops
(cantaloupe, cucumber, pumpkin, squash and watermelon). All samples were
tested by dot-immuno-binding assay (DIBA) against the antisera of seven
viruses, including cucumber mosaic virus (CMV), cucumber green mottle
mosaic virus (CGMMV), melon necrotic spot virus (MNSV), papaya ringspot
virus-watermelon strain (PRSV-W, squash mosaic virus (SqMV), watermelon
mosaic virus-2 (WMV-2) and zucchini yellow mosaic virus (ZYMV). The
highest incidence was recorded for PRSV-W, followed by WMV-2, and
ZYMV, which were contained in 67%, 17% and 15% respectively of the
collected samples. MNSV, SqMV, and CMV were detected in 6.0%, 5.2%
and 1.2% of the samples, respectively. None of the samples reacted positively
against the antiserum of CGMMV. Mixed virus infections were common
involving two or three viruses in various combinations. Triple and double
infections were found in 6.8% and 5.6% of samples, respectively. Some
symptomatic samples of watermelon, squash and pumpkin did not react with
the antiserum of the above tested viruses, indicating that other unknown
viruses may be infecting cucurbit crops.
Incidence and genetic variation of Tobacco mosaic virus through some
tomato fields of Tehran
A. ALISHIRI (1), F. Rakhshandehroo (1), H. Zamanizadeh (1)
(1) Department of Plant Pathology, Islamic Azad University, Science and
Research Branch, Tehran, IRAN
Phytopathology 100:S4
Tobacco mosaic virus (TMV) is a type member of the genus Tobamovirus.
This virus is one of the most destructive viral diseases of plants. The goal of
this study was to determine the incidence of Tobacco mosaic virus (TMV) on
tomato plants. During the years 2009 to 2010, a survey was conducted through
different tomato fields. A total of 256 leaf samples were randomly collected
from symptomatic and asymptomatic tomato plants in four zones of the
Tehran province of Iran and tested by the double antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA) using specific polyclonal
antibody (Agdia, U.S.A.). Results showed that 10.6% of the tested leaf
samples are infected with the TMV. Using PCR molecular method and
specific primers designed for the coat protein of the viral genome sequence,
presence of the TMV was confirmed for the ELISA positive-tested tomato
plants. All infected tested samples amplified a 694-bp fragment in PCR
reaction. The amplified fragments of five isolates have first been sequenced
and then aligned with the corresponding data available for other TMV isolates
in NCBI. Phylogenetic analysis revealed that one of all these Iranian isolates
together with some NCBI isolates were categorized in one cluster while all
other TMV isolates from NCBI were categorized in a separate cluster. Further
studies on the distribution of this virus in different provinces is, now
undertaken.
Effect of an at tassel fungicide application on yield and the foliar disease
complex in field corn in Mississippi
T. W. ALLEN (1), A. Henn (2), D. M. Ingram (3)
(1) Mississippi State University, Stoneville, MS, U.S.A.; (2) Mississippi State
University, Starkville, MS, U.S.A.; (3) Mississippi State University,
Raymond, MS, U.S.A.
Phytopathology 100:S5
Over the past few seasons foliar fungicide applications have drastically
increased across the United States in field corn in response to reports that an
automatic fungicide application will result in increased yield in the absence of
yield-limiting foliar diseases. Fungicides are suggested in the absence of
yield-limiting diseases and applications are timed for tassel (VT). In response
to this growing practice, research was conducted in Mississippi from 2007 to
2009 using three fungicides. Labeled rates of azoxystrobin + propiconazole
(as Quilt), propiconazole (as Propimax), and pyraclostrobin (as Headline)
were applied at VT at 27 locations, both in large (by air, n = 16) and small
plot trials (by ground, n = 11), over the three year period and replicated at
least 4 times at each location. In addition to collecting yield data at the end of
the season, plots were rated post-application for disease incidence and severity
as well as destructive plant sampling to determine potential impacts of the
fungicides on overall plant health. Plants destructively sampled were rated for
incidence of root and stalk diseases. In general, foliar disease incidence was
less than 5% at all locations for the three year study. Collectively, fungicide
application did not significantly increase yield compared to the untreated plots
for any of the chemistries utilized.
Characterization of promoter elements from plant pararetroviruses
associated with dahlia (Dahlia variabilis)
C. V. ALMEYDA (1), K. L. Druffel (1), H. R. Pappu (1)
(1) Washington State University, Pullman, WA, U.S.A.
Phytopathology 100:S5
Three distinct caulimoviruses have been reported from dahlia: Dahlia mosaic
virus (DMV), Dahlia common mosaic virus (DCMV) and an endogenous
plant pararetrovirus (DMV-D10). Based on sequence comparisons and
promoter prediction programs, the putative 35S promoter region from these
three viruses was identified. The promoter regions were independently cloned
into pCAMBIA1281Z. All constructs were introduced into Agrobacterium
tumefaciens by electroporation, and agroinfiltrations were done into N.
benthamiana. The activity and strength of the putative 35S promoter was
determined by transient expression of the beta-glucoronidase gene (GUS).
Results from qualitative GUS assays demonstrated that DMV, DCMV and
DMV-D10 promoter activity is similar to that of Cauliflower mosaic virus
35S promoter.
Metagenomic analysis of mycoviruses in grapevines
M. ALRWAHNIH (1), S. Daubert (1), A. Rowhani (1)
(1) University of California, Davis, CA, U.S.A.
Phytopathology 100:S5
Fungal viruses may be at least as numerous as plant viruses, but mycoviral
ecology and diversity is as yet largely unstudied. We have employed a
metagenomic approach to characterize the mycovirus populations on
grapevines. This approach was chosen to avoid the exclusion from our
analysis of viruses from non-culturable, non-purifyable fungal host species.
Total double stranded RNA was isolated from grapevine stem tissue and
subjected to analysis by 454 high-throughput sequencing and BLAST
screening. The analysis revealed a set of prevalent, diverse sequences related
to mycoviruses. Twenty four putative mycoviral groups were identified in the
samples, representing half of all known mycoviral families including the
Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and the Totiviridae.
The presence of the viral genomes identified by the BLAST screening was
confirmed by PCR tests of the starting stem tissue extract using primers
designed from the viral sequences identified in the informatic analysis. Three
of the putative mycovirus species were associated with Botrytis cinerea, a
fungal pathogen of grapes. But most of the species were found to be
undescribed mycoviruses and identified only through their distant sequence
relationships with known mycoviruses from non-grapevine fungal hosts.
Effect of temperature and UV radiation on survival of Coniothryrium
minitans and on efficacy in controlling lettuce drop caused by Sclerotinia
minor
M. ALVARADO-HERNANDEZ (1), B. M. Pryor (1)
(1) University of Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S5
Coniothyrium minitans has been previously reported to be more effective as a
biocontrol agent against Sclerotinia sclerotiorum than S. minor. It was
suspected that environmental factors played a major role in reducing C.
minitans survival and subsequent efficacy in controlling lettuce drop caused
by S. minor, which have higher reported scletorial densities in soil compared
to S. sclerotorium. Exposure of C. minitans spores to UV light (254nm)
revealed a negative effect on spore survival (<20% germination after 10 min).
Exposure of C. minitans to heat also revealed a rapid decline in spore
germination after 8 h exposure to high water or soil temperatures (<30%
germination after 8 h at 40°C). In field-based experiments, increased
application rates of commercial formulation of C. minitans - CONTANSWG
improved control of lettuce drop caused by S. minor. Further studies revealed
that one application of CONTANSWG through the sprinkler system (biogation)
resulted in a 25% decrease in the incidence of disease, compared to either
8lb/acre CONTANSWG or fungicide (Endura) treatments. Results suggest that
application of product via biogation may be a novel strategy for enhanced
performance of C. minitans in the biocontrol of lettuce drop caused by S.
minor.
Evaluating the resistance of plantain and banana genotypes to black
sigatoka (Mycosphaerella fijiensis m.) under greenhouse conditions
E. ALVAREZ (1), A. Cuellar (2)
(1) CIAT, Cali, COLOMBIA; (2) CIAT, Palmira, COLOMBIA
Phytopathology 100:S5
We evaluated, under greenhouse conditions, the reactions of plantain and
banana genotypes to the sigatoka fungus, Mycosphaerella fijiensis. To
inoculate plantain and banana seedlings of different genotypes, we used
aqueous suspensions of 5000 conidia mL–1 of each of 50 monosporic isolates
that had different pathogenicity levels and represented different production
areas in Colombia. Disease response according to genotype was assessed by
measuring the variables incubation period (IP), time of evolution of symptoms
(TES), area under the disease progress curve (AUDPC), and rate of disease
development (r). Isolates inoculated on cultivar ‘Dominico Harton’
demonstrated five levels of pathogenicity (very high, high, medium, low, and
very low), which had no relationship with their geographical origin or Musa
genotype. Isolates of different pathogenicity levels were present in the same
zone and for the same genotype. Plantain and banana genotypes reacted
differentially to M. fijiensis isolates, indicating the possible existence of
physiological races in the fungus. Disease severity was classified according to
three levels of reactions in Musa—resistant, intermediate, and susceptible—
where genotypes Topocho, Maqueño, FHIA 20, and FHIA 21 (plantain); and
Sedita and FHIA 23 (banana) presented the highest levels of resistance, that is,
disease was less severe and slower to progress.
Development of a Real-time PCR assay, to detect and quantify a 16SrIIIL phytoplasma associated with cassava frogskin disease (CFSD)
E. ALVAREZ (1), J. F. Mejia (2), J. M. Pardo (2)
(1) CIAT, Cali, COLOMBIA; (2) CIAT, Palmira, COLOMBIA
Phytopathology 100:S5
Cassava is a major source of carbohydrate in the tropics. It feeds millions of
people in America, Asia, and Africa. Yield losses of this root crop to CFSD in
Colombia and Latin America can be as high as 90%. Recently, this disease
was discovered to be associated with infection by a 16SrIII-L phytoplasma.
The disease is exponentially propagated through asexual seed, creating a
demand for disease-free planting materials. A TaqMan® probe was designed
for the microorganism, based on the rp gene (16Sr III-L phytoplasma). We
used qPCR to obtain a sensitivity that was 100- and 1,000-fold higher than
that obtained from nested. Detection levels were as sensitive as 4 × 102 copies
(to obtain the number of copies, we used relative quantification, using an
external standard). To validate the technique, we used field-harvested roots
showing typical disease symptoms, and obtained an average of 2.32 × 105
copies of the 16Sr III-L phytoplasma. A histological study of different tissues
in 3-month-old cassava plants showed that the best tissue for detecting the
microorganism is the pith in stems. With this technique, seed can be certified
as free of disease, the distribution of the associated microorganism in cassava
plants can be ascertained, and the potential for vector insect of the disease
discovered.
Vol. 100, No. 6 (Supplement), 2010
S5
Sensitivity of Botrytis cinerea isolates from strawberry to thiophanatemethyl and iprodione in Michoacan, Mexico
A. Alvarez-Medina (1), A. REBOLLAR-ALVITER (2), H. Silva-Rojas (3)
(1) Universidad Autonoma Chapingo, Morelia, MEXICO; (2) Univ Autonoma
Chapingo, Morelia, Mich, MEXICO; (3) Programa de Semillas, Colegio se
Postgraduados, Texoco, MEXICO
Phytopathology 100:S6
Grey mold caused by Botrytis cinerea is one of the most important pathogens
of strawberry in Mexico. Benzimidazole and dicarboxamide fungicides are
commonly used for controlling the disease during different times of the
growing season. The objective of this research was to determine the sensitivity
of several isolates of B. cinerea obtained from Zamora (ZV) and Maravatio
Valleys (MV), two of the most important regions producing strawberry in
Michoacan México, to the fungicides iprodione and thiophanate-methyl.
Sensitivity assays were conducted on PDA media amended with fungicide
concentrations ranging from 0.01 to 2000 µg/ml for tiophanate-methyl, and
from 0.1 to 50 µg/ml for iprodione. Each fungicide was tested against conidia
germination and mycelial growth. For iprodione, ED50 values for mycelial
growth ranged from 0.23 to 0.89 µg/ml in MV, and from 0.22 to 3.0 µg/ml in
ZV. For conidia germination ED50 values ranged from 0.47 to 16.8 µg/ml in
MV, and 0.59 to 22.6 µg/ml in ZV. For tiophanate-methyl, ED50 values for
mycelia in MV ranged from 0.11 to >2000 µg/ml, and 0.83 to > 2000 µg/ml
for ZV. ED50 values for conidia germination in this fungicide ranged from
0.04 to 3.3 µg/ml in MV, and from 0.97 to 3.53 µg/ml in ZV. Overall, ED50
values for both fungicides were higher in Zamora than Maravatio Valley.
UP-PCR analysis and UP-PCR cross-blot hybridization for grouping of
Rhizoctonia species isolated from turfgrass in Maryland and Virginia
B. S. AMARADASA (1), B. Horvath (2), D. K. Lakshman (3)
(1) Virginia Tech, Blacksburg, VA, U.S.A.; (2) University of Tennessee,
Knoxville, Knoxville, TN, U.S.A.; (3) USDA-ARS, Beltsville, MD, U.S.A.
Phytopathology 100:S6
Rhizoctonia brown patch is a serious disease of many turfgrass species in
warm regions of the U.S.A. Though hyphal anastomosis reactions have been
used to group Rhizoctonia species, it is time consuming and sometimes
difficult to interpret. Universally primed PCR (UP-PCR) analysis and UPPCR cross hybridization assay was used for grouping of genetically related
isolates. More than 400 Rhizoctonia isolates were collected from diseased
turfgrass leaves from five geographic areas in Virginia and Maryland. A
random sample of 54 isolates was selected and their anastomosis groups
(AGs) were determined by hyphal fusion reactions. The isolates were
amplified with a fluorescent UP primer to generate multiple PCR fragments
for each isolate. The fragment patterns were captured digitally and a
dendrogram was constructed. The cladistic analysis supported seven clades
including 3 clades of R. solani (AG1-1B, AG2-2IIIB and AG5), 1 clade of
binuleate Rhizoctonia like fungi (RLF) and 3 clades of Waitea circinata
(varieties zeae, oryzae and circinata). The UP-PCR analysis corresponded
well with traditional AGs by grouping isolates of similar AGs together. The
UP-PCR products were also spotted onto a nylon membrane, immobilized and
used for cross hybridization against PCR products from tester strains.
Genetically related isolates belonging to same AG subgroups cross hybridized
strongly while isolates of different AGs gave weak or no signal.
Relationship between pathogenicity and toxin production in Tangerine
pathotype of Alternaria alternata the causal agent of citrus brown spot in
Iran
N. AMINI (1), N. Kakvan (1), H. Zamanizadeh (1), S. Hajmansoor (1)
(1) Science and Research Branch, Islamic Azad University, Tehran, IRAN
Phytopathology 100:S6
Alternaria brown spot is one of the most important diseases of citrus
(Tangerine hybrids) all through the world. The disease is caused by Alternaria
alternata (Fr.) Keissl and makes serious economical losses. This pathogen
produces host specific toxins (HSTs) with the same host specificity as the
fungal isolates. In the present work, the pathogenic variability of the pathogen
was investigated. Furthermore, the possible relationship between the
pathogenicity and the ability to produce toxin were investigated for Iranian
isolates. A total of 40 isolates of Alternaria alternata from Tangerine hybrids
were collected from different regions of Iran. Pathogenic variability was
evaluated through in vitro conditions. The results revealed considerable
variation in aggressiveness of the isolates. Cluster analysis of the isolates
classified them into two categories: highly and moderate virulent groups. The
ability of each isolate to produce toxin was determined by measurement of
electrolyte loss from leaf disks. The results showed that culture filtrate
dilutions of pathogen were toxic to the natural host under experimental
condition. The pathogenicity of Alternaria alternata appears to be related to
the amount of toxin for each of the isolates tested. This method will hopefully
help us to produce more resistant Tangerine hybrids in Iranian citrus breeding
programs.
S6
PHYTOPATHOLOGY
Detection of Candidatus Liberibacter asiaticus associated with huanglongbing disease in the salivary glands and alimentary canal of Diaphorina citri
E. AMMAR (1), R. G. Shatters (1), D. G. Hall (1)
(1) ARS-USDA, Fort Pierce, FL, U.S.A.
Phytopathology 100:S6
Candidatus Liberibacter asiaticus has been strongly implicated as the
causative agent of huanglongbing (HLB), or citrus greening, which is the most
devastating citrus disease in Florida and other parts of the world. HLB is
transmitted in a persistent manner by psyllid vectors and in the U.S. and Asia
by the Asian citrus psyllid Diaphorina citri. We used quantitative polymerase
chain reaction (Q-PCR) to detect Ca. L. asiaticus in dissected organs of
individual D. citri adult males and females collected from HLB-infected citrus
trees in Florida between August and December 2009. The mean proportion of
PCR-positive organs was 13–24% for the alimentary canals, 12–16% for the
salivary glands, and 16–25% for the rest of the insect body. Percentage of
infection did not differ significantly between the three insect parts in males
but was significantly lower in the salivary glands than in the alimentary canals
of females. Our results provide the first PCR confirmation of Ca. L. asiaticus
in the alimentary canal and salivary glands of D. citri, and suggest that both
organs may constitute major transmission barriers to this bacterium in the
psyllid vector. We are currently testing other techniques including TEM,
immunolabeling, and in situ hybridization for the purpose of elucidating the
transmission barriers and cellular interactions of Ca. L. asiaticus in this
economically important vector.
Detection of Xylella fastidiosa in petioles is independent of sample storage
time and temperature
B. F. AMSDEN (1), P. Vincelli (1), J. R. Hartman (1)
(1) University of Kentucky, Lexington, KY, U.S.A.
Phytopathology 100:S6
Leaf petioles collected for isolation of Xylella fastidiosa are usually processed
within 12 hours of collection to optimize culturing the fastidious bacterium.
However, leaf samples collected from species of shade trees, horticultural
shrubs, and grape vines showing symptoms of bacterial leaf scorch are often
sent to our Diagnostic Laboratory several days after being collected. ELISApositive samples with weak to moderate ELISA scores will sometimes yield a
weak positive reaction by PCR, suggesting the possibility that sample
handling conditions may have detrimental effects on detection of X. fastidiosa.
Our sample storage study suggests that neither storage temperature (ambient
room temperature, 4°C, –20°C, or –80°C) nor duration of storage (≤24 hours
or 6 days) affects the ability to detect Xylella fastidiosa by real-time PCR in
petioles of shade trees, shrubs, and grape. The use of ELISA sample extract as
a source of X. fastidiosa DNA reduced the amount of time and effort required
to conduct PCR detection in bacterial leaf scorch suspects, compared to TE
bacterial release (pulverized infected tissue resuspended in TE buffer and used
directly in PCR reactions without DNA extraction) or total DNA purification
by QIAamp DNA Stool Kit methods.
Sporulation of Phomopsis viticola on infected grape tissues
D. J. ANCO (1), L. V. Madden (1), M. A. Ellis (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S6
Phomopsis viticola is the causal agent of Phomopsis cane and leaf spot on
Vitis spp. (grapes), which is a serious and economically important disease in
temperate regions. This disease is currently understood to be monocyclic, with
primary inoculum only being produced early in the growing season. The
objective of this study was to determine if secondary inoculum can be produced from primary infections on internodes and rachises during the growing
season. Infected first-year canes and rachises were collected throughout the
growing season of 2009 and observed for production of pycnidia and
sporulation after 48 h incubation in a mist chamber. Tissues were collected
from ‘Catawba,’ ‘Concord,’ and ‘Reliance’ vineyards and washed with
deionized water, and conidia were counted from these washings with a
hemacytometer. In 2009, the potential for lesions on infected grape canes to
produce conidia was not observed until after harvest. Very low amounts (e.g.,
1.5 alpha-conidia/mm2) of conidia were observed on some rachises during the
growing season; however, we were unable to isolate P. viticola from these
conidial suspensions. These results confirm that Phomopsis cane and leaf spot
is a monocyclic disease.
Effects of temperature and wetness duration on the sporulation rate of
Phomopsis viticola on infected grape canes
D. J. ANCO (1), L. V. Madden (1), M. A. Ellis (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S6
Phomopsis viticola is the causal agent of Phomopsis cane and leaf spot on
Vitis spp., a serious and economically important disease of grapes in
temperate regions. This disease is currently understood to be monocyclic, with
inoculum being produced early in the growing season. The objective of this
study was to examine the effects of temperature and wetness duration on the
sporulation rate of P. viticola in order to develop a predictive model for
sporulation in the vineyard. Infected first-year ‘Catawba’ canes were collected
in January, 2009, and stored at –20°C. Canes were placed in mist chambers
and incubated under various combinations of temperature and wetness
duration. Experimental design was a split-plot, with temperature (5, 12, 15,
18, 20, 22, 25, 28, and 35°C) as the main-plot and wetness duration (11, 23,
35, 47, and 71 h) as the sub-plot. Following incubation, canes were washed
with deionized water, and alpha-conidia were counted with a hemacytometer.
Results from 2009 indicate that sporulation of P. viticola on infected canes
can occur between 5 and 35°C, and is optimal near 22°C. Little to no
sporulation was observed after 11 h wetness duration from 5–35°C;
sporulation increased with increasing wetness duration at each temperature.
Regulatory role of c-di-GMP biosynthesis genes of Xylella fastidiosa’s
virulence factors
V. ANCONA (1), D. N. Appel (1), P. de Figueiredo (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S7
Xylella fastidosa (Xf) is a bacterial plant pathogen that has been recognized as
the causing agent of several plant diseases including Pierce’s disease of
grapevine (PD). Currently there are no commercial resistant varieties of grape
or an effective method for controlling PD. Therefore, this disease threatens the
U.S. grape and wine industries. Although Xf is known for causing PD, the
regulatory mechanisms that mediate virulence in the pathogen remain unclear.
Illuminating the molecular mechanisms mediating biofilm formation and
expression of virulence factors will promote the development of strategies for
controlling PD. Our project focuses on characterizing the role that cyclic
diguanylate (c-di-GMP) metabolic proteins play in regulating biofilm
formation, cell aggregation and virulence of Xf. c-di-GMP is a second
messenger that regulates aggregation, biofilm formation, and virulence in
several bacterial pathogens. This molecule is synthesized by diguanylate
cyclase enzymes (DGCs) and is degraded by phosphodiesterases (PDE). DGC
and PDE activities reside in the GGDEF, EAL or HD-GYP domains of
proteins respectively. A diverse array of bacteria harbor genes that encode
these enzymes. In fact, 5 genes have been identified in the Xf genome that are
predicted to encode proteins containing the conserved GGDEF, EAL and/or
HD-GYP domains. Mutations in these genes result in altered biofilm and
aggregation phenotypes suggesting a direct regulatory role on the expression
of Xf pathogenicity traits.
The soilborne pathogen, Phymatotrichopsis omnivora is the causal fungus of
cotton root rot of numerous dicots in the southwestern United States and
Mexico. Disease diagnosis depends on the presence of characteristic mycelial
cords on the roots of wilted plants, which are not always obvious. Early,
accurate and sensitive detection of P. omnivora in affected plant tissues is
needed by plant health officials for inspection of products from quarantined
states, and locally, by extension specialists to predict disease outbreaks and
identify reservoir hosts. Specific PCR primers recognizing conserved rDNAITS sequences were designed based on an alignment of 144 sequences from P.
omnivora isolates collected throughout North America. Three primer pairs,
PO1 (415 bp product), PO2 (499 bp product) and PO3 (146 bp product), were
validated in silico against published sequences and in vivo against infected
plant samples. PCR products were cloned and sequenced to confirm identity.
All primer sets allowed early detection of infected, asymptomatic plants. PO1,
PO2 and PO3 detected 5 × 10–7, 5 × 10–8 and 5 × 10–8 ng per µl of the P.
omnivora target DNA, respectively. PO3 also was compatible with qPCR and
thermostable helicase dependent amplification (tHDA) assays. The described
PCR assays should be useful for rapid, sensitive diagnosis, quantification of
infection in resistance breeding programs and agricultural biosecurity.
Highly sensitive molecular detection of five Pythium species
M. ARIF (1), F. Flores (1), F. M. Ochoa-Corona (1), C. Garzon (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S7
Detection and discrimination of Pythium species, the causal agents of root rot
and damping-off of seedlings, is difficult if based only on morphological
characteristics. Five sets of specific primers were designed from consensus
rDNA-ITS sequences of Pythium aphanidermatum, P. deliense, P. spinosum,
P. irregulare and P. cryptoirregulare retrieved from Genbank. The sequences
were aligned using ClustalX2 and were edited manually. Primer design was
accomplished using Web interface software Primer3, mFOLD and BLASTn
with validated thermodynamic parameters. Primers sets were validated in
silico against published sequences of the five species of Pythium, and in vitro
against isolates of the target species and related species. The amplified PCR
products were cloned and sequenced. Extensive PCR assays were conducted
to determine the sensitivity of each set of primers and optimal annealing
temperatures. The sensitivity of the primers for detection of P. aphanidermatum, P. deliense, P. spinosum, P. irregulare and P. cryptoirregulare is
of 10–5, 10–2, 10–6, 10–4 and 10–6 ng, respectively. This set of PCR assays
allows the rapid detection of, and discrimination among, five Pythium species
for applications in agricultural biosecurity and microbial forensics.
Biochemical and microscopical study of Phytophthora infestans process of
infection on Physalis peruviana
C. A. ANTOLÍNEZ (1), G. Danies (1), G. Peña (1), Á. M. Vargas (1), A. J.
Bernal (1), S. Restrepo (1)
(1) Univ De Los Andes, Bogota, COLOMBIA
Phytopathology 100:S7
Increased PCR amplification incorporating primer flap sequences and
free energy values near equilibrium
M. ARIF (1), I. Oikonomakos (1), D. Caasi (1), F. M. Ochoa-Corona (1)
(1) Oklahoma State University, Department of Entomology and Plant
Pathology, National Institute for Microbial Forensic and Food & Agricultural
Biosecurity (NIMFFAB), Stillwater, OK, U.S.A.
Phytopathology 100:S7
Phytophthora infestans is a plant pathogen that affects a great variety of crops
within the Solanaceae family. The pathogen has been described causing
disease in potato, tomato, lulo, tree tomato and in 2007 it was described
causing disease in Physalis peruviana (cape gooseberry). Since the report of
the disease, we have studied this particular interaction and we believe that the
infection process of cape gooseberry is different than in potatoes. The aim of
this work was to characterize the first defense reactions produced on cape
gooseberry leaves due to the infection with P. infestans. Detached Cape
gooseberry leaves were inoculated with a solution of 104 sporangia/ml.
Electron microscope photographs were taken at 0, 24, 48, 72 and 96 hours
post inoculation on the leaf abaxial surface up. Reactive oxygen species
(ROS) and induction of pathogen related proteins were measured at 0, 6, 12,
18, 24, 48 y 72 hours after inoculation and 6, 9, 12, 15 and 18 days after
inoculation. Phythopthora germinated and showed active aerial growing but
no evidence of penetration was found in the first days after the infection. It
seems that the cape goosberry ecotype used in this study is resistant to P.
infestans. To our knowledge this is the first study that characterizes the first
biochemical reactions caused by P. infestans on cape gooseberry. Our results
are fundamental for understanding how P. infestans affects other members of
the Solanaceae family.
Successful PCR amplifications rely on precise design of oligonucleotide
primers. Primer design still poses unresolved questions, particularly in studies
in which real time and/or end point PCR assays are resolved with SYBR
green. The aim of this study is to understand the effect on PCR yield of using
primers with minimal free energy (ΔG) and 5’ AT-rich overhanging
sequences. Specific primers for Pythium cryptoirregulare with ΔG=0 and a
second pair with ΔG=1 were designed based on consensus rDNA-ITS
sequences retrieved from Genbank. The sequences were aligned using
ClustalX2 and manually edited. Both set of primers were designed using
validated thermodynamic parameters and Web interface software Primer3,
mFOLD and BLASTn. Primers were validated in silico against published P.
cryptoirregulare sequences and the amplified products were cloned and
sequenced. No primer dimer formation was observed in PCR amplifications
using primers with ΔG=0. Dimer formation and SYBR green misleading
signals were observed with primers having ΔG=1 in real time PCR assays.
Primers with both minimal ΔG value and 5’ AT-rich overhangs showed
enhanced total PCR yields and accuracy. The described primer design
parameters have the potential for improving the efficiency of existing PCR
based assays and increasing the detection sensitivity of predetermined targets
in agricultural biosecurity and microbial forensics.
PCR detection and identification of Phymatotrichopsis omnivora
M. ARIF (1), S. M. Marek (1), F. M. Ochoa Corona (1), C. Young (2), C. D.
Garzon (1)
(1) Department of Entomology and Plant Pathology, Oklahoma State
University, Stillwater, OK, U.S.A.; (2) The Samuel Roberts Noble
Foundation, Ardmore, OK, U.S.A.
Phytopathology 100:S7
Expression profiling of host response of citrus to Candidatus Liberibacter
asiaticus infection
V. ARITUA (1), N. Wang (1)
(1) Citrus Research and Education Center, University of Florida, Lake Alfred,
FL, U.S.A.
Phytopathology 100:S7
Three species of Candidatus Liberibacter: C. Liberibacter asiaticus (Las), C.
Liberibacter africanus and C. Liberibacter americanus are associated with
Vol. 100, No. 6 (Supplement), 2010
S7
Huanglongbing disease of citrus. Of the three phloem-limited α-proteobacteria, Las has a worldwide distribution in citrus producing areas including
U.S. (Florida and Louisiana). A recent study indicates that Las infection
results into significant differences in response between citrus varieties or
relatives although none has been found to be resistant. Systemic infection
studies also show un-even distribution patterns of Las populations among
different organs and tissues of citrus. These results suggest that cellular response
to Las is genotype dependent and may be tissue specific as well. Since, the
molecular basis of the interaction is not yet fully elucidated, we are conducting transcriptional analyses of citrus varieties with varying degrees of response to Las infection at different infection stages in greenhouse and citrus
grove. We hypothesize that Las modulates different cellular processes in
citrus in time and space. Preliminary analysis of RNA from Las-infected
Valencia sweet orange (Citrus sinensis) roots using suppression subtractive
hybridization showed up-regulation of pathogenesis/resistance, biotic stress
related and cell wall re-modeling gene groups and transcriptional factors.
Some disease resistance genes homologous to nucleotide binding and Leucinerich repeat (NB-LRR) domain containing class of proteins were down regulated.
A bioinformatic study of pathogenicity factors in Xanthomonas
axonopodis pv. manihotis
M. L. ARRIETA (1), L. M. Rodriguez-R (1), R. Koebnik (2), S. Restrepo (1),
A. Bernal (1)
(1) Universidad de los Andes, Bogotá, COLOMBIA; (2) Institut de recherche
pour le développement, Montpellier, FRANCE
Phytopathology 100:S8
Xanthomonas axonopodis pv. manihotis (Xam) is a gram-negative bacterium
and the causal agent of Cassava Bacterial Bligth. Information about
pathogenicity determinants in this bacterium is currently limited in the
literature. The aim of this study was to increase the understanding of this
plant-pathogen interaction using genomics. A draft genome sequence of Xam
strain CIO151 was produced using second generation sequencing technology
and gene prediction was performed on the partial sequence. Based on
similarity analyses, putative protein annotation was performed. The G+C
content of the partial sequence (64.6%) was similar to that reported for other
species of Xanthomonas. Nevertheless, a significant part of genome sequence
(12.25%) showed an atypical G+C content, a fact that could suggest
horizontal gene transfer events. We report a preliminary set of effector
sequences in Xam, as well as the presence of sequences with similarity to
genes involved in regulation of pathogenicity factors (Rpf) and Xanthan
biosynthesis (gum). Also, putative proteins that could be involved in
pathogenicity, such as the ones involved in assembly of protein Secretion
Systems (Type I through III) were found. The partial genomic data here
suggest that Xam shares many pathogenicity factors with closely related
bacteria in the genus Xanthomonas. On the other hand, several changes in the
analyzed genes could account for host specificity differences with other
bacteria in the species X. axonopodis.
Effect of leaf age on primary infection and development of colonies of
strawberry powdery mildew
B. ASALF (1), A. Stensvand (2), D. M. Gadoury (3), R. C. Seem (3), A.
Tronsmo (2)
(1) Norwegian University of Life Sciences, Aas, NORWAY; (2) Aas,
NORWAY; (3) Geneva, NY, U.S.A.
Phytopathology 100:S8
Development of ontogenic resistance to powdery mildew (Podosphaera
aphanis) on strawberry leaves has been reported, however, the components of
resistance have not been elucidated. Five developmental stages of strawberry
leaves were identified and assigned numerical values from newly emerged and
unexpanded (S1) to fully expanded and dark green (S5) of cvs. Korona and
Senga Sengana. The upper and lower surface of the leaves were inoculated
from each of the five leaf developmental stages and incubated under
controlled conditions. The effect of leaf age on germination, infection
efficiency, latency period, and sporulation were later evaluated. All responses
were significantly (p = 0.05) affected by leaf age. Germination percentage,
infection efficiency, and sporulation were highest, and latent periods were
shortest on S1 leaves of both cultivars. On Senga Sengana, germinating
conidia produced fewer secondary hyphae during infection. Conidia produced
very few secondary hyphae and did not sporulate on S3 leaves, and no
infections established on S4 or S5 leaves. The high success of infection and
colonization of P. aphanis on S1 leaves indicates that disease is established
preferentially on emergent and expanding leaves and these should be the
target of management strategies.
Spinach seed a source of Verticillium dahliae in lettuce in coastal California
Z. K. ATALLAH (1), K. Maruthachalam (1), K. V. Subbarao (1)
(1) University of California-Davis, Salinas, CA, U.S.A.
Phytopathology 100:S8
S8
PHYTOPATHOLOGY
Fungal migrations in infested seed or vegetative material are cause for
concern and regulation. To clarify the mechanisms that led to the recurrent
epidemics of Verticillium wilt on lettuce in coastal California, we used 22
simple sequence repeat markers to retrace the evolutionary and migratory
histories of Verticillium dahliae. The markers were used on strains isolated
from: lettuce and other vegetable and small fruit plants growing coastal
California; tomato and lettuce seed from two inland California valleys;
spinach seed from northern Europe, Washington State and Chile; and
ornamentals from Wisconsin. There was no differentiation between the
spinach seed sub-populations and the other sub-populations, with the
exception of tomato, suggesting gene flow. Migration analyses also suggested
this gene flow from spinach seed sources. Haplotypes from coastal California
lettuce were assigned to spinach sub-populations, and similarly many spinach
seed haplotypes were assigned to non-self spinach sources. However, no
strains were assigned to the lettuce seed sub-population, including those
isolated from lettuce plants growing 60 Km away. Similarly, the structure of
the coastal California lettuce plant sub-population was similar to spinach seed
sub-populations, but not to other California sub-populations. This evidence of
migration corroborates the proposition that massive V. dahliae invasions in
germplasm shape population structures effectively regardless of the carrier
host germplasm.
Mitigation of aflatoxin contamination in Nigerian maize with atoxigenic
strain mixtures
J. ATEHNKENG (1), P. J. Cotty (2), R. Bandyopadhyay (1)
(1) International Institute for Tropical Agriculture, Ibadan, NIGERIA; (2)
University of Arizona, USDA ARS, Tucson, AZ, U.S.A.
Phytopathology 100:S8
In West Africa, aflatoxin contamination of maize is a frequent perennial problem resulting in serious impacts on health and trade. Competitive exclusion of
aflatoxin producers by atoxigenic strains of Aspergillus flavus is a viable
option for aflatoxin management. We evaluated the field efficacy of a mixture
of four atoxigenic strains in reducing aflatoxin contamination through displacement of aflatoxin producers in maize during the 2007 and 2008 seasons in four
states of Nigeria. Sterile sorghum grains were colonized independently by
each of four atoxigenic strains. After drying, colonized grain was mixed so
that the four strains were in equal proportions. The mixture was broadcast
over maize crops at 10 kg/ha 2–3 weeks before flowering. Grain from treated
and control fields were analysed for aflatoxins both at harvest and after
storage. In both years, the atoxigenic strain applications reduced both maize
contamination and the proportion of the crop infecting A. flavus population
composed of aflatoxin producers. Aflatoxin reductions of 67% to 95% were
associated with 74% to 80% displacement of aflatoxin producers. The results
demonstrate that effective atoxigenic strains native to West Africa can be
selected from fungal communities associated with maize production and successfully utilized in programs to mitigate aflatoxin exposure in human populations.
A flgB mutation in Xanthomonas axonopodis pv. glycines confers reduced
bacterial pustule disease of soybean
D. ATHINUWAT (1), T. J. Burr (1), S. Prathuangwong (2)
(1) Cornell University, Geneva, NY, U.S.A.; (2) Kasetsart University,
Bangkok, THAILAND
Phytopathology 100:S8
Xanthomonas axonopodis pv. glycines KU-P-SW005, the cause of bacterial
pustule disease on soybean, has a single monopolar flagellum that is
associated with swimming motility, biofilm formation, and virulence on
soybean. A targeted mutation in flgB, encode a flagella basal body rod protein
was generated with an EZ::TN transposome system. The flgB mutant lacked
flagellum and was swimming-minus. Furthermore, it generated an altered
biofilm (less robust than wildtype) and caused reduced disease severity on
soybean. Following inoculation of cv. Spencer, the flgB mutant gave a
virulence rating of 9.89% as compared to 51.98% for wildtype at 10 days post
inoculation. Complemented flgB mutant restored swimming motility, biofilm
and disease equal to the wildtype.
Control of peanut rust with fungicides in Nicaragua
J. AUGUSTO (1), T. B. Brenneman (1)
(1) University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S8
Peanut rust (caused by Puccinia arachidis) is an important disease that limits
peanut production in Nicaragua. Several fungicide programs were evaluated
from 2005 to 2009 in randomized block designs at six locations planted with
cv. Georgia Green. Four applications of tebuconazole formulations at 0.23 kg
a.i./ha (Tacora 25 EW, Folicur 25 EW, Orius 25 EW, and Tebuconazole 25%)
and chlorothalonil at 0.96 kg a.i./ha (Chlorothalonil 720) did not differ in rust
control, and all were effective in reducing disease severity. Tebuconazole
formulations had similar yields but higher than chlorothalonil. Pyraclostrobin
+ epoxiconazole (Opera, 0.14 kg a.i./ha), tebuconazole (Tebuconazole 25%),
epoxiconazole + carbendazim (Duett, 0.18 kg a.i./ha), azoxystrobin +
cyproconazole (Amistar Xtra, 0.10 kg a.i./ha), and chlorothalonil applied
twice either at night (3 to 5 a.m., when leaves were folded) or during the day
(10 a.m. to 12 p.m., when leaves were unfolded) all decreased rust severity
compared with the control, and application timing were not different. Night
fungicide applications generally had higher yields than day applications
presumably due to improved control of stem rot (Sclerotium rolfsii). Applying
pyraclostrobin + epoxiconazole, azoxystrobin + cyproconazole, picoxystrobin
(Acapela 25 SC, 0.10 kg a.i./ha), and trifloxystrobin + propiconazole
(Stratego, 0.16 kg a.i./ha) at applications 2 and 3, vs. 3 and 4, vs. 2 and 4 gave
similar rust control. Opera was generally the most effective fungicide for rust
control and yield increase.
Molecular characterization of resistance to boscalid and penthiopyrad in
Didymella bryoniae isolates collected from Georgia watermelon fields
H. F. AVENOT (1), A. Thomas (1), R. D. Gitaitis (1), D. B. Langston (1), K.
L. Stevenson (1)
(1) University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S9
In order to elucidate the genetic basis of resistance to the succinatedehydrogenase-inhibiting fungicides (SDHIs) boscalid and penthiopyrad in
field isolates of the gummy stem blight pathogen Didymella bryoniae, a 506bp fragment of the iron sulphur gene (DbSDHB) was amplified from a
fungicide-sensitive isolate of D. bryoniae. The deduced amino-acid sequence
showed high similarity with iron sulphur proteins (Ip) from other organisms.
A primer pair was designed from the obtained sequence and used to
specifically amplify the region containing the highly conserved histidine
residue known to confer resistance to SDHIs in several fungal species.
Subsequent comparison of the corresponding sequences of DbSDHB from
sensitive and resistant isolates revealed that the precise histidine residue
(codon CAC) was present in 12 sensitive isolates, but in 73 boscalid- and
penthiopyrad-resistant isolates the histidine codon was converted to tyrosine
(codon TAC). In 7 other isolates that were found to be resistant to boscalid,
but unpredictably sensitive to penthiopyrad, the histidine residue was replaced
by arginine (codon CGC). These findings seem to indicate that while the
histidine-tyrosine mutation conferred resistance to both fungicides, the
replacement of histidine by arginine conferred resistance to boscalid, but had
no impact on penthiopyrad sensitivity. The polymorphisms thus revealed in
DbSDHB sequences will be used to develop rapid molecular diagnostic assays
to detect SDHI resistance in this pathogen.
Validating environmental parameters for primary infection of grapes by
Erysiphe necator ascospores under Michigan conditions
L. L. AVILA (1), A. R. Sullenger (1), J. Kroll (2), N. L. Rothwell (2), A. M.
C. Schilder (1)
(1) Michigan State University, East Lansing, MI, U.S.A.; (2) Michigan State
University, Traverse City, MI, U.S.A.
Phytopathology 100:S9
Powdery mildew, caused by Erysiphe necator, is a common and widespread
disease of grapevines. In the spring, the fungus releases ascospores from
overwintering cleistothecia after 2.5 mm of rain and temperatures of 10°C or
above. To improve disease development predictions, ascospore release and
primary infection were monitored in unsprayed areas of research vineyards in
Clarksville and Traverse City, MI. Campbell weather stations were used to
monitor environmental conditions at the sites. Burkard spore traps (both
vineyards) and potted cv. Chardonnay bait plants (Clarksville only) were
placed between vines from before bud break until fruit set. Burkard reels and
plants were changed weekly and ascospores and powdery mildew colonies
counted. Ascospores were detected in the air for more than a week after rain
events from May until the end of June. Peak ascospore release occurred in
Clarksville on May 22–26 and in Traverse City on June 9, 2009. The presence
of powdery mildew colonies on bait plants also indicated ascospore activity
since early May. However, infections of field-grown vines did not become
evident until late June in Clarksville and late August in Traverse City,
indicating a discrepancy between the presence of primary inoculum and
disease development. This may be related to the exceptionally cool and rainy
2009 growing season. Further investigation is needed to better predict
powdery mildew development under Michigan weather conditions.
Screening for strobilurin (QoI) resistance in grape powdery mildew
populations in Michigan
L. L. AVILA (1), T. D. Miles (1), W. W. Kirk (1), A. M. C. Schilder (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S9
Powdery mildew, caused by Erysiphe necator, is the most common and
destructive disease of grapes worldwide. In Michigan, it is primarily
controlled with fungicides, including strobilurins (Quinone outside Inhibitors
[QoIs]). Resistance to this class of fungicides has been reported in E. necator
in New York and Virginia. To determine whether QoI resistance occurs in
Michigan, 12 E. necator isolates were collected from 5 vineyards in 2008 and
tested by PCR for the G143A single-nucleotide mutation responsible for QoI
resistance. The mutation was detected in one isolate, which was confirmed to
be resistant in a conidium germination assay on water agar amended with
trifloxystrobin at 0, 0.001, 0.01, 0.1, 1, 10, or 100 µg/mL and
salicylhydroxamic acid (100 µg/mL). The mutant was able to germinate at
100 µg/mL, whereas a representative wildtype isolate did not germinate
beyond 0.01 µg/mL. In 2009, 173 isolates were collected from 2 vineyards
with no fungicide application history, and 14 commercial and 6 research
vineyards. Isolates were screened for QoI resistance as described above.
Isolates in unsprayed vineyards had EC50 values mostly below 0.01 µg/mL,
while isolates that were highly resistant to trifloxystrobin (EC50>100 µg/mL)
occurred in 4 commercial and 5 research vineyards at frequencies of 11–50%
and 50–100%, respectively. These results suggest that fungicide resistance
may play a role in poor control of powdery mildew observed in some
Michigan vineyards.
Effects of extracts of some plants on the wet rot of Amaranthus cruentus L.
induced by Choanephora cucurbitarum and on the performance of the crop
A. N. AWURUM (1)
(1) Michael Okpara Univ of Agriculture, Umdike, Umuahia, Abia State,
NIGERIA
Phytopathology 100:S9
Amaranthus is one of the important leafy vegetable crops in Nigeria. The crop
sustains severe losses due to wet rot induced by Choanephora cucurbitarum.
Limitations in the use of synthetic chemicals in the control of horticultural
crop diseases continuously increase due to their negative environmental
impact. This has shifted attention to safer materials. The evaluation of the
efficacy of leaf extracts of Dennettia tripetala, Spondias mombin and
Bryophyllum pinnatum in controlling the growth of Choanephora
cucurbitarum was carried out in the greenhouse. The experiment was a 3 × 5
factorial in a completely randomized design (CRD) replicated 5 times. The
treatments comprised 3 methods of inoculation of the pathogen; un-inoculated
seeds, inoculated seeds and inoculated plants at 6 weeks. The chemicals were
benomyl a synthetic fungicide, extracts from different plants and sterile water
as control. From the result of the experiment D. tripetala significantly (P <
0.05) reduced the severity of the wet rot induced by C. cucurbitarum more
than the other plant extracts having a severity score of 5.8 in a 10 – point
scale, but the effect was not significantly (P < 0.05) different from that of
benomyl. Among the plant extracts, B. pinnatum was the least effective with a
severity score of 8.0. Plants in the control pot had the highest severity score of
9.8. The plants treated with benomyl and the plant extracts grew better and
had significantly higher total plant dry matter than plants in the control pots.
Downy mildew of basil in Illinois
M. BABADOOST (1)
(1) University of Illinois, Urbana, IL, U.S.A.
Phytopathology 100:S9
Downy mildew of basil, caused by Peronospora belbahrii, first occurred in
Illinois in 2009. The disease was detected in commercial fields throughout the
state. The first report of the disease in the United States was from Florida in
2007. P. belbahrii infects leaves, rapidly develops and spreads, and can cause
total crop loss. A trial was conducted in a commercial basil field at Momence,
Illinois, during September-October 2009 to evaluate the efficacy of selected
fungicides for control of downy mildew of basil. The trial included 12
treatments, which were performed in a randomized complete block design
with four replications. The field had been planted in April 2009 and plants
were actively growing with moderate infection of downy mildew on leaves.
Four spray-applications of the fungicides were made at 7-day intervals and
disease severity in the plots was assessed 7 days after the last spray
application. Severity of downy mildew was less than 4% in the plots sprayed
with chlorothalonil (Bravo Weather Stik), dimethomorph (Forum), zaxomide
+ mancozeb (Gavel), fluopicolide (Presidio), azoxystrobin (Quadris),
cyazofamid (Ranman), mandipropamid (Revus), and famoxadone +
cymoxanil (Tanos), when these fungicides were mixed with potassium
phosphite (ProPhyt). Severity of the disease was the lowest (1%) in plots
sprayed with Quadris; was highest (38.1%) in control plots. Severity of the
disease was 18.1% in the plots sprayed with an organic mix.
The Egestion-Salivation Hypothesis: Evidence for the role of vector saliva
in the inoculation mechanism of Xylella fastidiosa
E. A. BACKUS (1), K. Andrews (2), J. M. Labavitch (3), C. Greve (3)
(1) USDA ARS, Parlier, CA, U.S.A.; (2) Australian Dept. of Primary Industries,
Attwood, AUSTRALIA; (3) University of California-Davis, Davis, CA, U.S.A.
Phytopathology 100:S9
Despite ca. 40 years of study, the mechanism of inoculation of the Pierce’s
Disease bacterium, Xylella fastidiosa (Xf), by vectors such as the glassyVol. 100, No. 6 (Supplement), 2010
S9
winged sharpshooter (GWSS) is still unknown. Research on the EgestionSalivation Hypothesis for Xf inoculation will be presented. Two important
steps in this hypothesis are uptake of saliva containing the cell wall-degrading
enzyme beta-1,4 glucanase into the precibarium where Xf colonies develop,
followed by injection of this enzyme-containing saliva into the xylem prior to
ingestion. To directly test the role of saliva in inoculation, immunohistology
was used to study interactions between Xf and GWSS saliva in grapevine.
Adult GWSS were confined in small cages on grapevine stems for 24 hours
and allowed to probe, leaving salivary deposits in the plant. Xf was then
needle-inoculated into the same stem area; 1 hour later, the tissue was excised
and prepared for immunohistology using a commercial Xf probe. Xf bacteria
observed in xylem cells penetrated the semi-viscous saliva deposited during
GWSS probing prior to Xf inoculation. Therefore, Xf bacteria have the ability
to infiltrate gelled saliva containing salivary glucanase. This suggests that, in a
natural GWSS inoculation, Xf could potentially migrate out of injected saliva
and into xylem fluid. Implications for the mechanism of inoculation are
discussed.
Tospoviruses cause serious diseases in several important crop plants. The
genome of tospoviruses consists of three RNAs, large (L), medium (M) and
small (S). The L RNA is organized in negative sense orientation, whereas M
and S RNAs are in ambisense. The S RNA codes for a non structural protein
(NSs) in sense direction which was shown to function as viral suppressor of
gene silencing in plants. We used datura (Datura stramonium) as a differential
host for two distinct tospovirus species, Iris yellow spot virus (IYSV) and
Tomato spotted wilt virus (TSWV). Following mechanical inoculation of
datura, TSWV causes systemic infection, whereas IYSV infection of datura
remains localized to inoculated leaves. We demonstrate that, in a mixed
infection, TSWV facilitates the systemic movement of only the NSs gene of
IYSV, and the systemic symptoms produced by TSWV in the presence of the
IYSV silencing suppressor are more severe than those caused by TSWV
infection alone. The selective movement of the silencing suppressor gene of
one tospovirus species into younger, uninoculated leaves of an otherwise restrictive host suggests complementation between two distinct tospovirus species.
Production of mycotoxins by members of the Aspergillus section Nigri
isolated from peanuts and maize in the United States
C. Bacon (1), E. PALENCIA (2)
(1) USDA ARS, Athens, GA, U.S.A.; (2) Department of Plant Pathology,
University of Georgia, Athens, GA, U.S.A.
Phytopathology 100:S10
DNA microarray based universal plant virus detection and identification
B. BAGEWADI (1), D. C. Henderson (2), R. L. Jordan (2), K. L. Perry (3), U.
Melcher (4), D. Wang (5), K. Fischer (6), J. Hammond (2), C. M. Fauquet (5)
(1) Donald Danforth Plant Science Center, St. Louis, MO, U.S.A.; (2)
Beltsville, MD, U.S.A.; (3) Ithaca, NY, U.S.A.; (4) Stillwater, OK, U.S.A.; (5)
St. Louis, MO, U.S.A.; (6) Salt Lake City, UT, U.S.A.
Phytopathology 100:S10
Fungi of the Aspergillus section Nigri (black aspergilli) are pathogenic to
maize, grapes, onions, garlic, apples, mangoes, and peanuts. Although some
black aspergilli are reported as opportunistic pathogens, other species are able
to colonize maize seedlings as symptomless endophytes, which under stress,
can develop symptoms of seedling blight or later on symptoms similar to
Fusarium ear rot disease. The main concern for crops infected by black
aspergilli is the production of toxic secondary metabolites. Ochratoxin A, the
fumonisins, and penicillic acid are examples of these metabolites that are
carcinogenic to animals and are thereby classified as mycotoxins. The aim of
this research was to screen 60 field black Aspergillus strains, isolated as
asymptomatic endophytes from peanut and maize, for production of these
mycotoxins. HPLC-MS/MS analysis detected the production of ochratoxin A,
fumonisin B1, and penicillic acid when strains were cultured on maize, wheat,
and rye seeds. Our results indicated that the most dominant species isolated
was A. niger var. niger, and less than 20% of the field isolates were able to
produce ochratoxin A, while less than 10% produced fumonisins B1. Pencillic
acid was produced in high amounts (> 10 ppm) by these isolates, which is the
first report for the production of this mycotoxin by members of the Nigri
section. The fumonisins and penicillic acid are also phytotoxic and might play
roles in diseases of peanuts and maize.
To keep pace with the ever increasing number of plant viruses an effective
detection and identification system is essential to prevent the introduction and
spread of potentially devastating plant epidemics. DNA microarray methods
based on oligonucleotide probes offer cheap, rapid, reliable, and parallel
detection of plant pathogens including viruses. Previously published studies
have focused either on a single crop or family of plant viruses. To prototype
the development of taxonomy based DNA microarray diagnostic system for
all the plant virus species and sub-viral entities, we selected conserved
multiple probes for 54 plant viruses representing 17 virus families/groups and
printed on poly-lysine coated glass slides. For both RNA and DNA plant
viruses we used 2–5 µg of total RNA extracted from virus infected N.
benthamiana or host plants to post label with Cy3 fluorescent dye. Slides were
scanned using Axon 4000B scanner and spots were quantified using Genepix
Pro software. Using the prototype DemoPlantVirusChip a total of 22 plant
viruses (e.g., ACMV, TGMV, CMV, PVX, PVY and TMV) were detected
over a wide dynamic range with a sensitivity of 5 ng of amino-allyl labeled
DNA. As a pilot test, we are now testing this chip with 200 samples from a
potato gene bank from CIP and with 140 plant infected samples from ATCC.
In the phase II, we are now developing the full version of the plant virus chip
with 60-mer probes for every taxon/node of all plant viruses.
Relationship of substrate and surfactin production by Bacillus mojavensis
strains and their antagonistical response to Fusarium verticillioides
C. W. BACON (1), D. M. Hinton (2), T. Mitchell (2), M. Snook (2)
(1) USDA ARS, Athens, GA, U.S.A.; (2) USDA, ARS, Russell Research
Center, Athens, GA, U.S.A.
Phytopathology 100:S10
RT-qPCR analysis of genes associated with chestnut blight in susceptible
American and resistant Chinese chestnut trees
K. BAIER (1), C. A. Maynard (1), W. A. Powell (1)
(1) SUNY ESF, Syracuse, NY, U.S.A.
Phytopathology 100:S10
The endophytic bacterium, Bacillus mojavensis, RRC 101 controls fungal
diseases in maize and other plants. The bacterium and its cultural extracts
have been shown to be antagonistic to the pathogenic and mycotoxic fungus,
Fusarium verticillioides. An antifungal lipopeptide produced by B. mojavensis
strains in culture was identified as surfactin, a biosurfactant. HPLC-MS
spectra analyses indicated that B. mojavensis, RRC 101, produced Leu7surfactin as the major surfactin, although in the surfactin complex C-14 and
C-15 isoforms dominated. Bacterial strains and culture media can have a
direct effect on antifungal antagonism, surfactant production, and metabolic
utilization by strains of B. mojavensis. In this investigation, B. mojavensis
strains were screened to determine the effects of media on antagonism to F.
verticillioides, surfactant production, and metabolic utilization of key
substrates. The data indicated that the bacterial strains showed zero to high
levels of antagonisms, which were not correlated with total surfactin
production. Thus, the data suggest that either there are synergistic effects from
specific isoforms of surfactins produced on agar media or there are also some
unidentified biosurfactants or there areother inhibitory compounds yet
determined.
Genetic complementation between two viruses facilitates the systemic
movement of a gene silencing suppressor in an otherwise restrictive host
S. BAG (1), N. Mitter (2), H. R. Pappu (1)
(1) Department of Plant Pathology, Washington State University, Pullman,
WA, U.S.A.; (2) Agri-Science Queensland, Department of Employment,
Economic Development and Innovation, Queensland Agricultural Biotechnology Centre, St. Lucia, Qld 4067, AUSTRALIA
Phytopathology 100:S10
S10
PHYTOPATHOLOGY
American chestnut (Castanea dentata) is highly susceptible to infection caused
by the necrotrophic fungus, Cryphonectria parasitica. Closely related Chinese
chestnut (C. mollissima) exhibits a natural resistance to blight. Genes
commonly induced during plant defense response to infection, including genes
for −1,3−glucanase, cinnamyl alcohol dehydrogenase (CAD) and laccase,
were preliminarily identified as differentially expressed between Chinese and
American chestnut using suppression subtractive hybridization (SSH). Fulllength cDNA sequences of these genes have been identified in both chestnut
species using the Fagaceae genomic website (http://www.fagaceae.org/home).
Genes from the two species are very similar in DNA sequence, therefore it is
likely the expression patterns of specific genes is important to conferring the
blight-resistance in Chinese chestnut. Reverse transcription quantitative
polymerase chain reaction (RT-qPCR) was used to confirm and quantify
differential gene expression between American and Chinese chestnut stem
tissues. Results indicate there is significantly higher expression of some
defense related genes in stems of Chinese chestnut than in American chestnut.
Laccase, for example, had several hundred fold higher expression in Chinese
chestnut seedlings. Genes more highly expressed in Chinese chestnut would
be good candidates for use in genetic modification of American chestnut to
determine if they can enhance resistance to chestnut blight.
Evaluation of nitric oxide detoxifying flavohaemoglobin in the Fusarium
verticillioides – maize interaction
T. T. BALDWIN (1), A. E. Glenn (1)
(1) USDA, ARS, Toxicology & Mycotoxin Research Unit, Athens, GA, U.S.A.
Phytopathology 100:S10
Fusarium verticillioides is a non-obligate pathogen causing a number of maize
diseases. Apart from these diseases, F. verticillioides is also known to
asymptomatically infect most tissues of the plant. The production of the
mycotoxin fumonisin B1 by F. verticillioides and other complexities of the
interactions with maize may contribute to the dual nature of this symbiont.
One possible determinate of pathogenesis in the F. verticillioides – maize
interaction could be the regulation and signaling by Reactive Nitrogen Species
(RNS), specifically nitric oxide (NO). Detoxification of NO has been shown
to be a pathogenicity factor for the fungal human pathogen Candida albicans
and the bacterial plant pathogen Erwinia chrysanthemi. Both possess a
flavohaemoglobin, encoded by CaYHB1 and HmpX, respectively, that was
determined to be responsible for this detoxification. BLASTP search of the
Fusarium comparative genomes (Broad Institute) using these two genes
revealed two putative homologs in F. verticillioides, denoted NOD1 and
NOD2 (for Nitric Oxide Dioxygenase). To determine the function of NOD1
and NOD2, each gene was individually deleted in F. verticillioides using PEG
mediated transformation and homologous recombination. Mutants will be
evaluated for their ability to detoxify NO and for virulence against maize
seedlings. Understanding the function of these genes will give insight into the
role of NO in the F. verticillioides – maize interaction.
Use of lesioned mutants to characterize the genetic network underlying
control of the maize hypersensitive response
P. BALINT-KURTI (1), C. Weil (2), S. Chintamanani (3), R. Dhawan (4), A.
Negeri (4), A. Garg (3), B. Venkata (3), J. Green (5), J. Harnsomburana (5), J.
Palmer (2), V. Chaikam (2), C. Shyu (5), G. Johal (3)
(1) USDA-ARS, North Carolina State University, Raleigh, NC, U.S.A.; (2)
Department of Agronomy, Purdue University, West Lafayette, IN, U.S.A.; (3)
Department of Botany and Plant Pathology, Purdue University, West
Lafayette, IN, U.S.A.; (4) Dept. of Plant Pathology, NCSU, Raleigh, NC,
U.S.A.; (5) Informatics Institute, University of Missouri-Columbia, Columbia,
MO, U.S.A.
Phytopathology 100:S11
The hypersensitive response (HR) is the most important defense response in
plants, but details of how it is controlled and executed remain patchy. We
used a novel genetic technique called MAGIC (Mutant-Assisted Gene Identification and Characterization) to identify an HR-modulating locus in maize.
MAGIC facilitates identification of naturally-occurring alleles underlying
phenotypic variation from diverse germplasm using a mutant phenotype as a
“reporter”. In this case the reporter phenotype is caused by a partiallydominant autoactive disease resistance gene, Rp1-D21, which causes HR
lesions to form spontaneously. Genetic background profoundly affected the
Rp1-D21 phenotype. B73 and Mo17 partially suppressed and enhanced the
Rp1-D21 phenotype, respectively. By crossing the Rp1-D21 gene into a maize
recombinant inbred line (RIL) mapping population, we were able to map and
identify Hrml1 (HR-modulating locus 1), a locus responsible for modulating
the Rp1-D21 phenotype, on chromosome 10. Loci with smaller effects were
identified on chromosomes 1 and 9 (Genetics, in press). We are now
extending these studies with much larger, mapping populations to uncover
additional Hrml loci and to clone the underlying genes (funded by NSF grant
#0822495). Furthermore, we are using computational image analysis to
characterize the phenotypic expression of Rp1-D21 in diverse germplasm in
different environments. We expect these studies to lead to a deeper
understanding of the genetic network controlling the HR response in plants.
A protein localization and interaction map for Potato yellow dwarf virus,
a plant-adapted nucleorhabdovirus
A. BANDYOPADHYAY (1), K. Kopperud (1), G. Anderson (1), K. Martin
(1), M. Goodin (1)
(1) University of Kentucky, Lexington, KY, U.S.A.
Phytopathology 100:S11
The complete genome of the type species of the genus Nucleorhabdovirus,
Potato yellow dwarf virus (PYDV), was sequenced and functional protein
assays were used to determine the subcellular localization of the proteins
encoded by the virus. The antigenome of PYDV consists of 12,875
nucleotides and encodes 7 open reading frames (ORFs) equivalent to N
(nucleocapsid), X (unknown), P (phosphoprotein), Y (unknown), M (matrix
protein), G (glycoprotein) and L (polymerase) genes. The ORFs are separated
by conserved intergenic junctions and are flanked by leader and trailer
sequences, which are 149 and 91 nucleotides in length, respectively. When
expressed in plant cells, the PYDV N, P and M proteins localized to nuclei,
yet these proteins do not contain any predictable nuclear localization signals.
In addition, the M protein was shown to induce the intranuclear accumulation
of the inner nuclear membrane when expressed in the absence of any other
viral protein. Bimolecular fluorescence complementation was used to generate
the most comprehensive protein interaction map for a plant-adapted
rhabdovirus to date. Furthermore, phylogenic analyses of L proteins indicated
that PYDV is most closely related to the leafhopper-transmitted
rhabdoviruses, which formed a clade distinct from those transmitted by aphids
or planthoppers.
Mapping of genes for brown spot (Bipolaris oryzae) disease resistance in
rice
S. P. BANU (1), B. Meah (2), D. S. Brar (3), H. Leung (3), C. M. VeraCruz
(3)
(1) Bangladesh Agricultural Research Institute, Gazipur, BANGLADESH; (2)
Bangladesh Agricultural University, Mymensingh, BANGLADESH; (3)
International Rice Research Institute, PHILIPPINES
Phytopathology 100:S11
Brown spot (Bipolaris oryzae) is one of the major fungal diseases of rice and
distributed world wide. A combination of genetic, molecular and pathological
approaches was used in this study to identify and map novel, brown spot
resistant genes. Traditional japonica cultivar Dinorado (resistant) crossed with
semi dwarf modern indica cultivar IR 36 (susceptible). Phenotypic segregation
of 200 F3 progenies suggested that resistance to brown spot of rice is governed
by two recessive genes with 1 (homozygous resistant) : 8 (heterozygous) : 7
(homozygous susceptible) ratio. In addition, corresponding F2 progenies were
used as mapping populations to identify DNA markers associated with
resistance. Bulked segregant analysis was applied to analyze 186 F2 lines with
160 SSR markers distributed equally over each of the 12 chromosomes of rice
genome. Marker analysis showed significant association (<.0001) for 4
markers and explained 16.53–48.17% of the total phenotypic variation for
brown spot resistance. The interval analysis suggested that genes for
resistance to brown spot are located between 8.7 and 18.2 MB in chromosome
12. The two genes imparting resistance to brown spot in Dinorado are
designated as bs1 and bs2. The present findings would provide guidelines to
incorporate resistance from Dinorado to susceptible but otherwise high
yielding cultivars of rice. The closely linked DNA markers would be suitable
for use in marker-assisted selection in rice breeding programs.
Influence of temperature in the aquisition of ‘Candidatus Liberibacter
americanus’ by Diaphorina citri
J. C. BARBOSA (1), B. Eckstein (1), M. Gasparoto (1), R. da Silva (2), J.
Belasque Jr. (2), A. Bergamin Filho (1)
(1) Esalq-USP, Piracicaba, BRAZIL; (2) Fundecitrus, Araraquara, BRAZIL
Phytopathology 100:S11
Huanglongbing is one of the most destructive diseases in citris in the world.
Two species were detected in affected trees in Brazil: ‘Candidatus
Liberibacter asiaticus’ and ‘Ca. L. americanus’ (CAM). Both species are
transmitted by the psyllid Diaphorina citri. Studies carried out in Brazil
suggest that CAM is sensitive to higher temperatures, and this characteristic
have been associated to the reduction of the CAM infected plants incidence in
São Paulo state, Brazil. However, the influence of temperature in the
transmission of CAM by the psyllid is unknown. The objective of this work
was to verify the influence of temperature in the acquisition of CAM by
psyllids. In growth chamber, the acquisition of CAM by psyllids was analysed
under three conditions of temperatures (20/22°C, 25/27°C and 30/32°C) in a
12 h photoperiod. For each temperature condition, groups of adult psyllids
were caged on CAM infected citrus plants for an acquisition access period of
four days. Psyllids were then transferred to citrus test-plants (Citrus limonia
Osbeck). In each test-plant, one insect was confined for an inoculation access
period of 24 days. After that, insects were collected and individually analyzed
by PCR. Test-plants are kept in growth chamber at 25°C for future analyses.
Under 20/25°C, the acquisition efficiency of CAM by psyllids was higher
(49,21%) than at 25/27°C (40,97%) and 30/32°C (24,99%). Probably, these
results may help explain the low incidence of CAM infected plants in
Brazilian orchards nowadays.
Reliability and accuracy of visual methods used to quantify foliar
symptoms of bacterial spot of peach and nectarine
S. J. Bardsley (1), H. K. NGUGI (1)
(1) Penn State University, Biglerville, PA, U.S.A.
Phytopathology 100:S11
Bacterial spot caused by Xanthomonas arboricola pv. pruni is the most
important bacterial disease of peach and nectarine in Pennsylvania and severe
epidemics can result in 100% yield loss. Studies on bacterial spot
epidemiology rely on the quality of visual estimates of disease severity. The
objective of this study was to assess the reliability and accuracy of visual
estimates of bacterial spot severity compared to those of computer image
analysis. Three sets of leaves (n = 103, 103, and 104 leaves) with disease
severity levels ranging from 0 to 100% were assessed twice by one
experienced rater using direct visual estimation of percent leaf area covered
by symptoms. The leaves were also rated on a 1 to 7 rating scale (1 = 0%
symptomatic area, and 7 = >45%) by the same rater. The same leaves were
also assessed with the APS Assess image analysis software. Based on Lin’s
concordance analysis, direct estimation was more accurate than the use of the
rating scale with Lin’s concordance coefficient (ρc) values of 0.962, 0.957,
and 0.945, respectively, for the three sets of leaf samples compared with
0.865, 0.921, and 0.816 for estimates based on the rating scale. Direct
Vol. 100, No. 6 (Supplement), 2010
S11
estimation was more reliable, than the rating scale with the precision
coefficient (r) values of 0.986, 0.990, and 0.948 compared with 0.962,
0.965, and 0.919 for the rating scale. Bacterial spot severity estimates based
on direct estimation are more accurate and reliable than those based on the
rating scale.
Effects of crop and environmental variables on sugarcane brown rust
epidemics in Louisiana
W. BARRERA (1)
(1) Louisiana State University, Baton Rouge, LA, U.S.A.
Phytopathology 100:S12
Brown rust, caused by Puccinia melanocephala, is one of the most important
diseases of sugarcane worldwide. In Louisiana, yield losses exceeding 20%
have been documented. Resistance has been the major means of disease
control. However, pathogen adaptability can adversely affect resistance
durability. Replacing cultivars during periodic disease outbreaks can be
difficult due to a multiple year crop cycle and limited seedcane availability.
The use of fungicides to limit losses has shown promising results. However,
their use requires adequate understanding of the conditions leading to disease
development to maximize the economic benefit. The objective is to determine
the combination of crop growth characteristics and environmental factors that
result in severe brown rust epidemics. The goals are to develop a model that
describes disease progress and a forecasting system that provides for timely
fungicide applications. Variables monitored at two locations during 2009
included leaf wetness, relative humidity, rainfall, ambient temperature,
temperature at the leaf surface, wind direction, wind speed, solar radiation,
plant height, shoot population, number of leaves per shoot, and crop canopy
cover. Disease was assessed weekly as area exhibiting disease symptoms on
selected leaves and percentage of pustules with sporulation. Variables most
highly correlated with disease severity were shoot population, leaf wetness,
and temperature inside the canopy. Additional results will be presented and
discussed.
Chilling injury in tomatoes exposed to low temperatures in the field
J. A. BARTZ (1), M. A. Ritenour (2), M. Elkahky (3)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) University of
Florida/Indian River Research and Education Center, Fort Pierce, FL, U.S.A.;
(3) Plant Pathology Dept./University of Florida, Gainesville, FL, U.S.A.
Phytopathology 100:S12
Winter tomato production in 2010 was marked by prolonged periods of low
temperatures in the field. Harvested fruit exhibited surface pitting, puffiness
and cat-facing. Ripened fruit developed high decay incidences (>20%) of
Rhizopus rot and black rot (Alternaria). More decay was observed among fruit
stored at higher humidity (>95% versus 90%). The final color of such fruit
was marred by severe white/gold speckling in the periderm, which has been
identified as calcium crystals (likely calcium oxalate). At least one tomato
shipment was reported to have been rejected at a receiving point due to severe
speckling. White speckling was reported in green fruit and gold in red fruit.
We observed that removal of the surface tissue layers over the crystals
changed the perceived color from gold or yellow to white. Calcium oxalate
crystals are pyramids or needle shaped raphides. The latter cause damage to
adjacent parenchyma cells as the fruit are jostled during harvest and handling.
The damage promotes postharvest decays. Speckled fruit were reported to
have a reduced shelf-life. Previously, speckles in tomato fruit was associated
with reduced transpiration, high humidity, and warmer temperatures in the
field or greenhouse and fertilizer ratios that increased calcium uptake by the
plant. This is the first report that speckles can accompany chilling or near
chilling temperatures during fruit production.
Controlling of fire blight on popular apple cultivars with M9 rootstock
K. BASTAS (1)
(1) Selcuk University Faculty of Agriculture, Konya, TURKEY
Phytopathology 100:S12
The shoot blight phase of fire blight caused by Erwinia amylovora is highly
destructive within the current and subsequent growing seasons. This study
gives an assessment of the effect of different preparats, harpin protein (Hp),
aminobutryric acid+L-glutamic acid (ABA), copper oxychloride, copper
sulphate pentahydrate, phosphoroz acid, Ca-nitrate, foshetyl-Al, prohexadione-Ca (PC), Bacillus subtilis, B. subtilis+coralline (Bsc), potassium+
sulphur (KS) on shoot growth and fire blight disease on 10 years old apple
trees, Fuji, Breauburn, Royal Gala, Golden Delicious grafted on M9 rootstock
in 2008 and 2009 years. Whilst PC treatments early in the season considerably
decreased the length of shoots, Hp, Bsc, ABA, KS treatments caused
increasing of shoot growth. Disease incidence in apple trees treated with PC,
Hp and inoculated was about 18%, markedly contrasting with 62% in the
untreated plants. The use of resistance-inducing substances during the early
phase of shoot growth may offer a means of managing the shoot blight of fire
blight disease on apples.
S12
PHYTOPATHOLOGY
Characterization of extracytoplasmic function sigma factors in plant
pathogenesis by Pseudomonas syringae pv. syringae B728a
P. BASU THAKUR (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S12
Pseudomonas syringae pv. syringae B728a, an aggressive bacterial pathogen
of bean, utilizes large surface populations and extracellular signaling to
initiate a fundamental change from an epiphytic to a pathogenic lifestyle.
Extracytoplasmic function (ECF) sigma (σ) factors serve as important
regulatory factors in responding to various environmental signals.
Bioinformatic analysis of the B728a genome has revealed ten ECF σ factors,
five of which have high levels of sequence similarity to the FecI-type of ECF
σ factors and play a known role in the regulation of various iron transport
systems. Because iron is essential for the induction of major virulence factors
in B728a, we hypothesize that these FecI-type σ factors may play a critical
role in the bacterium’s transition between lifestyles. Deletion mutants of two
FecI-type σ factors in B728a have been created using homologous
recombination based on the phage Red recombinase method, and phenotypic
analysis is being performed. In this report, we characterize the function,
regulatory network and signal transduction mechanisms of these proteins to
help describe the adaptation of B728a to a pathogenic lifestyle.
A new potyvirus infecting cantaloupe (Cucumis melo) in the Imperial
Valley of California
O. BATUMAN (1), E. T. Natwick (2), R. L. Gilbertson (1)
(1) University of California, Davis, CA, U.S.A.; (2) University of California
Cooperative Extension Imperial County, Holtville, CA, U.S.A.
Phytopathology 100:S12
In 2008, mosaic, veinal banding and leaf distortion symptoms were observed
in cantaloupe in the Imperial Valley of California. Potyvirus infection was
confirmed with PCR and degenerate primer pairs that target the HC-Pro and
CI genes. The virus was sap-transmitted to Chenopodium quinoa (Willd.), C.
amaranticolor L. and Cucurbita pepo cv. Small Sugar, but not to Nicotiana
benthamiana, Datura stramonium, tomato, pepper and common bean.
Sequence analysis of the complete viral RNA genome (~9.6 kb) revealed a
typical potyvirus genome organization. The highest complete nucleotide
sequence identity, 78%, was with Zucchini yellow mosaic virus (ZYMV). The
capsid protein and 3′-untranslated regions were 92% identical to those of
ZYMV, whereas HC-Pro and CI genes were 79% identical. Based on these
results, this is apparently a new melon-infecting potyvirus species, which is
most closely related to ZYMV.
Current status of benzimidazole resistance of Erysiphe necator in Virginia
A. B. BAUDOIN (1)
(1) Virginia Tech, Blacksburg, VA, U.S.A.
Phytopathology 100:S12
Benomyl was used on grapes in the U.S.A. since the early 1970s, against
Botrytis, black rot, and powdery mildew (E. necator). Benomyl resistance of
grape powdery mildew was documented in New York and existed but was
rare in California in 1993–95. Benomyl was withdrawn in 2001, and
thiophanate methyl received a grape tolerance in 2002 for control of the same
diseases, but probably has received little use on Virginia grapes. No
documentation of the presence and extent of benzimidazole resistance in
Virginia E. necator could be found, although it is considered likely to be
present. Bioassays were conducted with thiophanate methyl (Topsin M 70WP)
to determine if it might be useful for occasional use. Data were obtained for
55 isolates from 19 Virginia and 4 nearby locations. Fifty-one of 55 E.
necator isolates grew well on leaf tissue treated with 50 mg/liter a.i. of
thiophanate methyl, two did not grow, and two had an intermediate reaction.
A number of isolates were also tested against a different formulation of
thiophanate methyl (Cleary 3336 Plus) and against an old sample of benomyl
(Benlate 50DF). Cleary performed similarly to Topsin M, but several isolates
were more strongly inhibited by benomyl. There appeared to be at least two
different levels of benomyl resistance: isolates inhibited by 250 mg/liter a.i.
but not by 50 mg/liter, and isolates inhibited by neither. E. necator resistance
to thiophanate methyl appears to be widespread in Virginia.
Succinate Dehydrogenase Inhibitor resistance risk assessment studies on
Mycosphaerella graminicola the causal agent of Septoria Leaf Blotch
C. Bayon (1), M. W. Fraaije (2), H. J. Cools (1), S. L. Rogers (1), J. A. Lucas
(1), B. A. FRAAIJE (1)
(1) Rothamsted Research, Harpenden, UNITED KINGDOM; (2) University
of Groningen, Groningen, NETHERLANDS
Phytopathology 100:S12
Mycosphaerella graminicola, causal agent of Septoria Leaf Blotch (SLB), is
highly adaptable and has shown an ability to overcome host resistance and
develop fungicide resistance. UK survey data from 1998 and 2004 has shown
that losses can exceed £40 million per year despite application of fungicides.
Due to resistance development to MBC and QoI fungicides and reduced
efficacy of some DMIs, SLB control is heavily dependent on robust rates of
curative triazoles with chlorothalonil (a multi-site inhibitor) or boscalid (a
Succinate Dehydrogenase Inhibitor (SDHI)) often added as mixing partner to
ensure a high level of disease control and to reduce resistance risk. Several
new SDHIs with some curative properties are expected to enter the market.
Our aim is to develop tools to predict and monitor resistance development to
SDHI fungicides in a range of cereal pathogens. We have generated and
characterised a collection of carboxin-resistant UV-mutants of M.
graminicola. A range of mutations (>13) located in Sdh subunits B, C and D
have been identified and genotype-to-phenotype relationships established.
Protein modelling and inhibitor docking studies were conducted to establish if
SDHI inhibitors have different binding properties. Further diagnostic
development, enabling detection of resistant alleles at low frequencies, and
cross-resistance studies will aid implementation of anti-resistance strategies to
prolong the cost-effectiveness and lifetime of SDHI fungicides.
Evaluation of seed treatments for management of Rhizoctonia dampingoff in lettuce
F. BAYSAL-GUREL (1), S. A. Miller (1)
(1) The Ohio State University, Wooster, OH, U.S.A.
Phytopathology 100:S13
Rhizoctonia damping-off can result in significant losses in lettuce transplant
and direct seeded field production. Experiments were performed in a
greenhouse to determine the efficacy of seed treatments against Rhizoctonia
damping-off in lettuce. Treatments were Coronet, Coronet+Thiram and
FarMore. Inoculum of R. solani was prepared on chopped potato/soil medium
and incorporated into potting mix at the rate of 0.5 g/100 ml mix. The
numbers of emerged healthy and diseased seedlings were counted 8, 15 and
22 days after seeding. The experiment was repeated twice. Emergence was
significantly increased compared to the non-treated, inoculated control by all
seed treatments in both experiments. The Coronet treatment alone and
combined with Thiram increased the percentage of emerged and healthy
seedlings to that observed in the non-inoculated control in the first
experiment. These treatments were also significantly more effective than
FarMore in increasing emergence. However, there were no significant
differences between the treatments in the second experiment. Pre-emergence
damping-off was moderate (24.5% and 14.6% in the non-treated, inoculated
control in the first and second experiments, respectively). All of the treatments
significantly reduced pre-emergence damping-off compared to the inoculated
control. The fresh weight of lettuce seedlings was also significantly increased
compared to the non-treated, inoculated control by all treatments.
Effect of disinfectants on transmission of Clavibacter michiganensis subsp.
michiganensis during grafting
F. BAYSAL-GUREL (1), X. Xu (1), G. Rajashekara (1), S. A. Miller (1)
(1) The Ohio State University, Wooster, OH, U.S.A.
Phytopathology 100:S13
Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis
(Cmm), is a serious disease of tomato produced in open field and protected
environments worldwide. Typical symptoms are stunting, wilting and death of
plants, foliar and stem necrosis and unmarketable fruit. Cmm is transmitted
from infected seed to seedlings, and mechanically from plant to plant during
grafting. Commercial disinfectants were examined for efficacy in eliminating
Cmm from grafting tools that dispensed the disinfectants onto the cutting
surface during grafting. A bioluminescent Cmm strain (BL-Cmm 17) was
used to facilitate the study of Cmm movement through grafted plants. The
movement of BL-Cmm17 up and down the stem from the graft union was
determined by tissue imprinting the cut surface of both scion and rootstock on
semi-selective medium at specific distances from the graft 0 and 7 days after
grafting. KleenGrow, bleach and Virkon S disinfectants were effective in
preventing Cmm transmission to grafted tomato seedlings without obvious
phytotoxicity. These disinfectants may prevent grafting-mediated Cmm
transmission when used in a grafting tool that delivers the disinfectant directly
to the cutting surface.
Copper resistance in Xanthomonas citri subsp. citri (Xcc) and X. alfalfae
subsp. citrumelonis (Xac) and comparison with other xanthomonads
F. BEHLAU (1), B. I. Canteros (2), J. H. Graham (3), J. B. Jones (1)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) INTA-EEA, Bella
Vista, ARGENTINA; (3) University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S13
Copper resistance genes (CuR) genes from Xcc strain A44 and Xac strain
1381 were cloned, sequenced and compared with xanthomonads from
different locations. The genes copL, copA, copB, copM, copG, copC and copD
were identified in Xcc A44. The same cop genes except copC and copD
occurred in Xac 1381. In addition, copF was found downstream of copG in
Xac 1381. X. vesicatoria 1111 and Stenotrophomonas maltophilia k279a had
the same set of cop genes found in Xcc A44 and copF, located downstream of
copD. A44 clone ends at copD and the cop genes in 1111 and k279a share
high homology (>95%) with cop genes found in A44. Thus, we presume that
copF is also present in A44. Primers based on the A44 gene sequences were
used to PCR amplify copL, copA and copB from other CuR xanthomonads.
Although sequence alignment of PCR products revealed high homology
(>90%) for copL, copA and copB among different xanthomonads,
phylogenetic analysis indicated variation. Cop genes in Xac strains from
Florida were more diverse than in Xcc strains from Argentina. Cop genes in
one Xcc strain were closely related to X. vesicatoria BV5-4 from Argentina
while the cop genes in the remaining strains were closely related to X.
gardneri from Costa Rica. Xac strains were not highly similar to one another
and were grouped with other Xanthomonas spp. CuR genes in xanthomonads
may have a common origin and have been exchanged by horizontal transfer.
Molecular analysis of turfgrass rusts reveals the widespread distribution
of Puccinia coronata as a pathogen of Kentucky bluegrass
L. A. BEIRN (1), B. B. Clarke (1), J. Crouch (1)
(1) Rutgers University, New Brunswick, NJ, U.S.A.
Phytopathology 100:S13
Rust is a common disease of cultivated turfgrasses that can cause extensive
damage in heavily infested areas. Over the past ten years, increased
susceptibility has been observed among several Kentucky bluegrass (Poa
pratensis L.) cultivars. To test whether increased disease in previously
resistant cultivars could be the result of new rust species and/or shifts in rust
race composition, wide-scale sampling of symptomatic grasses was conducted
to identify the primary rust species associated with turfgrass hosts.
Phylogenetic analysis of rDNA ITS sequences identified Puccinia coronata,
P. graminis, and P. striiformis from the tissue sampled. P. coronata was the
most prevalent species (68% of the samples) followed by P. graminis (27%)
and P. striiformis (5%). These species frequencies contradict what has
typically been reported by turfgrass breeders in the field based on phenotype
and disease symptoms. Not only was P. coronata found to be the predominate
species in the samples, but was also routinely found in association with
Kentucky bluegrass, indicating that the most common traits used to identify
these pathogens in the field - uredium/spore pigmentation and host plant
association - are inadequate to accurately identify rust species. We used the
ITS dataset to develop a real time PCR protocol as a tool for the accurate
discrimination of these rusts from turf. AFLP analysis is currently in progress
to evaluate genetic diversity in turfgrass rust populations.
Evaluation of synergistic effect between rhizobacterial strains Fusarium
oxysporum biological control agents
J. C. Bejarano (1), S. Franco (1), P. JIMENEZ (1)
(1) Univ Militar Nueva Granada, Bogota, COLOMBIA
Phytopathology 100:S13
Using a bacteria consortium for biological control is a strategy that allows to
explore posible synergistic relationships between the members of the
consortium. Also, it has been debated that consortia stimulate the ecosystem
diversity. Besides that, our model pathosystem (Physalis peruviana- Fusarium
oxysporum) is economicaly important and with no pesticide residue tolerance
in fruit. From a previous work, a collection of 5 rhizobacteria (2 Bacillus
subtilis, 2 Pseudomonas fluorescens and 1 Pseudomonas sp.) was obtained.
These 5 bacteria were distributed in consortia of 1, 2, 3, 4, 5 bacteria, and their
effect on F. oxysporum radial growth, the number of macro and micro conidia
produced per colonial area, and on P. peruviana germination rate, were
evaluated. Results showed that different bacterial combinations present
synergistic effect for an activity but they did not work well facing other
challenges. Concerning the results of the combinations, we found that
increasing the number of members in the consortium could have an inhibitory
effect among the bacteria especies. This effect is probably due to the fact that
metabolite production, such as antibiotics, is stimulated by processes such as
competence for nutritional resources, resulting then in an antagonistic effect.
PCR-RFLP analysis on genetic diversity of Fusarium spp. isolates
collected from sugarbeet fields of Iran
S. Beladi (1), S. REZAEE (1), B. Mahmoodi (2)
(1) Dept. of Plant Pathology, College of Agriculture and Natural Resources,
Science and Research Branch, Islamic Azad University, Tehran, IRAN; (2)
Sugar Beet Seed Institute, Karaj, IRAN
Phytopathology 100:S13
The amplified ITS region of rDNA, digested with three restriction
endonucleases; EcoR1, Taq1 and HaeIII, to analyze the genetic diversity
among 28 Fusarium spp. isolates from diseased sugarbeet with wilting and
yellowing symptoms from nine different regions of Iran. Based on
morphological features, 13 Fusarium oxysporum, seven Fusarium solani and
eight Fusarium proliferatum isolates were identified. Using PCR method two
Vol. 100, No. 6 (Supplement), 2010
S13
fragments of 550 and 570 bp were amplified for ITS1 and ITS4 sequences of
all isolates. Digestion patterns of ITS amplification products with EcoR1
revealed one restriction site in this region of DNA. No clear rDNA
polymorphism was observed after digestion with EcoR1. Digestion with Tag1
and HaeIII showed genetic diversity among different species of Fusarium.
Digestion with HaeIII resulted in three banding patterns with two or three
restriction sites. Digestion with Taq1 exhibited three banding patterns with
three or four bands with different sizes. The cluster analysis separated all
isolates into three groups based on their species. According to these results,
Iranian Fusarium isolates can be separated easily into different species on the
basis of ITS-RFLP method.
Sensitivity of Alternaria solani populations in Idaho to commonly used
fungicides and its effect on potato early blight management in Idaho
A. R. BELCHER (1), E. Wood (1), P. S. Wharton (1)
(1) University of Idaho, Aberdeen, ID, U.S.A.
Phytopathology 100:S14
Early blight, caused by the fungus Alternaria solani, is an important disease
on potato in Idaho. The strobilurin (QoI) fungicides (e.g. azoxystrobin and
pyraclostrobin) are currently favored as effective and safe methods for the
control of early blight. QoI fungicides were registered for use on potato in the
U.S. in the late 1990s. Soon after, in the early 2000s, isolates of A. solani with
reduced sensitivity to the QoIs were detected across the Midwest. In 2007 and
2008 many Idaho potato growers reported the failure of these fungicides to
control early blight. Thus, the goal of this project was to assess the prevalence
of A. solani isolates with reduced sensitivity to the QoI fungicides in
southeastern Idaho. In 2009, 77 isolates were collected from leaves and tubers
of potatoes grown in experimental and commercial fields. The isolates were
tested for sensitivity to azoxystrobin, pyraclostrobin, famoxodone plus
cymoxanil, and boscalid. Fungicide solutions were applied to PDA in a 2.5log dilution in a continuous radial concentration gradient using a spiral plater.
Fungal inoculum was placed in radial lines across the gradient. Fungicide
sensitivity was expressed as an EC50, the fungicide concentration at which a
fungal isolate’s radial growth was equal to 50% of the average growth of the
isolate on non-amended PDA. The results, to be presented, will be used to
develop a more sustainable potato early blight control program for Idaho
growers.
The effect of foliar fungicides on yield across Iowa in the 2008 and 2009
growing seasons
N. R. BESTOR (1), D. S. Mueller (1), A. E. Robertson (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S14
Interest in the use of foliar fungicides of soybean has increased in the past five
years. Reasons for this include higher soybean prices and the threat of foliar
diseases. In Iowa, diseases such as brown spot (Septoria glycines), Cercospora
leaf blight (Cercospora kikuchii) and frogeye leaf spot (C. sojina) can
potentially reduce yields. The effect of a foliar fungicide applied at either
growth stage R1 or R3 on disease severity and yield of soybean was evaluated
at five locations in Iowa in both 2008 and 2009. Fungicides belonging to
strobilurin, triazole and carboxamide groups were evaluated. Percent foliar
disease of brown spot, Cercospora leaf blight, frogeye leaf spot, and downy
mildew were assessed at growth stage R6. Brown spot was the most prevalent
disease at all locations, but disease pressure was low in both 2008 and 2009
(15% severity and 5.5% severity in the lower canopy at growth stage R6,
respectively). In general, yields were greatest with an R3 application of a
fungicide containing a strobilurin. In 2008, the average of all fungicides at all
five locations applied at R3 (54.3 bu/ac) yielded higher than the non-treated
control (50.2 bu/ac). However, in 2009, the average of all fungicides at all five
locations applied at R3 (60.7 bu/ac) yielded similar to the non-treated control
(60.1 bu/ac).
Identification and characterization of fungal communities associated with
soybean roots in Minnesota
J. C. BIENAPFL (1), D. K. Malvick (1), J. A. Percich (1)
(1) University of Minnesota, St. Paul, MN, U.S.A.
Phytopathology 100:S14
Root diseases of soybean cause substantial yield reduction in the U.S.
However, information regarding the distribution and pathogenicity of fungal
communities associated with soybean roots is limited. In 2007 and 2008,
soybean root samples were collected in July from 15 fields in 10 counties
representing major soybean production areas in Minnesota (MN).
Symptomatic and asymptomatic root tissue was plated onto four different
media. Although Fusarium was the most frequently isolated genus in all
locations, additional fungal genera and species were recovered. Thirty
representative non-Fusarium isolates were selected and identified to species
using morphological characteristics and sequences of the internal transcribed
spacer region. Pathogenicity of these isolates to soybean seedlings was
S14
PHYTOPATHOLOGY
evaluated in a greenhouse. The 30 fungal isolates included the genera
Arthrographis, Bionectria, Cylindrocarpon, Exophiala, Neonectria,
Mortierella, and Rhizoctonia. Results indicated a diversity of fungi is
associated with soybean roots in the early reproductive stages in MN,
including some that have not been previously reported from soybean roots.
Isolates of Arthrographis, Bionectria, Cylindrocarpon, Exophiala,
Neonectria, and Rhizoctonia species produced root rot symptoms ranging
from discrete lesions to extensive taproot necrosis on seedlings and may cause
root rot in MN soybean fields. Additional isolates are currently being
identified and tested for pathogenicity on soybean.
Development of a multiplex assay for genus and species-specific
detection of Phytophthora based on differences in mitochondrial gene
order
G. BILODEAU (1), F. N. Martin (1), M. D. Coffey (2), C. L. Blomquist (3)
(1) United States Department of Agriculture-Agricultural Research Service
(USDA-ARS), Salinas, CA, U.S.A.; (2) Department of Plant Pathology and
Microbiology, University of California Riverside, Riverside, CA, U.S.A.; (3)
California Department of Food and Agriculture, Plant Pest Diagnostics
Branch, Sacramento, CA, U.S.A.
Phytopathology 100:S14
The genus Phytophthora contains more than one hundred described species
and given their importance to agriculture accurate and rapid detection tools
are essential. A range of markers have been developed for this genus, usually
based on polymorphisms at primer annealing sites that rely on accurate
control of annealing temperature for specificity. An alternative approach for
enhanced specificity is to design markers based on differences in the location
of annealing sites. We have looked at gene order differences in the
mitochondrial genome of Phytophthora compared to Pythium and plants for
developing a single amplification assay for genus as well as species specific
detection (single amplification primer pair with TaqMan probes for genus and
species-specific ID). Three conserved gene order differences have been
identified with conserved regions suitable for genus specific detection
adjacent to variable regions for species-specificity. Two of these should allow
for design of species-specific probes for more than 65 species. The
amplification primers and genus specific probe were effective when evaluated
against a wide range of isolates representing all formally and provisionally
described Phytophthora spp. as well as a number of Pythium spp. and plants.
Multiplex amplifications with species-specific probe combinations P.
ramorum-kernoviae, P. fragariae-citricola-cactorum and P. alni were also
effective and are under evaluation with field samples.
Quantification and rapid detection of Verticillium dahliae in soil
G. BILODEAU (1), P. Uribe (1), S. T. Koike (2), F. Martin (1)
(1) United States Department of Agriculture-Agricultural Research Service
(USDA-ARS), Salinas, CA, U.S.A.; (2) Univ. Calif. Cooperative Extension,
Monterey County, Salinas, CA, U.S.A.
Phytopathology 100:S14
Verticillium wilt is caused by the fungus Verticillium dahliae, which survives
in the soil for long periods of time as microsclerotia. In strawberry production
inoculum densities of 3-5 ms/g are high enough to cause disease losses. A
soil-plating assay is currently used to assess inoculum density but takes 6–8
weeks to get results. The development of a rapid and accurate detection
method using a molecular diagnostic assay like TaqMan Real-Time PCR is
desirable. We have approached this objective by focusing on optimizing
procedures for DNA extraction from the soil, developing post extraction
procedures to remove PCR inhibitors, and creating a highly sensitive marker
system specific for V. dahliae. Soil quantification with molecular techniques
is challenging because PCR inhibitors can influence amplification kinetics,
making accurate quantification across samples or soil types difficult. To
account for this an internal control assay multiplexed with the V. dahliae
assay was developed. To correlate the results of the PCR assay with soil
population densities, soil samples were collected from infested fields and
analyzed by traditional plate count technique and DNA extraction using the
optimized real-time PCR assay. The results obtained with the two methods
were plotted using regression analysis and the correlation between the Ct
value and the plate count was high (R2 = 0.85). Pathogen densities as low as 2
ms/g soil were accurately detected with a final Ct less than 34.
Effect of temperature on Bacterial wilt, caused
solanacearum, incidence in tobacco cultivars
R. J. BITTNER (1), A. Mila (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S14
by
Ralstonia
Bacterial wilt is a destructive disease of many crops, including tobacco. Use
of resistant cultivars is one of the most effective means to reduce losses from
R. solanacearum, but little is known about the mechanism of resistance in
tobacco cultivars. Temperatures above 28°C have been shown to increase the
severity of bacterial wilt in resistant tobacco cultivars. In this study, we
examined the effect of different temperatures on resistance of six tobacco
cultivars to R. solanacearum. Highly resistant cultivars K346 and Sp168 and
low resistant cultivars K326, NC71, RJR15, and RJR75 were compared under
six temperatures (35, 30, 25, 20, 15, and 10°C). Four strains of R.
solanacearum (race 1, biovar 1) were used. The highest disease incidence was
observed in all cultivars at 30 and 35°C, 18 days post-inoculation. In contrast,
no disease symptoms were observed when plants were incubated at 10 and
15°C. Plants were placed in a 30°C incubator for an additional 18 days and
disease was observed on all cultivars. Temperature (P < 0.0001), cultivar (P =
0.03), and strain (P < 0.0001) were significant factors explaining disease
incidence. We are currently further investigating if the temperature affects the
host (cultivar), the pathogen strain, or their interaction. Understanding host
parasite interactions and temperature effects offers information to advance
breeding and disease management strategies.
Diversity of sooty blotch and flyspeck fungi from apples in northeastern
Turkey
J. M. BLASER (1), A. Karakaya (2), D. A. Mayfield (1), J. C. Batzer (1), M.
L. Gleason (1)
(1) Iowa State University, Ames, IA, U.S.A.; (2) University of Ankara,
Ankara, TURKEY
Phytopathology 100:S15
Fungi in the sooty blotch and flyspeck (SBFS) complex are epiphytes that
infest apples and other tree fruit crops. In regions with humid climates, SBFS
fungi blemish the fruit cuticle, reducing market value of the crop. In this
study, SBFS isolates were obtained from apples grown in Rize, Turkey. SBFS
colonies with subtending apple cuticle were excised, pressed, photographed,
and shipped to Ames, Iowa for isolation. Of 592 primary isolates from 148
apple peels, 50 isolates were selected for further study. The internal
transcriber spacer (ITS) and large subunit (LSU) regions of ribosomal DNA
were amplified, sequenced and compared to previously identified fungi.
Isolates were placed into genera using parsimony analysis of the LSU.
Putative species were delineated from ITS sequences as well as morphology
on apple and in culture. A total of 17 putative species were delineated; 14
were placed in the Capnodiales, two were placed in the Chaetothyriales, and
one could not be placed to order but LSU parsimony analysis grouped it in
Dothideomycetes. Eleven putative SBFS species had not been described
previously, whereas previously described species included Peltaster
fructicola, Zygophiala wisconsinensis, Pseudocercosporella spp. RH1 and
RH3.1, Zygophiala sp. FS6, and Stomiopeltis sp. RS4.1. These findings
expand the documented range of genetic diversity within the SBFS complex
and are the first information about the taxonomic classification of these fungi
from Turkey.
The activity of citrus canker lesions on grapefruit in Florida, June 2009–
January 2010
C. H. BOCK (1), P. E. Parker (2), T. R. Gottwald (3), J. H. Graham (4)
(1) USDA ARS, Fort Pierce, FL, U.S.A.; (2) USDA-APHIS-PPQ, Edinburg,
TX, U.S.A.; (3) USDA, ARS, USHRL, Fort Pierce, FL, U.S.A.; (4) University
of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S15
Lesions of citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), on
citrus fruit preclude sale of the fruit to the fresh market. Assessing lesion
activity in orchard-grown grapefruit provides information on the population
dynamics of fruit lesions in a commercial situation and helps gauge risk
associated with infected fruit entering fresh markets. To quantify the
proportion of active lesions, and Xcc production, we collected eighty lesions
from the rind of grapefruit once a month from June 2009–January 2010 from
an orchard in East Florida and assessed activity of each lesion by dilution
plating on nutrient agar. Linear regression analysis indicated a slight decline
in the proportion of active lesions (R2 = 0.45). In June 88% of lesions
produced Xcc, and by January, at the time of harvest, 69% of lesions were
active. However, the average number of bacteria produced was greatest in
November (3.9 × 105 Xcc/mm2 of lesion), and least in August (2.8 × 104
Xcc/mm2 of lesion). In January, 2.0 × 105 Xcc/mm2 lesion was produced.
The maximum quantity of Xcc produced was in December (5.2 × 107
Xcc/mm2 of lesion). These data suggest that in Florida there was little change
in the activity of canker lesions on fruit from shortly after lesion development
to the point of harvest. Foliar lesions are also reported to remain fully active
for at least six months. This reinforces the need to focus on post harvest
approaches to deactivating lesions of citrus canker on fresh fruit.
Distribution of canker lesions on grapefruit in Florida
C. H. BOCK (1), P. E. Parker (2), T. R. Gottwald (3)
(1) USDA ARS, Ft Pierce, FL, U.S.A.; (2) USDA-APHIS-PPQ, Edinburg,
TX, U.S.A.; (3) USDA, ARS, USHRL, Fort Pierce, FL, U.S.A.
Phytopathology 100:S15
Citrus canker, caused by the plant pathogenic bacterium Xanthomonas citri
subsp. citri (Xcc) is an important disease of grapfruit in Florida. To establish
disease distribution on fruit, six samples of 24 diseased grapefruit were
collected from two groves in East Florida. A plane was sliced through the
middle of the fruit such that the vertical dimension of the fruit (the diameter,
with peduncle at the apex) was split into equal sections. The surface area of
each fruit half (hemisphere) was assumed to be the same. For all six samples
of 24 fruit each the lesions were enumerated on the upper (peduncle end) and
lower (flower scar) halves. On four of the samples the fruit sliced along three
planes and the number of lesions enumerated on each of the four slices. On
the six samples, 70–82% of all lesions were found on the upper half of the
grapefruit. Sequentially on the four quartered samples, 40–47% of all lesions
were found on the upper quarter of the fruit, and 28–39%, 9–16% and 7–10%
of lesions were found on the lower three quarters, respectively. GLM analysis
showed significant differences in lesion counts from each section; the highest
count consistently being on the upper portions of the fruit. A logistic model
described the relationship between lesion count and vertical distance from the
fruit apex. Presumably the upper surfaces of the fruit are more prone to
infection as they have greater exposure to splash born inoculum.
Proteomics analysis of Ralstonia solanacearum identifies candidate
proteins that contribute to pathogenicity on tomato plants at low
temperatures
A. BOCSANCZY (1), U. C. Achenbach (2), D. J. Norman (1)
(1) Department of Plant Pathology, University of Florida, IFAS-MREC,
Apopka, FL, U.S.A.; (2) Institute of Molecular Plant Physiology and
Biotechnology of Plants (IMBIO), Bonn, GERMANY
Phytopathology 100:S15
Ralstonia solanacearum is a common bacterial plant pathogen in tropical and
subtropical areas of the world with a wide host range which include
economically important crops such as potato and tomato. The pathogen
species is very diverse and complex. R. solanacearum strain UW551 which
has been classified as a race 3 biovar 2 (R3B2) has the ability to infect tomato
and potato plants at low temperatures as we previously determined comparing
the pathogenicity of R3B2 strains with other races of Ralstonia in
environmental chambers at 18°C and at 30°C. The model strain GMI1000
however does not infect its host plants at 18°C. We hypothesized that either
UW551 has genes not present in GMI1000 or their expression is differentially
regulated at low temperatures. The objective of our study is to identify
determinants at molecular level of the pathogenicity of UW551 at low
temperatures. In order to identify candidates for genes/ proteins we designed
experiments where we compared protein levels of R. solanacearum UW551
and GMI1000 at 18°C and 30°C, when the pathogenic strains are in contact
with tomato plants roots. We identified a list of potential candidate genes that
might contribute to the virulence of UW551 under cool weather conditions.
Currently we are confirming expression of the candidate genes using real time
PCR techniques and we are working towards producing deletion mutants of
selected candidates in order to characterize their function in pathogenicity.
Field detection of Phytophthora ramorum DNA within 30 minutes
R. C. BOHANNON (1), P. Russell (1)
(1) Agdia Inc., Elkhart, IN, U.S.A.
Phytopathology 100:S15
Agdia, Inc. has developed a method to detect plant pathogen DNA in less than
30 minutes, start to finish, using a new isothermal DNA amplification system.
Initial tests were developed to detect Phythophthora ramorum as the
prototypic target analyte, owing to its importance in the ornamental field and
association with Sudden Oak Death syndrome…responsible for killing
millions of trees in California and Oregon. The new system successfully
demonstrated 100% specificity for P. ramorum in over 70 samples tested from
Pythophtora and Pythium lineages and demonstrated extremely low levels of
detection. Tests were developed against other relevant pathogens and a system
developed to give users an answer in the field, or lab, in less than 5 minutes
without the need for instrumentation for selected analytes. Discussion of this
breakthrough and technology will be presented.
Modeling Fusarium head blight and deoxynivalenol content in barley in
response to field temperature and wetness durations
K. D. BONDALAPATI (1), J. M. Stein (1)
(1) South Dakota State University, Brookings, SD, U.S.A.
Phytopathology 100:S15
Fusarium head blight (FHB), caused by the fungus Gibberella zeae
(anamorph: Fusarium graminearum), continues to be a serious problem for
barley producers in the U.S. Northern Great Plains and elsewhere. Field
experiments were conducted during the 2005–9 growing seasons to evaluate
the combined effects of temperature and wetness durations (relative humidity
> 90%) prior to full head emergence on disease development and
deoxynivalenol (DON) accumulation in malting barley. Disease incidence
Vol. 100, No. 6 (Supplement), 2010
S15
(number of diseased spikes/total), average severity (number of disease
spikelets/total), and DON content in the grain (mg/kg) were collected from 51
location*years for three varieties, ‘Conlon’, ‘Robust’ and ‘Tradition’. A
binary DON response variable, eDON, was created based on the DON content
for each variety at every location*year where ‘0’ and ‘1’ represent values
below or above a threshold of 0.5 mg/kg. A Weibull function was calculated
to predict the probability of infection using the average temperature and
weighted wetness durations in the field during the 10-day prior and including
the full head emergence day. Disease incidence, severity, DON content and
eDON were significantly correlated to the Weibull variable calculated from
the field weather data (p < 0.001). The sensitivity, specificity and total
prediction accuracy obtained from the confusion matrix after dividing the
observations at a cut-off of 0.5 of Weibull variable in comparison with eDON
were greater than 80%.
Effects of water vapor, liquid water, and their interaction on the
germination of urediniospores of Phakopsora pachyrhizi
M. R. BONDE (1)
(1) USDA ARS, Frederick, MD, U.S.A.
Phytopathology 100:S16
“Cold-induced dormancy” has been described as a phenomenon in which
urediniospores, including those of P. pachyrhizi, after having been frozen at
ultra-low temperatures, require either a heat shock (e.g. 40°C for 5 min) or
over night hydration in a water-saturated atmosphere to germinate. To better
understand this phenomenon in P. pachyrhizi, following liquid nitrogen
storage urediniospores were subjected to specific treatments including over
night hydration in a water-saturated atmosphere, a 40°C-heat shock for 5 min,
and submersion in liquid water (0.02% Tween 20), individually and in every
treatment combination. Fresh urediniospores from plants in a containment
greenhouse served as controls. In all experiments, fresh and previously frozen
urediniospores behaved the same. Heat shocks had no effect on germination,
whereas hydration in a water-saturated atmosphere was required for high
germination for all samples. Submersion of urediniospores in liquid water
without prior hydration in a water-saturated atmosphere reduced germination
by 84 to 96%, whereas floating non-hydrated urediniospores on liquid water
reduced germination by only 45% compared to hydrated urediniospores
placed directly on water agar. When hydrated urediniospores were submerged
for 2 min to 60 min in liquid water, germination was reduced only by 11 to
23%, respectively. The interaction of water vapor and liquid water in the
laboratory was highly significant and may play an important role in nature.
An assessment of sensitivity to fungicides in Tennessee isolates of the
cucurbit powdery mildew pathogen, Podosphaera xanthii
S. C. BOST (1)
(1) University of Tennessee, Nashville, TN, U.S.A.
Phytopathology 100:S16
Seven Tennessee isolates of the cucurbit powdery mildew pathogen,
Podosphaera xanthii, were tested for sensitivity to several fungicides in 2009.
Comparison of the results to two earlier surveys (1998 and 2004) indicates
that the incidence of high levels of resistance to the strobilurins increased
from zero in 1998 to 100 percent in 2009. Systemic fungicides had not been
used in any of the fields sampled in 2009, and two of the fields were organic
production. High levels of resistance to the benzimidazoles were found in all
isolates in all three surveys. All isolates tested in the 2009 survey were highly
sensitive to myclobutanil, boscalid, sulfur, cyprodinil plus fludioxonil,
fenthiopyrid, and quinoxyfen. However, the survey results conflicted with
recent anecdotal field reports indicating undesirable levels of control by
myclobutanil. The powdery mildew population present in a fungicide efficacy
field trial did not change in sensitivity to any of the tested products, based on
the results of greenhouse tests conducted on samples collected before the first
application and after the final application.
Sorghum as a bioenergy crop in Alabama: Disease and yield evaluations
K. L. BOWEN (1), A. K. Hagan (1), A. C. Rocateli (2), R. L. Raper (3), E. B.
Schwab (4), D. Bransby (2), F. J. Arriaga (5), K. S. Balkcom (5)
(1) Auburn University, Auburn, AL, U.S.A.; (2) Agronomy and Soils, Auburn
University, AL, U.S.A.; (3) USDA-ARS Dale Bumpers Research Center,
Booneville, AR, U.S.A.; (4) USDA-NRCS, Auburn, AL, U.S.A.; (5) USDAARS National Soil Dynamics Lab, Auburn, AL, U.S.A.
Phytopathology 100:S16
In Alabama, in 2009, several studies were monitored to evaluate the impact of
various production practices on disease occurrence and the effect of diseases
on yields of sorghum. Sweet sorghum cultivars, as well as grain and forage
sorghum, were included in these studies. Fresh (biomass) and dry weights as
well as juice and Brix (% sucrose) yields were recorded. In south-central
Alabama (Brewton), the best cultivars, including ‘M81-E’ and ‘Dale’, yielded
in excess of 80 tons per hectare fresh weight biomass in the absence of
diseases. Zonate leaf spot (Gloeocercospora sorghi) was the dominant disease
S16
PHYTOPATHOLOGY
in south Alabama (Baldwin Co.); however, anthracnose (Colletotrichum
graminicola) occurred primarily on the sweet sorghum M81-E. Anthracnose
also predominated on sorghum in east-central AL (Macon Co.). In one Macon
Co. trial, anthracnose was lower in conventionally tilled plots than those under
conservation tillage. Brix was negatively related to anthracnose intensity on
M81-E in south AL, while this dry matter yield was negatively related to
anthracnose in east-central AL on forage sorghum. Increasing nitrogen rates
had little if any impact on disease severity or any yield parameter in south AL.
However, more severe anthracnose was found with higher nitrogen rates and
with later planting dates in sweet sorghum in Macon Co.
Evaluating the impact of nutritional treatments on Xylella fastidiosa in
grapevine
J. BRADY (1), J. Faske (1), T. Faske (2), D. McGahan (2)
(1) Texas AgriLife Research, Stephenville, TX, U.S.A.; (2) Tarleton State
University, Stephenville, TX, U.S.A.
Phytopathology 100:S16
Xylella fastidiosa is a bacterial pathogen that causes leaf scorch in a wide
array of plant species including grape, where it causes Pierce’s disease. X.
fastidiosa colonizes the nutritionally poor environment of xylem elements,
utilizing dilute salts and amino acids in xylem fluid. The bacterium exhibits
fastidious nutritional requirements, a slow growth rate, and is sensitive to
perturbations in its growth medium. Plant nutritional regimes were examined
for the potential to alter bacterial proliferation in planta. Potted Cabernet
Sauvignon grapevines were mechanically inoculated with X. fastidiosa and
subjected to weekly soil drench treatments in which levels of selected
micronutrients were varied, either alone or as a component of Hoagland’s
solution #2. X. fastidiosa proliferation in stem tissue was measured by QRTPCR. Grapevines treated with increased levels of zinc, manganese, or copper
had fewer X. fastidiosa than grapevines treated with Hoagland’s solution or
water. Grapevines treated with Hoagland’s solution minus zinc had higher X.
fastidiosa levels than grapevines treated with complete Hoagland’s solution,
while bacterial levels in manganese deprived grapevines were not different
than those treated with complete Hoagland’s solution. Experiments are
ongoing to test the utility of altered plant nutritional regimes as a management
tactic in field plots.
Phytotoxicity danger of phosphorous acid generating fungicides and
fertilizer products applied to blueberry and grapes
P. M. BRANNEN (1), J. Garner (2), J. Smith (3)
(1) University of Georgia, Athens, GA, U.S.A.; (2) University of Georgia,
Blairsville, GA, U.S.A.; (3) University of Georgia, Alma, GA, U.S.A.
Phytopathology 100:S16
Phosphorous acid generating fungicides and fertilizers have seen a recent
increase in use for either direct of indirect management of multiple diseases of
small fruits, especially Phytophthora root rot and downy mildew. Labels differ
markedly between phosphorous acid products, and though these products are
known to have some phytotoxic issues, label warnings and use patterns vary
between products; labeled spray intervals and rates are highly variable for the
same commodities, though near-equivalent amounts of phosphorous acid
might be utilized. In some cases, overuse of these materials by producers has
been observed. To confirm potential phytotoxic responses with phosphorous
acid applied to blueberry, studies in Georgia with weekly applications of
phosphorous acid indicated that damage did not necessarily occur gradually,
but damage, as indicated by scorched leaves and leaf drop, was observed
rapidly following five applications of the material. Likewise, similar damage
was observed in Vitis vinifera grapes in two years of testing, with 100% plant
mortality occurring in the second year of testing after six applications.
Similarly, scorch and plant mortality did not occur gradually, but was only
observed after an apparent critical plant stress was achieved. Phosphorous
acid generators are excellent tools for disease management, but their
phytotoxic potential should be further tested, clearly communicated, and
potentially standardized across products.
Aggressiveness of Rhizoctonia solani AG 2-2 on sugar beet and rotation
crops
J. R. BRANTNER (1), C. E. Windels (1)
(1) University of Minnesota, NWROC, Crookston, MN, U.S.A.
Phytopathology 100:S16
Rhizoctonia crown and root rot of sugar beet caused by Rhizoctonia solani
AG 2-2 intraspecific groups (ISGs) IV and IIIB is increasing in Minnesota and
North Dakota. Of 1,000 cultures of AG 2-2 isolated from diseased sugar beet,
24 of each ISG were selected to represent a wide geographic area and
previous crops. They were tested for aggressiveness on adult sugar beet roots
and seedlings of sugar beet and rotation crops. Adult sugar beet roots were
inoculated when 8-wk old; in seedling tests, a commercial greenhouse soil
was infested with inoculum before planting. Disease was assessed at 12 days
after inoculation. Both ISGs were equally aggressive on adult sugar beet roots;
root rot indices (RRI = 0-7 scale) averaged 5.0 (3.3-5.6) for IV and 4.9 (3.85.9) for IIIB. On seedlings of sugar beet and rotation crops, range of
aggressiveness for both ISGs overlapped, but IIIB caused significantly more
disease than IV. Sugar beet RRI (0-100 scale) averaged 78 (42-100) for IIIB
and 51 (5-100) for IV. Corn RRI (1-5 scale) averaged 3.1 (1.8-4.1) for IIIB
and 2.1 (1.2-3.0) for IV. Pinto bean RRI (1-5 scale) averaged 4.4 (3.5-5.0) for
IIIB and 2.7 (1.8-4.5) for IV. Soybean RRI (1-5 scale) averaged 4.1 (2.7-5.0)
for IIIB and 3.2 (1.9-4.6) for IV. Aggressiveness of AG 2-2 cultures on adult
sugar beet roots was unrelated to pathogenicity on seedlings of any crop but
there were significant correlations (P < 0.001) for aggressiveness of cultures
on seedlings between crops.
The response of the energy crop Miscanthus to fungal pathogens: A
preliminary study
J. M. BRENNAN (1), E. Glynn (2), K. McDonnell (3)
(1) Plant Health Laboratory, Seed Certification Division, Department of
Agriculture, Fisheries and Food, Backweston, Celbridge, Kildare, IRELAND;
(2) Department of Agriculture, Fisheries and Food, Backweston & UCD
School of Agriculture, Food Science and Veterinary Medicine, UCD College
of Life Sciences, Belfield, Dublin 4, Kildare, IRELAND; (3) UCD School of
Agriculture, Food Science and Veterinary Medicine, UCD College of Life
Sciences, Belfield, Dublin 4, Dublin, IRELAND
Phytopathology 100:S17
There is increasing interest in the use of biomass crops for energy production.
Miscanthus is a perennial rhizomatous C4 grass that has remarkable
adaptability to different climatic environments. As the acreage of the biofuel
crop Miscanthus expands in Ireland and the EU, we can expect that pests and
pathogens will have a significant impact on both biomass yield and biomass
quality. However, little is known about diseases of novel biofuel crops such as
Miscanthus. The aim of this research was to assess the response of Miscanthus
x giganteus towards fungal pathogens including some of the main Irish cereal
pathogens using an in vitro detached leaf assay and in vivo whole plant test.
Visual disease symptoms observed on the Miscanthus x giganteus leaves in
this experiment varied among the 19 pathogens (37 isolates) assessed and
symptoms included brown lesions, premature necrosis, presence of pynidia
and mycelial growth. In general, all 19 pathogens (37 isolates) assessed
caused some level of disease in vitro. The pathogen Rhizoctonia solani caused
the greatest visual disease symptoms on the Miscanthus x giganteus leaves
(mean LGR = 3.5 cm day -1), while R. secalis caused the least visual disease
symptoms (mean LGR = 1.5 cm day -1) (P < 0.05). While of the 6 pathogens
assessed in the in vivo whole plant test only F. culmorum, F. graminearum
and M. nivale caused visual disease symptoms (mean LGR = 0.3 – 0.5 cm day
-1).
Phytophthora ramorum and Phytophthora kernoviae in Ireland: The
current situation
J. BRENNAN (1), D. Cummins (1), S. Kearney (1), G. Cahalane (1), S. Nolan
(1), J. Choiseul (1)
(1) Department of Agriculture, Fisheries & Food, Kildare, IRELAND
Phytopathology 100:S17
Phytophthora ramorum is a serious pathogen of trees and ornamental plants,
causing a disease known as sudden oak death (SOD) in the U.S.A. Plants
affected by P. ramorum show a range of symptoms including stem canker and
tip dieback. Phytophthora ramorum was first detected Ireland in 2003.
Phytophthora ramorum is reported to infect over 64 plant species, including a
number which have significant commercial and amenity value in Ireland,
particularly Rhododendron and Viburnum spp. The closely-related P.
kernoviae causes similar symptoms to P. ramorum and was first discovered in
the UK in 2003, New Zealand in 2006 and Ireland in 2009. To date P.
ramorum and P. kernoviae have not been detected on tree species in Ireland,
however there is strong concern however that Irish trees could become
infected. Extensive surveys have been carried out by the Department of
Agriculture, Fisheries & Food (DAFF) from 2003 to present. Since 2003
nearly 6000 samples were collected around Ireland and P. ramorum was
detected in all years: positive samples: 8% (2003), 2% (2004), 19% (2005),
10% (2006) & 16% (2007), 12% (2008) & 11% (2009)]. In 2003, P. ramorum
was only found on Rhododendron and Viburnum spp., by 2009 the presence
of P. ramorum was confirmed on six plant genera (Rhododendron, Viburnum,
Camellia, Photinia, Magnolia & Leucothoe). Phytophthora kernoviae was first
detected in Ireland in 2008 on Rhododendron spp. and confirmed in 2009.
Eradication & containment measures are being implemented in accordance
with EU legislation.
Discovering single nucleotide polymorphisms (SNPs) in an
uncharacterized fungal genome using the software EagleView to evaluate
454 sequencing data
K. D. BRODERS (1), P. J. SanMiguel (2), R. P. Westerman (2), K. E. Woeste
(3), G. J. Boland (1)
(1) School of Environmental Sciences, University of Guelph, Guelph, ON,
CANADA; (2) Department of Horticulture and Landscape Architecture,
Purdue University, West Lafayette, IN, U.S.A.; (3) USDA Forest Service,
Hardwood Tree Improvement and Regeneration Center, Department of
Forestry and Natural Resources, Purdue University, West Lafayette, IN,
U.S.A.
Phytopathology 100:S17
Benefits from high-throughput sequencing technology, such as 454
pyrosequencing, is most apparent for species with high societal or economic
value but few genomic resources. However, it is questionable how well the
sequencing of large numbers of short reads, for species with essentially no
prior genome sequence information, will support SNP discovery. The focus of
this research was to develop a method for identifying SNPs in a fungal
genome using 454 pyrosequencing when no reference sequence is available.
To discover SNPs, genomic DNA from eight isolates of Sirococcus
clavigignenti-juglandacearum were bulked in one four-region sequencing run
on a Roche 454 GS_FLX. This yielded 71 million total bases comprising 217
thousand reads, 80% of which collapsed into 16,125,754 bases in 30,339
contigs upon assembly. By aligning the reads from multiple isolates we
detected 298 SNPs using Roche’s gsMapper. However, with no reference
sequence available, it was difficult to detect true polymorphisms. EagleView
software was used to manually examine each contig that contained one or
more putative SNPs. The program confirmed 45 of the original 298 putative
SNPs. Of those 45 SNPs, 16 were validated using standard sequencing. This
research provides the framework for the rapid and cost-effective discovery of
SNP markers for non-model organisms and proves to be especially useful in
the case of asexual or clonal fungi with limited genetic variability.
Reclassification of the butternut canker fungus, Sirococcus clavigigentijuglandacearum, into the genus Ophiognomonia
K. D. BRODERS (1), G. J. Boland (1)
(1) University of Guelph, Guelph, ON, CANADA
Phytopathology 100:S17
Sirococcus clavigignenti-juglandacearum (Sc-j), which causes a canker
disease on butternut, is largely responsible for the trees decline in the United
States and Canada. The original description of the species was based largely
on anamorphic characters as the teleomorph is unknown. Recent phylogenetic
investigations found that Sc-j is not a member of the genus Sirococcus, and
proper taxonomic classification is required. The aim of this study was to use
sequence data to determine the phylogenetic placement of Sc-j within the
Gnomoniaceae. Twenty-eight isolates of Sc-j from Ontario and across the
eastern United States, in addition to representatives of the major lineages
within the Gnomoniaceae, were included in the analysis. A portion of each of
the genes coding for -tubulin, actin, calmodulin, internal transcribed spacers
1 & 2, and the translation elongation factor 1-alpha were sequenced and
evaluated. There were no differences in the sequences of the five genes among
the isolates of Sc-j studied, providing evidence for a recent introduction into
North America, followed by asexual reproduction and spread via conidia. The
phylogenetic analyses clearly demonstrate the butternut canker fungus does
not belong in the genus Sirococcus; and provided strong support (99% MP
and 100% NJ bootstrap values) for its inclusion in the genus Ophiognomonia,
thereby, supporting a reclassification of the fungus to Ophiognomonia
clavigignenti-juglandacearum.
Revising the high temperature threshold for the Gubler-Thomas grape
powdery mildew risk index
J. C. BROOME (1), E. K. Hand (1), P. Backup (1), C. N. Janousek (1), W. D.
Gubler (1)
(1) University of California Davis, Davis, CA, U.S.A.
Phytopathology 100:S17
Powdery mildew, caused by Erysiphe necator, is an important disease of
grapes. A temperature-driven disease risk model was developed by Gubler &
Thomas (GT). Two years of detached leaf co-culture studies were conducted
with single high temperature treatments at a range of durations and showed
that E. necator continues to grow and reproduce in the lab at higher
temperatures than previously reported. In 2009 we tested how consecutive,
multiple heat treatments affected fungal growth parameters. We found that
higher temperatures are increasingly lethal to the pathogen, reduce colony
survival, and delay and reduce spore production. Temperature alone had a
more pronounced effect than did the number of consecutive heat treatments
(1, 2 or 3). Repeated consecutive exposures of 4 hrs at 36 and 38°C up to 3
days in a row resulted in less colony death and higher spore production, than
one continuous exposure of 12 hrs at the same temperature. We are field
testing several revisions of the GT model; raising the high temperature
threshold and lengthening its duration from 35°C for 15 min, to 36°C and
38°C for 4 and 2 hrs, respectively. We adjusted how the index accounts for
observed delays in fungal growth and reproduction. Future work will involve
Vol. 100, No. 6 (Supplement), 2010
S17
integrating information on early season vineyard inoculum density and host
varietal resistance into the model.
A tag-array minisequencing-based system for detecting and genetic fingerprinting Wheat streak mosaic virus: Implications for plant pathogen
forensics
T. BROWN (1), U. Melcher (1), J. Fletcher (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S18
The U.S. Department of Homeland Security has identified several gaps in the
security of the agricultural sector, indicating that one of the nation’s largest
economic contributors is vulnerable to a biological attack. Validated forensic
tools that could be applied during a plant pathogen forensic investigation are
needed for a balanced U.S. forensic capability. The goal of this project is to
develop and validate a tool to simultaneously identify and forensically profile
plant pathogens during the attribution of a biocrime. A tag-array
minisequencing system was employed to profile a panel of single nucleotide
polymorphisms (SNPs) from the model pathogen Wheat streak mosaic virus.
Tailed primers terminating directly upstream from the SNPs were extended by
one fluorescently labeled ddNMP during the minisequencing reactions.
Products were subsequently hybridized to the tag array for detection and
genotyping. Using synthetic targets, base misincorporation was found to be
negligible and the mean values between technical replicates (n = 3) were
indistinguishable using one-way ANOVA. The specificity of the assay has yet
to be determined, however the primer design parameters are expected to yield
strain specificity. The universality of this tool will provide a foundation for
the development of similar systems for other plant and human pathogens. This
tool also will be useful for rapid diagnostics and epidemiology studies during
natural plant disease outbreaks.
Commercial extracts of the brown seaweed Ascophyllum nodosum and
silicon reduce plant death due to Fusarium solani and increase yields of
cucurbits
G. E. Brust (1), R. E. ROSS (2), J. Jayaraj (3)
(1) University of Maryland, Upper Marlboro, MD, U.S.A.; (2) Acadian Sea
Plants LLC, Monroe, NC, U.S.A.; (3) The University of the West Indies, St.
Augustine, TRINIDAD
Phytopathology 100:S18
Crop losses due to Fusarium spp. are important to cucurbit growers along
with an increasing interest in natural ways to improve disease resistance.
Extracts of the brown seaweed, Ascophyllum nodosum and products
containing silicon have both been shown to promote disease resistance on
many crops. In a 2008 watermelon trial located in Upper Marlboro, MD,
Fusarium solani symptoms were suppressed by extracts of Ascophyllum
nodosum. At the final rating, 30% of the watermelon plants were dead from
this pathogen in the control plots vs. 10% in Ascophyllum extract treatments.
A second study was implemented in 2009 on Gladiator Pumpkins. Calcium
silicate and Ascophyllum seaweed extract were applied to pumpkins grown in
a field known to have Fusarium spp. infected squash three years prior. At the
final rating, 24.6% of the pumpkin plants were dead in the control plots vs.
19.2% in the silicon plots, 13.6% in the Ascophyllum extract treatment, and
just 6.1% in the plots with both calcium silicate and Ascophyllum extract.
These field studies were further supported by two greenhouse studies where
applications Ascophyllum extract to cucumber plants reduced incidence of
Fusarium oxysporum and enhanced the activities of plant defense-related
enzymes including chitinase, -1,3-glucanase, peroxidase, polyphenol
oxidase, phenylalanine ammonia lyase and lipoxigenase as well as elevated
levels of total phenols compared to the control.
The effects of soil steaming on the abundance of bacterial microflora in
the rhizosphere and roots of Chrysanthemum
M. BURKETT-CADENA (1), E. van Santen (1), J. Kloepper (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S18
Soil steaming is a substitute for methyl bromide for managing soil-borne
pathogens. In the cut-flower industry, negative effects of soil steaming have
been reported as growers have noticed a reduction in flower size and increase
in deleterious rhizobacteria in chrysanthemum. To examine the effects of
steaming on the rhizosphere microflora, greenhouse experiments were
conducted which documented changes in populations of total culturable
bacteria, fluorescent pseudomonads, and aerobic spore forming bacteria
(AEFB) after repeated steam applications. In the first cropping cycle,
treatments included steamed soil, steamed soil + PGPR, and non-steamed soil.
For the next cycle, half of the steamed soil was steamed, adding a fourth
treatment. Rhizosphere bacteria were isolated, and populations determined by
direct plate count. In the first cycle, bacterial populations from unsteamed soil
were significantly lower than populations from steamed soil and steamed soil
+ PGPR. In the second cycle, steamed soil + PGPR retained higher
S18
PHYTOPATHOLOGY
populations of total bacteria and AEFB than unsteamed soil. Soil that was
steamed twice had significantly higher populations of fluorescent
pseudomonads than all other treatments, but had significantly lower
populations of total bacteria than all other treatments. Populations in soil that
was not resteamed for the second cycle differ from the control, suggesting that
populations will not return to control levels even if steam is not reapplied.
Evaluation of inoculation methods for screening of rapeseed materials for
resistance against Sclerotinia sclerotiorum
P. BURLAKOTI (1), V. Magnusson (2), W. D. Dai (2), L. E. del Rio
Mendoza (1)
(1) Dept. Plant Pathology, North Dakota State University, Fargo, ND, U.S.A.;
(2) Dept. Plant Science, North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S18
Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is an economic
fungal disease affecting rapeseed (Brassica napus L) worldwide. Since
expression of SSR symptoms shows much variability and the trait is
quantitative in nature, reliable phenotypic evaluation methods for
characterization of SSR resistance are needed. Six different inoculation
methods were compared for their reliability to discriminate between S.
sclerotiorum-resistant and susceptible materials. The methods were evaluated
using two S. sclerotiorum isolates (WM031 and WM192) collected in North
Dakota and 326 clonal plants derived from double haploid resistant (PI458940
x Ames 26628) and susceptible (Westar) B. napus materials. The methods
involved mycelial inoculation on detached leaves and stems, petiole
inoculation, straw inoculation, stem-piercing with toothpick, and mycelial
spray. The experiment was conducted using a randomized complete block
design with four replications and was repeated once. Detached materials were
inoculated and incubated in laboratory at 16 hour light daily and 22°C
temperature while inoculated plants were incubated in greenhouse at similar
conditions. Detached stem and the petiole inoculation techniques produced the
most consistent results and are considered more reliable to evaluate materials
for their reaction to SSR.
Identification and analysis of Fusarium verticillioides genes differentially
expressed during conidiation
A. M. BURNHAM (1), D. W. Brown (2), A. E. Glenn (3)
(1) Department of Plant Pathology, University of Georgia, Athens, GA,
U.S.A.; (2) USDA, ARS, Bacterial Foodborne Pathogens & Mycology
Research Unit, Peoria, IL, U.S.A.; (3) USDA, ARS, Toxicology & Mycotoxin
Research Unit, Athens, GA, U.S.A.
Phytopathology 100:S18
Fusarium verticillioides, an endophytic maize pathogen, is the causal agent of
diseases such as ear rot, seedling blight, and stalk rot, resulting in major
economic losses in maize production. This fungus can also cause certain
diseases in animals due to consumption of feed contaminated with fumonisin
mycotoxins. F. verticillioides produces abundant microconidia in long chains
from the apex of phialides. These conidia are essential for the infection of the
crop. Upon mating two previously characterized conidia-producing strains,
MRC 826 and NRRL 25029, spontaneous mutations occurred resulting in half
of the progeny being unable to produce conidia. These mutants produced germ
tube-like growths from the tips of phialides instead of normal enteroblastic
production of conidia. Based on microarray data comparing MRC 826 (wild
type) and AEG 3-A3-5 (aconidial mutant), thirteen candidate genes having at
least 10-fold change in expression in the wild-type strain were chosen for
further analysis. One of the thirteen, FVEG_10983, has been targeted for gene
deletion because of its 72-fold change. FVEG_10983 has no homology to
previously characterized proteins and no obvious protein domains. BLAST
searches showed significant similarity only to other filamentous fungi
including one homolog from F. oxysporum and three from F. graminearum.
Further analysis of FVEG_10983 and the other genes may identify novel
characteristics of sporulation in F. verticillioides.
Differential expression of putative polyketide biosynthetic gene clusters in
Fusarium verticillioides
R. BUTCHKO (1), D. Brown (1), M. Busman (1), B. Tudzynski (2), P.
Wiemann (2)
(1) USDA, Peoria, IL, U.S.A.; (2) Westfälische Wilhelms-Universität Münster
Institut für Botanik, Münster, GERMANY
Phytopathology 100:S18
The maize pathogen Fusarium verticillioides can produce a number of
polyketide derived secondary metabolites, including fumonisins. Fumonisins
cause diseases in animals, and show epidemiological correlation with
esophageal cancer and birth defects in humans. The F. verticillioides genome
contains numerous polyketide synthase (PKS) genes, including FUM1 which
encodes the PKS required for fumonisin production. Only a small number of
these PKS have been described with regard to the polyketide molecules they
produce. Regulation of the expression and activity of fungal secondary
metabolite genes clusters can be effected by multiple factors including both
pathway specific and nonspecific transcription factors. In Aspergillus species,
LaeA has been shown to regulate the expression of secondary metabolite gene
clusters at a higher hierarchical level, acting as a master regulator at the
chromatin level. Here, the effects of the deletion of a putative laeA homolog in
F. verticillioides are described. Toxin analysis revealed a decrease in fumonisin
production, as well as other secondary metabolites. Microarray analysis
indicates that the expression of the other putative secondary metabolite gene
clusters is down- regulated in the F. verticillioides laeA deletion strain.
Exploring cover crop carbon sources for anaerobic soil disinfestation
D. M. Butler (1), E. N. ROSSKOPF (1), N. Burelle (1), J. Muramoto (2), C.
Shennan (3)
(1) USDA ARS, Fort Pierce, FL, U.S.A.; (2) Center for Agroecology and
Sustainable Food Systems, University of California, Santa Cruz, CA, U.S.A.;
(3) Department of Environmental Studies and Center for Agroecology and
Sustainable Food Systems, Santa Cruz, CA, U.S.A.
Phytopathology 100:S19
In Florida field trials, a raised-bed plastic mulch vegetable production system
was developed using anaerobic soil disinfestation (ASD) for bell
pepper/eggplant double crop production. Pathogen, weed, and nematode
control was equivalent or better than methyl bromide. Molasses was used as
the carbon source for supporting microbial generation of anaerobicity. To test
warm season cover crops as replacements for molasses, a greenhouse trial was
conducted using field soil in which tropical legumes and grasses and brassica
species were grown and incorporated with or without composted broiler litter.
Pathogen inoculum packets, yellow nutsedge tubers, and root-knot nematode
eggs were introduced at cover crop incorporation. Anaerobicity (Eh) was
monitored using oxidation-reduction probes and pathogen survival was
assessed. All cover crops resulted in cumulative Eh values that were equal to
the molasses treatment. Litter did not affect cumulative Eh in the greenhouse,
but an interaction occurred between cover crop and litter with regard to
survival of Fusarium oxysporum f. sp. lycopersici (Foxy) and Sclerotium
rolfsii (SR). Foxy survival was reduced by more than 97% in all cover crops
and the fallow treatment containing molasses when compared to the
unamended control. Survival of SR was lowest in the molasses only treatment
and in sorghum-sudan, pearl millet, and cowpea mixed with pearl millet or
mustard-arugula with no litter.
Foliar fungicides reduce anthracnose top dieback of maize
E. BYAMUKAMA (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S19
Infection of maize by Colletotrichum graminicola may result in leaf blight,
top dieback or stalk rot symptoms. Foliar applied fungicides have been
reported to reduce stalk rot and top die back under high foliar disease severity.
The effect of fungicide application on anthracnose top die back of maize was
investigated at three locations in Iowa in 2009. At two locations, ISU
Southeast Research Farm (SERF) and ISU Southwest Research Farm
(SWRF), three fungicides, Headline (6oz/acre), Quilt (14oz/acre), and
Stratego (4oz/acre), were applied at either tasseling, blister or milk growth
stages to one hybrid. At Sorensen, Headline (6 oz/acre) was applied at
tasseling to four hybrids with varying stalk rot susceptibility. No fungicide
application served as the check. In all trials, the incidence of top die back at
early dent, mean stalk rot severity at physiological maturity, yield, and grain
moisture data were collected. Fungicide application significantly (P < 0.0001)
reduced top die back incidence compared to the check at all the three sites. An
application of a fungicide at VT reduced disease the greatest. No differences
between fungicides were detected for top die back incidence. Fungicide
treated plots had lower stalk rot severity at SERF and SWRF but not at
Sorensen. There was a negative but significant correlation between grain yield
and top back incidence (r = –0.72, P = 0.04) at Sorensen. Further work is
needed to determine the effect of anthracnose top die back on yield of maize.
Assessment of new biomaterials for sample collection and nucleic acid
recovery
D. J. CAASI (1), M. Arif (1), F. M. Ochoa-Corona (1)
(1) Oklahoma State University, Department of Entomology and Plant
Pathology, National Institute for Microbial Forensic and Food & Agricultural
Biosecurity (NIMFFAB), Stillwater, OK, U.S.A.
Phytopathology 100:S19
Collecting and archiving nucleic acids (NA) are key steps in detection and
diagnosis when using PCR for agricultural biosecurity or microbial forensics
applications. Paper-based technologies for collection offer several advantages,
such as storage of NA at room temperature. However, the recovery of NA
from this technology requires few steps and direct PCR is hampered by the
residual paper matrix itself. We tested five biomaterials with soluble
properties having different thicknesses (0.05 mm to 0.17 mm) for
effectiveness in PCR amplification of NA. Scanning electron microscopy of
pore spaces and crevices of the biomaterials either dry or wet, and with or
without bacteria (Pseudomonas syringae pv. tomato) were retained after
wetting. The absorbance of residual materials was measured in solution at 260
and 280 nm, and ranged from 0.014 to 0.034 O.D. at 260 nm, and from 0.013
to 0.030 O.D. at 280 nm. Optically measured residues were highest in the
thickest biomaterial (0.17 mm) and lowest in the thinnest (0.05 mm). The five
biomaterials were also blotted with DNA from Pythium spinosum or dsRNA
from Citrus leprosis virus C and then subjected to PCR. Blotted DNA and
RNA were successfully retrieved from all materials used; and residues were
not inhibitory to PCR amplification. The assessed biomaterials have a
potential application for streamlining both PCR-based protocols and NA
storage during the collection and recovery of microbial NA.
New biocontrol strategy against the sexual fruiting bodies of grapevine
powdery mildew
T. CAFFI (1), S. Legler (1), V. Rossi (1), L. Kiss (2), A. Pintye (2)
(1) Univ Cattolica del Sacro Cuore, Piacenza, ITALY; (2) Hungarian
Academy of Science, Budapest, HUNGARY
Phytopathology 100:S19
Erisyphe necator is the casual agent of grapevine powdery mildew and its
control is essentially based on chemicals. Potential BCAs (Bio Control
Agents) were tested for their anti-powdery mildew effect and some
commercial biofungicides were developed. Nevertheless, reports on their
efficacy show that they cannot control E. necator efficiently. A new strategy
was studied within the “BCA_grape project” (funded by EU Commision, 7th
Framework Programme of Research) aimed at inserting biocontrol in an
integrated disease management approach. This strategy includes i) new strains
of the mycoparasite Ampelomyces spp.; and ii) new targets their application,
i.e., the fruiting bodies of E. necator (chasmothecia). Ten different European
Ampelomyces strains were selected for their ability in producing abundant
conidia and germinating rapidly, under a wide range of environmental
conditions. Ability of these strains of parasitizing chasmothecia was tested
under controlled environmental conditions: they had different mycoparasitic
activity, but all of them were more effective on young than mature
chasmothecia. Late summer and fall applications of new Ampelomyces strains
in vineyard (when first immature chasmothecia were formed) were able to
significantly reduce the severity of ascosporic infections in the following
spring and to delay the beginning of the epidemic compared to the untreated
plots. A mathematical model was also developed to simulate chasmothecia
maturation, as a tool for timing BCA application.
A test of taxonomic and biogeographic predictivity: Resistance to potato
virus Y in wild relatives of the cultivated potato
X. CAI (1), D. Spooner (2), D. Halterman (2), A. Charkowski (3), R. Groves
(4), S. Jansky (2)
(1) University of Wisconsin-Madison, Madison, WI, U.S.A.; (2) USDA/ARS,
Madison, WI, U.S.A.; (3) University of Wisconsin-Madison, Plant Pathology,
Madison, WI, U.S.A.; (4) University of Wisconsin-Madison, Entomology,
Madison, WI, U.S.A.
Phytopathology 100:S19
A major justification for taxonomic research is its assumed ability to predict
traits in a group for which the trait has been observed in a representative
subset. In this study, we evaluated potato virus Y resistance using 135
accessions of potato to determine whether we can predict the distribution of
resistance in wild Solanum species based on taxonomic or biogeographic data.
Tremendous variation for PVY resistance was found within and among
species. There is no consistent association between resistance and taxonomic
series, clades, ploidy, and breeding system. However, the correlation
coefficient between endosperm balance number (EBN) and PVY resistance
was –0.22. The five species with the highest percentage of resistant plants
were 1 EBN. A Chi-square test indicated that the 1 EBN species contain a
higher percentage of resistant plants than expected. Our study identified new
germplasm with resistance: S. albornozii, S. andreanum, S. bukasovii, S.
bulbocastanum, S. cardiophyllum, S. hjertingii, S. iopetalum, S. jamesii, S.
kurtzianum, S. paucijugum, S. pinnatisectum, and S. schenckii.A correlation
between resistance and elevation at which the individuals were collected was
high and significant. A Chi-square analysis revealed that there was a much
higher than expected proportion of resistant plants in accessions collected at
elevations below 2100m. This relationship may be related to the distribution
of aphids, which act as vectors for PVY.
Genus Alternaria species as pathogens of Vaccinium meridionale in
Colombia
C. Calderon (1), C. Socha (1), S. Restrepo (2), P. JIMENEZ (3)
(1) Bogotá, COLOMBIA; (2) Univ de Los Andes, Bogotá, COLOMBIA; (3)
Univ Militar Nueva Granada, Bogotá, COLOMBIA
Phytopathology 100:S19
Vol. 100, No. 6 (Supplement), 2010
S19
In previous studies, Alternaria sp. was identified as a pathogen of Vaccinium
meridionale. Its attack induces a variable type of symptoms and is frequently
recovered from the shoot. To know more about which species are involved in
this pathosystem, different organs showing red spots, and necrotic spots were
sampled in 3 different farms in Boyacá department, Colombia. After surface
sterilization, the samples were cultured on PDA. Alternaria sp. was recovered
from all of the samples. Taxonomic identification and Koch’s postulates
corroboration on detached organs and complete plants, were performed.
Results showed that A. alternata and A. tenuissima, are the causal agent of
disease. However, A. alternata induced red spots, that grew very slowly and
showed a necrotic center. This species was more aggresive when a lesion was
present on the tissue, then the invasion generated a fast growing necrotic area,
and the conidiophores arranged in concentric circules. Meanwhile, A.
tenussima showed a cottony mycelium, extended on the organ’s surface and,
suddenly, the tissue under the mycelium became necrotic. These results
suggest that A. alternata is a mild pathogen, able to take advantage,
agressively, of open courts in a more effective colonization. Differently, it
seems clear that A. tenuissima is a strong pathogen able of penetrate the
tissues in a direct way, without requiring open courts.
PGPR potential of Bacillus isolated from potato rhizosphere in the
Andean highlands of Peru
P. CALVO-VELEZ (1), E. Ormeño-Orillo (2), E. Martínez-Romero (2), A.
Oswald (3), D. Zuñiga-Davila (4)
(1) Auburn University, Auburn, AL, U.S.A.; (2) Universidad Nacional
Autónoma de México, Cuernavaca, MEXICO; (3) International Potato Center,
Lima, PERU; (4) Universidad Nacional Agraria La Molina, Lima, PERU
Phytopathology 100:S20
Potato is an essential crop in Peru, contributing both to commercial and
subsistence agriculture; however, there have been no comprehensive studies
of its rhizosphere diversity. The aim of this investigation was to isolate, screen
and identified bacteria of the genus Bacillus from the potato rhizosphere.
Rhizosphere samples were collected from 21 fields in two highlands regions,
representing several native varieties of potato. In a first screening of these
isolates, their capacity to antagonize Rhizoctonia solani was evaluated. The
strains which tested positive were further screened for Fusarium solani
antagonism, IAA production, and phosphate solubilization. Finally a
greenhouse trial was conducted and the strains were identified using
molecular characterization with the 16s r RNA technique. We obtained a total
of 63 isolates of Bacillus spp.; 43 (68%) of those showed positive control of
R. solani ; and 91% of the selected strains also inhibited the growth of F.
solani. IAA was produced at some level by 81% of the strains, and 58%
solubilized tricalcium phosphate. In the greenhouse experiment a significant
increase in number and dry weight of tubers, and total dry weight of the plant
with 23 strains. Phylogenetic analysis revealed that the majority of the strains
were B. amyloliquefaciens. Hence, the rhizosphere of native potatoes growing
in the Andes is a rich source of antagonistic Bacillus spp. with potential future
use as PGPR inoculants to improve potato production.
Sowing the Seeds of Science
A. R. Camp (1), H. W. Lange (1), S. Reiners (1), C. D. SMART (1)
(1) Cornell University, Geneva, NY, U.S.A.
Phytopathology 100:S20
Plant pathologists and other scientists at Cornell University’s New York State
Agricultural Experiment Station (NYSAES) are involved in an ongoing
partnership with the Geneva City School District in Geneva, NY. The goals of
this program are to (i) engage elementary aged students in a hands-on,
inquiry-based study of science; (ii) generate awareness of how food is grown
and where it comes from; and (iii) increase student exposure to agricultural
sciences and related careers. Beginning in the spring of their third grade year,
students are visited by Cornell faculty and learn about seeds, take a field trip
to NYSAES to see the research that happens there, and help plant a vegetable
and flower garden at their elementary school. During the summer, they have
the opportunity to participate in a 5-week science camp run by school district
faculty and scientists from Cornell. In the fall of their fourth grade year they
share their new knowledge with the school community at a fall Harvest
Festival. We have seen several positive outcomes from this program,
including increased student knowledge, based on pre- and post-tests given
during the Summer Science Camp; increased enrollment in local 4-H
programs; and an increase in the proportion of students who pass the 4th grade
science test mandated by New York State. In addition, this program helps
plant pathologists and other scientists from Cornell foster good relationships
with, and increase their impact in, their local community.
Phytophthora capsici in New York State: Resistance to mefenoxam and
population structure
A. R. CAMP (1), M. G. Milgroom (2), J. C. Meitz (3), A. McLeod (3), W. E.
Fry (2), M. T. McGrath (4), H. R. Dillard (1), C. D. Smart (1)
S20
PHYTOPATHOLOGY
(1) Cornell University, Geneva, NY, U.S.A.; (2) Cornell University, Ithaca,
NY, U.S.A.; (3) University of Stellenbosch, Stellenbosch, SOUTHWEST
AFRICA; (4) Cornell University, Riverhead, NY, U.S.A.
Phytopathology 100:S20
More than 250 isolates of Phytophthora capsici were collected in 2006, 2007,
and 2008 from sweet peppers, hot peppers, summer squash, winter squash,
pumpkins, zucchini, tomatoes, and eggplants grown at 22 sites in four regions
of New York State (western, central, Capital District, and Long Island).
Isolates were assayed for mefenoxam resistance and assigned to multilocus
genotypes (MLGs) based on mating type and five microsatellite loci.
Mefenoxam-resistance was common in the Capital District and on Long
Island, but not in western and central New York. Both A1 and A2 mating
types were found at 12 of the 22 sites. At seven of the 11 sites from which at
least ten isolates were sampled the ratio of A1 to A2 isolates did not differ
significantly from 1:1. Of the 126 distinct MLGs identified, 117 were each
restricted to only one of the 22 sites, and nine were detected at two or three
sites each. From analysis of pairwise comparisons, it was learned that
populations at nearly all sites were significantly different (P ≤ 0.05) from each
other. Much of the variation in the state-wide population could be attributed to
differences either among regions or among sites. These results indicate that P.
capsici in New York is highly diverse, but gene flow among different regions
and fields is very restricted.
Impact of cropping sequence on diseases, nematodes, and yield of peanut,
cotton, and corn in central Alabama
H. L. CAMPBELL (1), A. K. Hagan (1), K. L. Bowen (1), K. S. Lawrence
(1), S. P. Nightengale (2)
(1) Auburn University, Auburn, AL, U.S.A.; (2) EV Smith Research Center,
Plant Breeding Unit, Tallassee, AL, U.S.A.
Phytopathology 100:S20
A study was established at the Plant Breeding Unit, Tallassee, AL in 2003 to
assess the impact of crop rotation on severity of diseases in peanut and corn as
well as cotton root-knot nematode in corn, cotton, and peanut and to define
the agronomic benefits or limitations of corn as a rotation partner with peanut
and cotton. Peanuts were rated for leaf spot diseases, soil-borne diseases, and
TSWV. Soil samples were taken for nematode assays shortly after each crop
was harvested. In 2009, rotation and the Counter 15G soil insecticide
treatment had a significant impact on corn yield and yield was significantly
higher for the Counter 15G treated corn. Occurrence of CBR and peanut yield
was significantly influenced by crop sequence but stem rot and leaf spot were
not. TSWV levels were so low that only a few symptomatic plants were
observed. CBR incidence was highest in plots maintained in continuous
peanuts and yield was significantly lower than that of peanuts cropped behind
one or two years of cotton or corn. Generally, yields for the one year out
cropping sequence where cotton followed corn were higher compared with
cotton followed by cotton and corn. Crop sequence had a significant impact
on the yield of corn as well as on CBR and yield of peanut but not the severity
of foliar diseases of corn or peanut.
Multiple effects of grafting on tomatoes and associated microbial
communities
C. CAO (1), F. Baysal-Gurel (2), S. A. Miller (2), T. L. Graham (3), M. D.
Kleinhenz (2), D. M. Francis (2), B. B. McSpadden Gardener (2)
(1) The Ohio State University, Wooster, OH, U.S.A.; (2) The Ohio State
University, OARDC, Wooster, OH, U.S.A.; (3) The Ohio State University,
Columbus, OH, U.S.A.
Phytopathology 100:S20
In order to determine if grafting affects the physiology, health, and associated
microbial communities of tomato, rootstocks were left ungrafted, self-grafted
or cross-grafted to cv. Celebrity before transplanting into the field in a
randomized complete block design. Overall, ungrafted plants had more fresh
and dry weight biomass than self-grafted regardless of rootstock (P < 0.01 for
all comparisons). Nitrogen was 3–20% higher in leaves of self-grafted than
ungrafted plants in 3 of 4 rootstocks tested (P < 0.05). Additionally, grafting
was shown to influence levels of two secondary products present in apical
leaves (P < 0.05 each). Comparisons of foliar disease incidence, i.e. late blight
and Septoria leaf spot, revealed that self-grafted plants had less disease than
ungrafted plants in three of four comparisons. In contrast, no significant
variation in disease incidence was observed in cross-grafted plants, indicating
that root stock genotype did not affect host resistance status. Terminal
restriction fragment length polymorphism (T-RFLP) analyses of 16S and ITS
sequences were performed to determine whether grafting or rootstock affected
rhizosphere community structure. However, no clear patterns of graftinginduced changes in microbial community structure were observed. These
results indicate that grafting-induced changes in host physiology, and not the
root-associated microbial communities, can influence plant health several
weeks after transplanting.
Fusarium stem blight of blueberry in Argentina
M. Caprara (1), E. R. WRIGHT (1), M. C. Rivera (1), B. A. Perez (2)
(1) Facultad de Agronomía, Universidad de Buenos Aires, Ciudad de Buenos
Aires, ARGENTINA; (2) IMYZA-INTA, Hurlingham, Buenos Aires,
ARGENTINA
Phytopathology 100:S21
In Argentina, Buenos Aires province is one of the major producing areas of
blueberry (Vaccinium corymbosum L.). Disease surveys conducted in 2009 in
the Indacochea area (Chivilcoy county) allowed to observe blueberry plants
cv. O’Neal with dieback and stem blight. This disease affected 5% of the
plants. Small pieces of the diseased branches were superficially disinfected
and placed on 2% WA medium at room temperature. After purification,
fungal morphological traits were studied in CLA, SNA and PDA. A
pathogenicity test, with the Fusarium isolate obtained, was conducted by
spraying a conidial suspension on the aerial part of unwounded and previously
wounded one year-old blueberry plants cv. O’Neal. After 10 days, stem and
leaf blight was recorded in unwounded and wounded plants but not in
blueberry fruits. Studies are underway to confirm the section and species to
which this Fusarium isolate may be considered.
Phytophthora infestans oospores: Production and viability in Colombia
M. E. CÁRDENAS (1), M. C. Céspedes (1), A. J. Bernal (1), S. Restrepo (1)
(1) Univ De Los Andes, Bogota, COLOMBIA
Phytopathology 100:S21
Phytophthora infestans is the causal agent of the late blight disease in potato
(Solanum tuberosum) and other members of the Solanaceae family (S.
phureja, S. lycopersicum, S. betaceum, S. melongena, S. quitoense and
Physalis peruviana among others). In Colombia, the incomes of many people,
mainly farmers, depend on most of these crops, mainly potato, tree tomato and
cape gooseberry. Recently, the A2 mating type was reported for the first time
in this country and opened the possibility to a higher genetic variation in the
P. infestans population that could lead to changes in its fungicides
susceptibility patterns, an increased virulence or a broadening of host range.
However, last year, a P. infestans population survey showed that in Colombia
this pathogen is clonal despite the presence of the A2 mating type. In order to
understand the population dynamics of this pathogen in Colombia crosses
between A1 mating type from different hosts and the Colombian A2 mating
type from cape gooseberry were tested for the production, viability and
germination of oospores. Apparently, the host adaptation is not an explanation
to the reduced c.f. inexistent sexual reproduction of this pathogen in
Colombia. Besides the isolation low frequency of the A2 mating type a postmating incompatibility is suggested according to the low viability and
therefore the germination percentages obtained.
Effects of pH on genes involved in oxalic acid production in the brown rot
fungus Postia placenta
B. CARLSON (1), J. Jellison (1)
(1) University of Maine, Orono, ME, U.S.A.
Phytopathology 100:S21
Brown rot fungi are the organisms primarily responsible for the
biodegradation of soft, in-service wood. During the decay process, brown rot
decay fungi such as Postia placenta release large amounts oxalic acid (pKa1 =
1.27, pKa2 = 4.28) which rapidly dissociates into H+ and oxalate (C2O42–). To
characterize the relationship between glucose consumption, oxalic acid
production and pH, a time course study was run over 21 days on P. placenta
grown in 50 ml of modified Highley’s media. Shake cultures with 1 mM
asparagine and ammonium-L-tartrate and 5% glucose were harvested after 1,
3, 5, 7, 13, 17, 19, 22 days following inoculation and were analyzed for
glucose, oxalic acid, pH, and biomass. As available glucose in culture
decreased, pH decreased from 4.73 ± 0.03 to 2.81 ± 0.06 and oxalic acid
increased from below detection limits to 0.51 ± 0.10 mg/ml. To determine the
effect pH has on gene expression of enzymes involved in oxalic acid
synthesis, static cultures were grown and pH was adjusted to 7 with 2M
NaOH. 17 hours after treatment, the pH of the treated culture dropped and
mycelia were harvested for real time PCR. Preliminary results (n = 1) show a
9.4 fold increase in glyoxylate oxidase but no significant change in oxalate
decarboxylase, oxaloacetase, isocitrate lyase gene expression.
Monitoring latent Colletotrichum acutatum infections in strawberry using
a bioassay and real time PCR in organic and conventional systems
M. E. CARNES (1), M. Rahman (1), F. J. Louws (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S21
Strawberry anthracnose fruit rot, caused by C. acutatum is an economically
important disease in the southeastern United States. Latent infections (LI) can
lead to wide-spread pathogen dispersal due to the shipment of infected plant
material. In 2009, strawberry leaves were sampled from 40 plants weekly at
an organic and conventional farm and LI was evaluated using a paraquat dip
bioassay. LI incidence was 15% in leaves sampled at the organic farm on 10
March, and increased to 67% by 1 May. In contrast, at the conventional farm
where fungicide was applied as a preventative measure, LI incidence reduced
throughout the monitoring period from 20% on 17 Mar to 5% by 23 April.
The paraquat assay is effective, but requires handling of toxic chemicals and a
7 day incubation period. A quantitative PCR (qPCR) protocol was developed
for rapid and more sensitive screening. During the 2009–10 growing season,
infection incidence was compared using qPCR and paraquat dip assays on
leaves sampled from the same plants. Compared to the paraquat assay,
utilization of the qPCR TaqMan protocol increased detection of latent
infections by 39%. Equally efficient results were obtained using a SYBR
green protocol. Compared to very young or old leaves, greater sensitivity was
observed by using middle-aged leaves to evaluate LI on foliage. Modification
of the qPCR protocol for high throughput screening of LI incidence may
provide an additional tool in integrated disease management strategies.
Additional sources of broad-spectrum resistance to Puccinia coronata f.
sp. avenae in Canadian accessions of Avena barbata
M. CARSON (1)
(1) USDA ARS, St. Paul, MN, U.S.A.
Phytopathology 100:S21
Crown rust (Puccinia coronata f. sp. avenae) is the most damaging disease of
oat and race-specific seedling (Pc) genes for resistance have been the primary
means of control. As resistance genes have been deployed in cultivars,
corresponding virulence in the crown rust population increased rapidly, such
that the effective lifespan of a resistant cultivar in the U.S. is now five years or
less. Introgression of resistance from diploid and tetraploid Avena species into
hexaploid oat has been difficult due to differences in ploidy levels and the
lack of pairing of homeologous chromosomes. The wild tetraploid A. barbata
has been a source of powdery mildew and stem rust resistance in cultivated
oat, but has largely been unexploited for crown rust resistance. Tests of 1099
A. barbata accessions from the Canadian Plant Gene Resources Center, not
represented in the USDA collection, revealed that 11.4% were at least
moderately resistant at the seedling and adult plant stages when tested with a
highly diverse bulk inoculum derived from the St. Paul buckthorn nursery.
Eighteen accessions were rated as highly resistant or a mix of highly resistant
and resistant plants in both seedling and adult plant tests. Three accessions
displayed a unique ‘blotchy’ resistant reaction as adult plants. Resistant
accessions were found from throughout much of the natural range of A.
barbata, but the Western Mediterranean and Lebanon had the highest
frequency of accessions with broad-spectrum resistance.
Development of a quantitative real-time PCR protocol for the analysis of
early colonization of sugarcane plantlets with Leifsonia xyli subsp. xyli
G. Carvalho (1), R. S. de Faria (1), F. R. Oliveira (1), A. T. Ramos (1), C. B.
Monteiro-Vitorello (1), L. E. CAMARGO (1)
(1) University of São Paulo, Piracicaba, BRAZIL
Phytopathology 100:S21
The bacterium Leifsonia xyli subsp. xyli (Lxx) causes the ratoon stunting
disease (RSD), one of the most important diseases of sugarcane worldwide.
The objective of this study was to develop a quantitative real time PCR assay
and use it to study the dynamics of early colonization of sugarcane with Lxx
in a susceptible and a resistant variety. Primers specific for Lxx were designed
based on its available genome sequence and used to quantify Lxx in leaves of
young plants. In vitro grown plantlets were transferred to pots and inoculated
60 days after by cutting them just above the apical meristem and placing a 30
µL volume of a liquid culture (OD = 0,8) on the cut surface. Bacterial
populations were estimated in leaf DNA extracts 10, 20, 40 and 80 days after
inoculation. The results indicated a rapid in planta growth of Lxx in the
susceptible variety that sharply contrasts with its fastidious behavior in
artificial medium. This suggested that a sizeable colonization of sugarcane
tissues occurs well before the manifestation of external symptoms, which
happen at least 9 months after inoculation. In the resistant variety, no
significant changes in bacterial titers were detected over time. Further studies
should be pursued to determine if this rapid inoculation and quantification
method reflects the level of resistance displayed by sugarcane varieties under
field conditions. If so, the methodology described could be used as a rapid
screening method to select resistant genotypes.
Isolation and molecular identification of Fusarium oxysporum f. sp.
vasinfectum race 2 present in Alabama cotton
J. D. CASTILLO (1), K. S. Lawrence (1), L. F. Cruz (1), K. M. Glass (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S21
Fusarium oxysporum f. sp. vasinfectum (FOV) causes Fusarium wilt of cotton
and was first reported in Alabama by Atkinson in 1892. Currently, eight races
of FOV have been described around the world. Historically races 1 and 2 have
Vol. 100, No. 6 (Supplement), 2010
S21
been reported in the U.S., but recent studies in the southeastern U.S. reveal the
presence of four novel genotypes, and the presence of races 3 and 8 outside of
California. Therefore, the objective of this study was to identify the races of
FOV present in the Auburn University Fusarium wilt research test field. FOV
from thirty cotton lines with varying root-knot nematode susceptibility were
isolated from symptomatic cotton plants and confirmed as FOV based on
morphology. Race was determined based on the sequences of three different
genes and enzyme restriction digestion analysis previously reported. Partial
sequences of translational elongation factor (EF-1α), phosphate permease
(PHO), and beta-tubulin (BT) genes were analyzed, and a maximum
parsimony tree was generated from these gene sequences. Additionally, a
restriction enzyme digestion analysis of the intergenic spacer (IGS) region of
nuclear rDNA was performed. Fragments amplified were 561, 404, and 810
bp for EF-1α, PHO, and BT, respectively. Preliminary results confirm that the
FOV race 2 is present in Alabama, and the FOV isolates fit in the Lineage II
(composed by race 1, 2, and 6) in the maximum parsimony tree.
Multilocus analysis of Rhizoctonia solani and related species associated
with rice sheath blight in Arkansas
V. L. CASTROAGUDIN (1), R. D. Cartwright (1), J. C. Correll (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S22
Sheath blight (SB) is one of the most important diseases of rice in the southern
United States and Rhizoctonia solani AG 1-IA is considered the primary
causal agent. However, R. solani AG 11, R. oryzae, R. oryzae-sativae, R. zeae,
and Sclerotium hydrophilum can also be isolated from rice with SB symptoms.
More information about the molecular diversity of R. solani and related
species affecting rice is needed. In this study, the phylogenetic relationship of
a diverse collection of Rhizoctonia spp. from rice in Arkansas was examined
by a multilocus sequencing approach, simple sequence repeats (SSR), and
universal primed PCR (UP-PCR) analysis. For multilocus-sequence based
analysis, the targeted loci were the internal transcribed spacer (ITS) region of
ribosomal DNA, cytochrome oxidase 1, the first and second largest subunits
of RNA polymerase II, beta-tubulin, and six anonymous nuclear markers. The
data analysis indicates the presence of inter- and intra-specific diversity at the
nuclear loci examined. Limited sequence variation was observed within the
ITS region of R. solani AG 1-IA. The population diversity of AG 1-IA and
AG 11 isolates also was examined with SSR and UP-PCR markers, and
isolates of R. solani AG 1-IA could be distinguished into eight and thirteen
haplotypes, respectively.
A comparison of standard and high-fidelity PCR in the detection of
Sclerotium rolfsii and a Dickeya sp. from Phalaenopsis orchids
R. A. Cating (1), M. A. Hoy (2), A. J. PALMATEER (1)
(1) University of Florida, Homestead, FL, U.S.A.; (2) University of Florida,
Gainesville, FL, U.S.A.
Phytopathology 100:S22
The polymerase chain reaction (PCR) has become widely used in
phylogenetic and genomic analyses, plant-disease diagnoses, and to examine
pathogen diversity. However, standard PCR usually does not amplify
sequences of more than 5 kb and can be inhibited by plant cellular contents
including host genomic DNA when used for the detection of plant pathogens
directly from plant tissue. The high-fidelity PCR has been used to detect a
number of microbes while in the presence of host genomic DNA and is more
efficient than the standard PCR. In this study, high-fidelity and standard PCRs
were used to detect S. rolfsii and a Dickeya sp. directly from inoculated
orchids. The high-fidelity PCR could detect the presence of the pathogen in all
inoculated plants, while the standard PCR could detect only the positive
controls. These results indicate that the high-fidelity PCR may enable a
dramatic improvement in the detection of a wide range of plant pathogens,
especially when low template concentration, contaminating or competitor
DNA, or amplification inhibitors exist in a given sample.
Molecular and morphological characterization of a Phytophthora
infestans population in the Colombian Andean region
M. C. CÉSPEDES (1), M. E. Cárdenas (1), A. M. Vargas (1), A. J. Bernal (1),
S. Restrepo (1)
(1) Univ De Los Andes, Bogota, COLOMBIA
Phytopathology 100:S22
Late blight caused by Phytophthora infestans is one of the most devastating
diseases of potato in the world. This pathogen can also attack economically
important crops in Colombia such as Cape gooseberry, tomato and naranjilla.
To establish an effective control to prevent this disease, it is necessary to
know the genetic structure of the population. In the present study we collected
23 isolates from different hosts in two departments, Cundinamarca and
Boyacá. The objectives of the study were i) to characterize the resistance level
of the isolates to Mefenoxam ii) to establish the baseline of sensitivity of the
isolates to two fungicides (Mefenoxam and Cymoxanil) iii) to determine the
S22
PHYTOPATHOLOGY
physiological races present in the population and iv) to characterize the
molecular diversity of the isolates using one avirulence gene (Avr3a), the tubulin gene and two single copy genes with an RXLR motif. We found that
52% of the population showed sensitivity to mefenoxam. Results obtained
from the molecular diversity suggested that the population is clonal in this
Colombian region. These results along with the current study of the
physiological races provide new insights in the characterization of this
population. The gather information will help in the development of durable
management strategies for the future, managing fungicide resistance and
contributing for wider studies on global diversity.
Cmm-tomato interactions: Visualization during infection, biofilm
formation and epiphytic fitness
L. CHALUPOWICZ (1), O. Dror (2), M. Cohen-Kandli (2), R. Eichenlaub
(3), E. Zellermann (3), K. Gartemann (3), G. Sessa (4), I. Barash (4), S.
Manulis-Sasson (2)
(1) Tel Aviv University, Ramat Aviv, ISRAEL; (2) ARO, The Volcani
Center, Beit Dagan, ISRAEL; (3) University of Bielefeld, Bielefeld,
GERMANY; (4) Tel-Aviv University, Tel Aviv, ISRAEL
Phytopathology 100:S22
The Gram-positive actinomycete Clavibacter michiganensis subsp.
michiganensis (Cmm) is the causal agent of tomato wilt disease, an
economically important disease worldwide. Pathogenicity of Cmm is
associated with two plasmids, pCM1 and pCM2 and a chp/tomA pathogenicity
island. Cmm colonization of tomato plant by microscopic methods, as well as
epiphytic fitness on tomato leaves and biofilm formation were examined.
Confocal laser scanning microscopy revealed that GFP-labeled Cmm
extensively colonized the xylem vessels with preferential attachment to spiral
rings and tracheary elements of wilted leaves. Scanning electron imaging
showed aggregates of Cmm cells between the spiral elements at 7days postinoculation, while after 15 days Cmm densely colonized the whole xylem
vessels. Moreover, Cmm cells were observed on remnants of cell wall which
appeared perforated and might result from cell wall degrading enzymes.
Crystal violet staining assay showed that biofilm formation of Cmm grown in
xylem sap was significantly higher as compared to Luria Broth and minimal
medium by 2.5 and 1.5 fold, respectively. In addition of being an efficient
endophyte, Cmm showed high epiphytic fitness on tomato as compared to
bean leaves. These results were supported by the observation that GFP-labeled
Cmm cells densely colonized tomato leaf surfaces. This is the first study in
which GFP-labeled Cmm was employed to demonstrate endophytic and
epiphytic characteristics of this phytopathogen.
Description and evaluation of an education and outreach program for
best management of Ralstonia solanacearum race 3 biovar 2
P. G. CHAMPOISEAU (1), J. B. Jones (1), C. Allen (2), T. M. Momol (3)
(1) University of Florida, Department of Plant Pathology, Gainesville, FL,
U.S.A.; (2) University of Wisconsin-Madison, Department of Plant Pathology,
Madison, WI, U.S.A.; (3) University of Florida, District Directors Office,
Gainesville, FL, U.S.A.
Phytopathology 100:S22
Ralstonia solanacearum race 3 biovar 2 (R3b2) is considered a serious
quarantine pest in Canada and Europe and is listed as a Select Agent plant
pathogen in the United States, where it is subject to the strictest biosecurity
regulations. Due to quarantine regulations, it is critical to prevent reintroduction and possible spread of this pathogen in the U.S., and this
primarily involves exclusion, early detection, and unambiguous identification
of the pathogen; it also requires preparedness and effective training of official
regulators, diagnosticians, industry members, and other individuals responsible for first detection and response to a possible R3b2 discovery in the U.S.
To achieve this objective we have developed a 3-year integrated education
and outreach program, as part of a USDA-founded project, to target multiple
audiences in the U.S. This program included participation to multiple meetings and conferences, use of current web-based technologies, and development of e-learning modules for delivery of up-to-date educational materials.
Along with the presentation of these new educational materials, we describe
the development and use of various evaluation tools to assess program
effectiveness. Among these tools, monitored access of our Ralstonia/bacterial
wilt-dedicated website shows that stakeholders from diverse organizations
both within and outside the U.S. regularly use this resource to obtain updated
and accurate information on R3b2 and bacterial wilt disease management.
Possible functions of light-induced proteins in cercosporin biosynthesis by
Cercospora kikuchii
A. K. CHANDA (1), Z. Chen (1), R. W. Schneider (1)
(1) Louisiana State University, Baton Rouge, LA, U.S.A.
Phytopathology 100:S22
Cercospora kikuchii is the causal agent of leaf blight and purple seed stain of
soybean. C. kikuchii has established in the southern United States and caused
significant yield reduction in soybean. The pathogenicity of C. kikuchii has
been attributed to a polyketide photosensitizer, cercosporin. Cercosporin is a
non-host specific toxin produced by plant pathogens that belong to genus
Cercospora. There has been a great interest in understanding cercosporin
biosynthesis by many researchers, which can help in managing diseases
caused by Cercospora species and also to minimize the loss from the diseases.
We utilized two-dimensional protein gel electrophoresis (2DGE) to identify
possible proteins involved in cercosporin biosynthesis by comparing protein
profiles of C. kikuchii grown under cercosporin-favoring (light) and
cercosporin-suppressing (dark) conditions. We identified and sequenced
several proteins that were differentially expressed under light and dark. The
light up-regulated proteins showed high homology to proteins like
hydroxynaphthalene reducatse, S-adenosylmethionine synthetase, as well as to
the expressed sequence tags (ESTs) from Cercospora zeae-maydis. Their
corresponding genes have been cloned to determine their roles in cercosporin
biosynthesis and pathogenicity of C. kikuchii.
A survey of fungicide resistance in the Venturia inaequalis populations of
Indiana and Michigan
K. S. CHAPMAN (1), G. Sundin (2), J. Beckerman (1)
(1) Purdue University, West Lafayette, IN, U.S.A.; (2) Michigan State
University, East Lansing, MI, U.S.A.
Phytopathology 100:S23
Venturia inaequalis, the causal agent of apple scab, infects apple trees. Longterm and extensive fungicide use has led to multiple fungicide resistances
developing with varying frequencies in different orchards. To assess fungicide
resistance levels, isolates of V. inaequalis were collected from Indiana and
Michigan orchards. Single-spore derived isolates were evaluated using
mycelium growth assays with previously determined baseline concentrations
of fungicides and corresponding thresholds for growth. Fungicides tested
include: kresoxim-methyl, thiophanate-methyl, dodine and myclobutanil. We
identified isolates which were classified as resistant or shifting towards
resistant to each fungicide. Resistance to kresoxim-methyl and myclobutanil,
the primary fungicides used for apple scab management, was present in both
states. This is the first report of field resistance to kresoxim-methyl in the
United States. A total of 19% of isolates from Indiana and 44% in Michigan
were shifted to kresoxim-methyl. Resistance to myclobutanil occurred in over
55% of isolates. Isolates that tested resistant or shifted often tested this way
for multiple chemicals. Of 199 isolates tested, 38% were identified as resistant
or shifted to two fungicides and 12% were resistant or shifted to all four of the
fungicides. The presence of resistance to all major fungicides used for apple
scab management leaves growers with fewer options for control and a higher
risk for crop loss.
Molecular approaches for unraveling phytopathogenic fungi – Macrophomina phaseolina in cluster bean
A. CHAUDHURY (1)
(1) Department of Bio & Nano Technology, Giuru Jambheshwar University of
Science & Technology, Hisar, Haryana, INDIA
Phytopathology 100:S23
Soil borne phytopathogenic fungus Macrophomina phaseolina is prevalent in
over 500 crops species, especially, cluster bean, cotton, okra, soybean, in
India, Brazil, United Kingdom, Australia, North and South America, Africa
and parts of Europe by causing Charcoal rot disease is a major constraint for
higher yields. Genetic and phenotypic characterization of various isolates
obtained from several host plants using conventional techniques, PCR based
molecular markers: RAPD, PCR–RFLP of rDNA ITS region, Microsatellite
primed PCR and Rep-PCR conducted for evaluation of genetic diversity as an
initial step towards understanding population structure of charcoal rot
pathogen. Results reveal that it is a rapid, inexpensive technique, highly
reproducible and almost as discriminatory as MSP-PCR for genotyping M.
phaseolina isolates. It is quite suitable for understanding disease epidemiology
at molecular level, suggesting, thereby, that it is a robust technique employed
for genotypic and phylogenetic studies for determining taxonomical diversity
of commercially important cluster bean. Experiments on optical properties of
Macrophomina phaseolina impregnated Sol-gel-derived silica matrices
revealed that it is quite rapid as compared to conventional calorimetric
methods. Now efforts are underway for developing SCAR as a diagnostic tool
for detection of M. phaseolina. Data presented here will serve as platform in
designing strategies for breeding program, epidemiological and other
taxonomic studies in future.
Systemic acquired resistance for reducing bacterial wilt severity on
annual bluegrass
A. CHAVES (1), N. A. Mitkowski (1)
(1) University of Rhode Island, Kingston, RI, U.S.A.
Phytopathology 100:S23
Annual bluegrass (Poa annua L.) is a common grass species found on golf
courses. Although a weed, it is managed as the primary turf on many putting
greens in the Northeast. Poa annua is susceptible to numerous diseases
including bacterial wilt, caused by Xanthomonas translucens pv. poae. The
bacteria multiply in xylem vessels, causing wilting and necrosis. Few control
options exist for this disease. Copper compounds can be used but have a high
level of phytotoxicity. Antibiotics are not labeled for use on golf courses. We
have studied induced resistance as a possible control for this disease. Trials
were undertaken to study the effects of exogenous applications of salicylic
acid, 2,6-dichloroisonicotinic acid (INA), Actigard® (acibenzolar-S-methyl),
Aliette® (fosetyl-aluminum), beta-amino butyric acid (BABA), Messenger®
(purified harpin protein), and dihydrogen potassium phosphate (K2HPO4).
These chemicals have been shown to induce resistance in other plant species
by mimicking systemic acquired resistance. Our results demonstrate that INA,
SA, and Actigard are the most effective in inducing resistance, frequently
increasing survival to between 30 and 60% in greenhouse trials conducted
under high humidity (> 75%) and high temperatures (25°C). Some plant
survival was also seen using Aliette, K2HPO4, and BABA but at lower levels.
Harpin appears to be minimally effective. No previous work has been done on
the efficacy of induced resistance in controlling bacterial wilt on annual
bluegrass.
Functional analysis suggests evolutionary conservation of Pto and Rsb
Pseudomonas resistance phenotypes between tomato and potato
Y. CHEN (1), M. Stolz (2), K. Neu (3), D. Halterman (3)
(1) Department of Plant Pathology, University of Wisconsin-Madison,
Madison, WI, U.S.A.; (2) Memorial High School, Madison, WI, U.S.A.; (3)
U.S. Department of Agriculture-Agriculture Research Service, Vegetable
Crops Research Unit, Madison, WI, U.S.A.
Phytopathology 100:S23
Pathogen effector, AvrPtoB, from Pseudomonas syringae pv. tomato, has two
distinct avirulence determinants. One contains the first 307 amino acids
(AvrPtoB1-307), which is recognized by the Pto kinase and leads to
hypersensitive response (HR). The other contains an additional 80 amino
acids (AvrPtoB1-387) that are recognized by the Fen kinase and also leads to
the HR. The C-terminal region of AvrPtoB has E3 ligase activity,
ubiquitinating the Fen kinase, leading to its degradation and thus suppressing
host defense. Interestingly, although the Pto and Fen kinases share 80% of
amino acid identity, Pto is resistant to AvrPtoB-mediated degradation. A host
resistance phenotype, Rsb (Resistance suppressed by AvrPtoB C-terminus),
which is conferred by Fen, occurs more frequently than Pto-like resistance in
wild species of tomato. In our study, we tested multiple individuals of potato
from 10 different wild species by Agrobacterium infiltration. We found that
Rsb was also very common among wild potato species and we identified Ptolike resistance in two different species. Interestingly, we also observed several
different recognition patterns of truncated and full-length AvrPtoB. Pto-like
genes from these wild species of potato have been cloned and sequenced with
the expectation that elucidation of interactions between Pto-like genes from
potato and AvrPtoB will help us understand how Pto-mediated resistance
evolved in the arms race between host and pathogen.
High-throughput detection of Sclerotinia sclerotiorum from oilseed rape
by Taqman quantitative realtime PCR
C. CHEN (1), W. Zhao (1), M. Zhou (1), J. Wang (1)
(1) Nanjing Agricultural University, Nanjing, PRC PEOPLES REP OF
CHINA
Phytopathology 100:S23
Stem rot of oilseed rape is caused by Sclerotinia sclerotiorum, one of the most
damaging pathogens. In this study, beta-tubulin from S. sclerotiorum was
applied to develop a Taqman quantitative realtime PCR to detect populations
of the pathogen. One Taqman probe (5′-Rox-CGA GAA CTC TGA
CGAGAC CTT CTG TA-Tamra-3′) and one primer pair TYF (5′- ATA
TAACGCTACTCTCTCTGTTC -3′)/TYR (5′- AGCCAACTTTCGG
AGATTTG-3′) were developed and synthized according to the specificity of
the beta-tubulin gene (GenBank access number AY312374) of S.
sclerotiorum. Amplifications were conducted in 25 µl volumes containing
d2H2O 10 µL, buffer (10x) 2.5 µl, MgCl2 (25mM) 5 µL, dNTP (10 µM) 2 µL,
TYF (10 µM) 0.5 µL, TYR (10 µM) 0.5 µL, fluorescent probe (10 µM) 2 µL,
genome DNA from sclerotia (50 µg/mL) 2.0 µL, Taq DNA ploymerase (5
u/µL) 0.5 µL. The PCR amplifications were performed using the following
parameters: an initial pre-heat for 10 sec at 95°C, followed by 40 cycles at
95°C for 15 s, denaturation at 56°C for 60 s, extension at 72°C for 30 s.
Stardard curve equation Y = -3.30X+24.30 (R2 = 0.995) was set up to detect
the population of S. sclerotiorum. The technique appears suitable for the
epidemiological studies such as high-throughout detection of S. sclerotiorum
populations from diseased stems, leaves and petals of oil rape. All the
procedure of detection may be finished within 7 h. The real-time PCR assay
Vol. 100, No. 6 (Supplement), 2010
S23
was developed in this study could help growers make a timely decision on
fungicide application.
Guangdong (China) and Florida (U.S.) populations of “Candidatus
Liberibacter asiaticus” distinguished by a genomic locus with short
tandem repeats
J. CHEN (1), X. Deng (2), X. Sun (3), D. Jones (3), M. Irey (4), E. Civerolo
(5)
(1) USDA ARS PWA, Parlier, CA, U.S.A.; (2) South China Agricultural
University, Guangzhou, PRC PEOPLES REP OF CHINA; (3) Division of
Plant Industry, Department of Agriculture and Consumer Services,
Gainesville, FL, U.S.A.; (4) US Sugar Corp./S. Gardens Citrus, Clewiston,
FL, U.S.A.; (5) USDA-ARS, Parlier, CA, U.S.A.
Phytopathology 100:S24
presence of preformed compounds. The levels of antioxidant enzymes were
very different in calamondin, kumquat, or Mexican lime before or after Xac
challenge. In comparison with Mexican lime or calamondin, leaf extracts from
kumquat had lower superoxide dismutase (SOD), glutathione reductase (GR),
and glutathione peroxidase (GPx). However, these enzymatic activities in
kumquat promptly increased after inoculation. Thus, our results suggest that
both preformed and induced defense compounds of kumquat may have an
important role in cellular resistance to Xac.
Characterization of a putative Ustilago maydis pathogenesis gene, Upe
H. K. CHEUNG (1)
(1) Trent University, Peterborough, ON, CANADA
Phytopathology 100:S24
Huanglongbing (HLB, yellow shoot disease) is a highly destructive disease
that threatens citrus production worldwide. The disease was first observed in
Guangdong, P. R. China over 100 years ago, and was found in Florida, U.S.A.
in 2005. “Candidatus Liberibacter asiaticus” has been associated with HLB.
Understanding the global epidemiology of HLB is important for management
of the disease. In this study, we identified a genetic marker containing small
tandem repeats in the genome of “Ca. L. asiaticus” and comparatively
analyzed the tandem repeat numbers (TRNs) in bacterial populations from
Guangdong and Florida. The Guangdong population consisted predominately
of strains with TRN of 7 (TRN7) at a frequency of 47.6%. The Florida
population consisted predominately of strains with TRN of 5 (TRN5) at a
frequency of 84.4%. TRNs ranged from 3 to 16. The apparent absence of TRN
of 9, 10, 11, and 12 separated the bacterial strains into two groups: TRN less
than 10 (TRN<10) and TRN greater than 10 (TRN>10). In Florida, TRN<10
strains (103/109 or 94.5%) were widely distributed in all HLB-affected
counties. TRN>10 strains (6/109 or 5.5%) were found in central Florida. This
is the first report documenting the differentiation of “Ca. L. asiaticus”
populations between Asia and North America and the possible presence of
two differentially distributed genotypes of “Ca. L. asiaticus” in Florida.
Genome annotation of the corn smut pathogen Ustilago maydis revealed a
large number of genes potentially involved in the completion of meiosis. This
pathogen requires growth in the plant to become competent to undergo
meiosis and therefore we hypothesize that signals received from the host plant
stimulate U. maydis meiotic development. One such gene, an ortholog of the
transcription regulator Ume6, was investigated. Deletion of this gene did not
result in a discernable haploid cell phenotype. However infection with
compatible deletion strains resulted in increased pathogenesis and earlier
onset of disease symptoms. Teliospores that were produced from these
infections germinated and completed meiosis in a manner indistinguishable
from wild type cells. This indicates that this Ume6 ortholog is not required for
meiosis; however it suggests a role in pathogenic development. Since the
structure of the gene indicates that it is likely a transcription factor we have
named the gene Upe for unregulated pathogenesis gene expression. The
pattern of pathogenesis in the Upe deletion strains is consistent with Upe
being either a repressor of pathogenesis genes or an activator of genes that
suppress the host plant response. Further mutant strains in which the
expression of Upe is altered have been created and the impact of these
mutants on haploid cell growth and pathogenic development will be presented
along with progress on determining variation in gene expression responses.
Evaluation of tandem repeat polymorphisms between two pathogenically
similar strains of Xylella fastidiosa from almond and grape in California
J. CHEN (1)
(1) USDA ARS PWA, Parlier, CA, U.S.A.
Phytopathology 100:S24
Environmental regulation of stomate-based defense against bacterial
infection in Arabidopsis
R. CHITRAKAR (1), M. Melotto (1)
(1) University of Texas at Arlington, Arlington, TX, U.S.A.
Phytopathology 100:S24
Whole genome tandem repeat polymorphisms were evaluated between two
closely related Xylella fastidiosa strains, M23 and Temecula1, both cause
almond leaf scorch disease (ALSD) and grape Pierce’s disease (PD) in
California. Strain M23 was isolated from almond and the genome was
sequenced in this study. Strain Temecula1 was originally isolated from grape
and its genome was sequenced previously. Among the 48 identified tandem
repeat (TR) loci evaluated by sequence similarity flanking the TRs, two were
unique to strain M23, six were unique to strain Temecula1, and 40 were
shared by both bacterial strains with a similarity of >70%. Yet, the two strains
differ in TR numbers (TRNs) in all shared loci. Eight shared loci were
selected to evaluate TRN variation using additional 10 X. fastidiosa strains.
Results from our analyses indicate that TRN comparison could be highly
powerful for discriminating closely related bacterial strains. However, careful
selection and evaluation of TR loci is critical in order to avoid over estimating
of bacterial strain genetic diversity.
Stomata are natural openings in the plant epidermis responsible for gas
exchange between plant interior and environment. They are formed by a pair
of guard cells, which are able to close the stomatal pore in response to a
number of external factors including light intensity, carbon dioxide
concentration, and relative humidity (RH). The stomatal pore is also the main
route for pathogen entry into leaves, a crucial step for disease development.
Recent studies have unveiled that closure of the pore is effective in preventing
bacterial disease in Arabidopsis plants and the successful plant pathogen
Pseudomonas syringae pv. tomato (Pst) DC3000 is able to re-open stomata by
producing the phytotoxin coronatine. A major unanswered question is: “how
do stomata respond to combined effect of biotic and abiotic stresses?” We
found that coronatine can re-open dark-closed stomata three hours postincubation with purified coronatine or the coronatine producing Pst DC3000.
Same trend did not hold for the coronatine deficient mutants, Pst DC3118 and
Pst DB29. We also have evidence that high RH (95%) reduces bacteriumtriggered stomatal closure. The same effect was not observed under low RH
(60%). These results suggest that guard cells prioritize their response when
exposed to multiple stimuli. Understanding this process should help
elucidating the effectiveness of stomatal-based defense in nature where plant
experiences constant influx of external stimuli.
The mechanisms associated with cellular resistance of calamondin and
kumquat to citrus canker caused by Xanthomonas anxonopodis pv. citri
B. CHEN (1), L. Wang (2), K. Chung (3), M. Lee (1)
(1) Department of Plant Pathology, National Chung Hsing University,
Taichung, TAIWAN; (2) Plant Protection Department, Chiayi Agricultural
Experiment Branch, Agricultural Research Institute, Council of Agriculture,
Chiayi, TAIWAN; (3) Citrus Research and Education Center, Institute of
Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred,
FL, U.S.A.
Phytopathology 100:S24
Citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac), is one of
the most severe diseases on citrus in Taiwan. It has long been known that
calamondin (× Citrofortunella microcarpa) and kumquat (Fortunella spp.) are
highly tolerant to Xac compared to other citrus cultivars. Molecular and
physiological approaches were used to explore possible mechanisms attributed
to Xac resistance. Pathogenicity assays on leaves wounded prior to spray
inoculation revealed that Xac induced flatter necrotic lesions on calamondin
and kumquat, but promoted characteristic canker lesions with raised and corky
appearance on the susceptible variety, Mexican lime (Citrus aurantifolia).
When inoculated by infiltration, Xac propagated at lower rate and magnitude
in kumquat compared to Mexican lime or calmondin. Further analysis
revealed that the peel, but not leaf, extracts from calamondin or kumquat
displayed inhibitory effects to Xac as assayed in culture, implicating the
S24
PHYTOPATHOLOGY
Structure of Sclerotinia sclerotiorum within and among lettuce fields in
California
P. CHITRAMPALAM (1), Z. K. Atallah (1), B. Wu (2), K. V. Subbarao (1)
(1) University of California, Davis, Salinas, CA, U.S.A.; (2) Oregon State
University, Madras, OR, U.S.A.
Phytopathology 100:S24
Genetic structure of Sclerotinia sclerotiorum among and within lettuce fields,
as well as in individual lettuce plants was analyzed using 16 microsatellite
markers. Isolates were collected from four fields, including 2 that were
sampled twice at 3-weeks interval. Based on the lettuce drop incidence, one or
two 25 × 25 m2 plots with multiple diseased plants were sampled in each field
and all infected lettuce plants in were collected. In addition, 10 isolates were
obtained from four randomly chosen lettuce heads. Results from PCR with
SSR markers revealed high genetic variability in S. sclerotiorum populations
both among and within lettuce fields. Although, several haplotypes were
present in all four fields, their frequency varied significantly among fields. No
correlation was observed between MCGs and SSR haplotypes. Furthermore,
the analysis of S. sclerotiorum from single lettuce heads revealed high
genotypic variability, suggesting that inoculum from genetically different
sclerotial sources could infect the same lettuce head establishing a mechanism
for recombination. This hypothesis is currently being tested experimentally.
Fusarium avenaceum as causal agent of root rot in field peas and its
control
K. CHITTEM (1), L. Porter (2), K. McPhee (3), M. Khan (3), R. S. Goswami (3)
(1) North Dakota State University, Fargo, ND, U.S.A.; (2) Prosser, WA,
U.S.A.; (3) Fargo, ND, U.S.A.
Phytopathology 100:S25
Fusarium root rot is a serious disease in all field pea producing areas in the
United States and has become a major constraint in the North Central region
over the past years. Traditionally, Fusarium solani f. sp. pisi was considered
to be the primary causal agent of this disease, but recent surveys conducted in
North Dakota identified Fusarium avenaceum as the most prevalent pathogen
associated with pea root rot in this state. The objectives of our study were to
assess variation in aggressiveness of F. avencaeum isolates from field peas,
evaluate commercial varieties for resistance to F. avencaeum and F. solani f.
sp. pisi, and to estimate the ability of waste-lime, a by-product of the sugar
industry, as a soil amendment to control this pathogen. The sand corn-meal
layer method was used for studying the variation in aggressiveness, and for
screening cultivars for resistance to both pathogens under greenhouse
conditions. Significant variation in aggressiveness was observed among
isolates and some isolates of F. avenaceum were more aggressive than isolates
of F. solani f. sp. pisi. Twenty one cultivars of green and yellow peas were
screened for the resistance against both F. avenaceum and F. solani, and only
cv. Franklin was found to be partially resistant to both pathogens. Use of
waste lime resulted in significant reductions in growth of both root rot
organisms in laboratory tests and reduced the disease severity in inoculated
greenhouse trials.
RpfC control tctE/tctD expression and mutations in the genes reduced
virulence of Xanthomonas oryzae pv. oryzae KACC10859
J. CHO (1), M. Jeong (2), W. Kim (3), J. Cha (2)
(1) Chungbuk National Univ, Cheongju, SOUTH KOREA; (2) Cheongju,
SOUTH KOREA; (3) Iksan, SOUTH KOREA
Phytopathology 100:S25
The significantly down regulated genes in the rpfC- mutant, which was
screened by microarray analysis. The result was confirmed tctE/tctD genes
that were known as a two-component signal transduction system involved in
tricarboxylate transport in Salmonella typhimurium. Marker exchange
mutations in tctE/tctD reduced the lesion length to about 65% of wild type’s.
Virulence factor of Xoo such as xylanase, cellulase, motility and
polysaccharide (EPS) were also decreased in the mutants. Complementation
of the mutations restored the virulence and virulence-related phenotypes.
Reduction of tctE/tctD expression in the rpfC- mutant was confirmed by the
transcription real-time PCR and assay of tct promoter activity which was
fused to the GFP reporter. RpfC was known an important virulence regulator
in Xanthomonas campestris and the closely related bacterial pathogens. These
results suggest that RpfC controls tctE/tctD expression and the tctE/tctD are
involved in virulence expression in the Xanthomonas oryzae pv. oryzae
KACC10859.
Discovery of key transcription factor-encoding genes for pathogenicity in
the plant-pathogenic fungus Alternaria brassicicola
Y. CHO (1)
(1) University of Hawaii At Manoa, Honolulu, HI, U.S.A.
Phytopathology 100:S25
Necrotrophic fungi are responsible for about 80% of the annual economic
losses in agriculture worldwide, although they account for just 4% of fungal
diversity. The necrotrophic fungus Alternaria brassicicola causes black spot
disease of brassicaceous plants, including the oil-producing B. napus and
model plant Arabidopsis. In order to identify key transcription factors (TFs)
involved in pathogenesis, we have produced targeted gene disruption mutants
for 160 of the 400 TF genes in A. brassicicola. Our bioassays on cabbage
leaves, identified one pathogenicity factor with mutants that were
nonpathogenic, and six strong and two weak virulence factors whose mutants
showed respectively 50–90% and 20–49% reduction in disease symptoms
compared to the wild type. We also discovered a unique gene whose mutants
showed a 100% increase in virulence. We suspect the role of the group with
decreased virulence was as positive regulators and the group with increased
virulence was a negative regulator for downstream pathogenesis genes. Nine
of these ten genes discovered in this study were novel virulence factors and
only one gene (PacC) was previously identified as a pathogenicity factor in
other fungal species. The newly discovered genes associated with pathogenicity or virulence will lead to an understanding of the orchestrated regulation of fungal genomes during pathogenesis. Such detailed information can
be used to tailor efficient strategies for the management of necrotrophic fungi.
Application of Fungal Secretome Database to identify effector proteins in
the plant pathogenic fungi
J. CHOI (1), J. Park (1), D. Kim (1), K. Jung (1), S. Kang (2), Y. Lee (1)
(1) Department of Agricultural Biotechnology, CeFP, CFGR, and CAB, Seoul
National University, Seoul, SOUTH KOREA; (2) Department of Plant
Pathology, The Pennsylvania State University, University Park, PA, U.S.A.
Phytopathology 100:S25
Fungi secrete proteins which have various functions to adapt various niches.
Some of them in pathogens function as effectors that manipulate and/or
destroy host cells. To enhance prediction accuracy for secretory proteins in
fungi, we constructed Fungal Secretome Database (FSD; http://fsd.snu.ac.kr/)
with an ensemble pipeline using nine prediction softwares. Accuracies of the
pipeline showed 99.16% and 84.17% for 2,512 experimentally verified SPdb
proteins and the 1,093 characterized fungal secretory proteins; while SignalP
3.0 presents 89.81% and 75.30%, respectively. To present all information
intuitively, the FSD presents two main features: summary page and Favorite, a
personalized repository implemented in Comparative Fungal Genomics
Platform (http://cfgp.snu.ac.kr/). When 158 fungal/oomycete genomes were
applied to the FSD pipeline, 208,883 proteins (15.21%) were predicted as
secretory. The presence of putative effectors containing known host targeting
signals such as RXLR or RXLX[EDQ] was investigated. Much higher
frequency of RXLX[EDQ] was found in fungi compared to RXLR, suggesting
that the RXLX[EDQ] may be one of fungal-specific signatures of effectors.
FSD could serve as an integrated platform that supports researches on secretory proteins, especially in effector proteins, in the plant pathogenic fungi.
Generation of reactive oxygen species via NOXa is important in
pathogenicity in Mycosphaerella graminicola
Y. CHOI (1), S. B. Goodwin (1)
(1) USDA-ARS, West Lafayette, IN, U.S.A.
Phytopathology 100:S25
Mycosphaerella graminicola is an important wheat pathogen causing
significant economic loss. M. graminicola is a hemibiotroph, indicating that a
biotrophic stage with nutrient uptake and a necrotrophic stage associated with
a possible toxin or reactive oxygen species (ROS) are important to
pathogenicity. To better understand the pathogenic mechanisms of M.
graminicola, we employed over-expression strategies; selected target genes
for over-expression were CREA, AREA, and NOXa, which might function as
regulators in nutrient acquisition and ROS generation. Increased expressions
of CREA, AREA, and NOXa were confirmed via q-RT PCR and subsequently
used for pathogenicity testing. Among them, the NOXa over-expression strain,
NO2, resulted in significantly increased pathogenicity. Moreover, instead of
the usual filamentous growth, we observed a significant predominance of
yeast-like growth in NO2, which is correlated with ROS production. Our data
indicate that ROS generation via NOXa is important to pathogenicity as well
as development in M. graminicola.
Gene encoding a c-type cyclin in Mycosphaerella graminicola is involved
in melanin biosynthesis, stress response, and pathogenicity
Y. CHOI (1), S. B. Goodwin (1)
(1) USDA-ARS, West Lafayette, IN, U.S.A.
Phytopathology 100:S25
Mycosphaerella graminicola is an important wheat pathogen causing septoria
tritici blotch. To date, an efficient strategy to control M. graminicola has not
been developed. More significantly, we have a limited understanding of the
molecular mechanisms of M. graminicola pathogenicity. In this study, we
attempted to characterize an MCC1-encoding c-type cyclin, a homologous
gene to FCC1 in Fusarium verticillioides. Four independent MCC1 knock-out
mutants were generated via Agrobacterium-mediated transformation (ATMT).
All of the MCC1 mutants showed consistent multiple phenotypes. We could
observe significant reductions in radial growth on PDA in all of the MCC1
mutants. In addition, MCC1 gene deletion mutants produced less mycelium on
PDA, had increased melanin biosynthesis on agar plates, showed an increase
in their stress tolerance response, and were reduced significantly in
pathogenicity. These results indicate that the MCC1 gene is involved in
multiple signaling pathways including pathogenicity in M. graminicola.
Regulation and functional characterization of Monilinia fructicola
polygalacturonase genes MfPG1, MfPG2, MfPG3 and MfPG5
C. CHOU (1), R. M. Bostock (2), M. Lee (1)
(1) Department of Plant Pathology, National Chung Hsing University,
Taichung, TAIWAN; (2) Department of Plant Pathology, UC Davis, Davis,
CA, U.S.A.
Phytopathology 100:S25
Monilinia fructicola, the causal agent of peach brown rot, secretes a number
of endopolygalacturonases (endo-PGs) that can degrade pectin, a major
component of plant cell walls. MfPG1 encodes an endo-PG, the expression of
Vol. 100, No. 6 (Supplement), 2010
S25
which is up-regulated by the host phenol caffeic acid (CA) and downregulated by H2O2. However, brown rot development in inoculated peach fruit
and total PG activity in M. fructicola culture filtrates are inhibited by the
presence of CA. CA can act as an antioxidant to lower the intracellular redox
potential of fungal cells. H2O2, a pro-oxidant, is generated in response to
infection by pathogens and its production may facilitate disease development
caused by necrotrophic pathogens. The results suggest complex redox control
of endo-PG expression in M. fructicola, and possibly differential regulation of
other PG genes in response to changes in the redox environment during
infection. Three additional M. fructicola endo-PG genes – MfPG2, MfPG3
and MfPG5 – were isolated by degenerate and inverse PCR and sequenced.
Phylogenetic analysis revealed that these genes are highly related to endo-PG
genes of Botrytis cinerea and Sclerotinia sclerotiorum. Potential redoxsensitive transcription factor binding sites were identified within all MfPG
promoter regions, suggesting that their expression is also regulated by redox
changes. Studies on the role of MfPG1 in the M. fructicola-plant interaction
and the regulation of MfPG2, MfPG3, and MfPG5 will be presented.
collected using Burkard volumetric spore traps sited in two fields. The
quantification of M. graminicola by qPCR revealed the occasional presence of
airborne inoculum from January till December: peak release occurred not only
in the autumn and winter but also in the spring and summer. The comparable
temporal profile of airborne inoculum in the two fields, located 18 km apart,
indicated a comparable infection pressure due to this inoculum. Second, the
development of the teleomorph stage was monitored during the growing
season by collecting wheat and assessing the release of ascospores from their
leaves. Ascospores were obtained from the beginning of June 2009 on F4 and
F1 leaves, and then sporadically on F1 to F3 leaves until July. The airborne
inoculum detected in fields in the summer could therefore be explained by the
presence of mature pseudothecia produced in field. The sexual cycle thus also
plays a role not only by giving rise to secondary infection, but also by
allowing the fungus to complete many sexual cycles within a season. Control
strategies using fungicides have thus to be adjusted to limit the evolution of
fungicide resistance in M. graminicola populations due to recombination
occurring between the different treatments during the season.
Influence of row covers on muskmelon pollination in the absence of
bacterial wilt
X. CIBILS STEWART (1), E. Saalau Rojas (1), M. L. Gleason (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S26
Relationship of fungal and bacterial seed microflora to soybean seed
vigor
K. A. COCHRAN (1), J. Rupe (1), A. Steger (1), R. Holland (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S26
Yield losses of >80% can occur on muskmelon due to bacterial wilt, a disease
caused by Erwinia tracheiphila. The bacterium is transmitted by two species
of cucumber beetles; disease management requires suppression of the beetles.
Row covers placed over plants until 10 days after the start of bloom can
control bacterial wilt, but could potentially impede pollination if the row
covers blocks the access of pollinating insects. To test this possibility, we
compared muskmelon yield in two row-cover treatments (row cover ends
opened at anthesis, and bumble bees inserted under the row cover) with
adjacent control plots (row cover removed at anthesis, and row cover removed
10 days after anthesis). Rows were 30.5 m long. Incidence of bacterial wilt
was minimal during the study due to very low populations of cucumber
beetles. Yield was lower in the middle of the rows than at the ends, suggesting
that pollinators behavior was influenced by position in the row. Yield was
higher in control than treatment plots. Delaying row cover removal until 10
days after anthesis also delayed harvest. These preliminary results suggest
that, in the absence of bacterial wilt, delaying row cover removal until 10 days
after anthesis can delay and even reduce muskmelon yield.
Seedborne pathogens can greatly reduce soybean seed performance. To
determine the effect of seed infection on soybean seed germination and vigor
seven soybean cultivars with a range of reactions to Phomopsis longicolla and
Cercospora kikuchii were planted at Kibler, AR, in 2008 and 2009. Plots were
treated or untreated with azoxystrobin. In 2008, half of each plot was
promptly harvested and the other half was harvested three weeks later. In
2009, harvest was delayed due to rain. Vigor was accessed by standard
germination (SG), accelerated aging (AA), the Seed Vigor Imaging System
(SVIS), and plating of seed. In 2008, P. longicolla, and C. kikuchii was
negatively correlated with SVIS vigor for the first harvest. No relationship
was found between SG or AA and recovery of pathogens for the first or
second harvest. In 2009, there was a negative correlation between the
incidence of P. longicolla, and Bacillus subtilis with SG and AA. Seed
infection by P. longicolla and C. kikuchii were significantly affected by
cultivar. In 2008, cultivar and fungicide affected C. kikuchii recovery, and in
2009, they affected P. longicolla recovery. Germination and vigor were higher
in the second harvest in 2008 and vigor was higher in 2009 in response to the
foliar fungicide. Under low disease pressure (2008), soybean SG and AA were
not related to seed infection, but SVIS was. Under higher disease pressure
(2009), SG and AA were negatively correlated with increases in some
pathogens.
Factors influencing the production of Maize fine streak virus proteins in
Drosophila S2 cells
F. M. CISNEROS (1)
(1) The Ohio State University, Wooster, OH, U.S.A.
Phytopathology 100:S26
Maize fine streak virus (MFSV) is negative-sense RNA virus in the family
Rhabdoviridae. Our goal is to determine whether the viral proteins required
for synthesis of the MFSV genomic RNA can be produced in Drosophila S2
cells. We previously demonstrated that the MFSV nucleoprotein (N) and
phosphoprotein (P) could be expressed from pMT/V5-His-Topo vector in S2
cells for at least 4 days. In contrast, expression of the MFSV replicase protein
(L) was not detected in S2 cells under the same conditions. This could be due
to the frequent changes in its sequence observed when the plasmid was
propagated in E. coli. To avoid this problem, we are testing the expression of
the L protein using linear DNA fragments (LDF) instead of circular plasmids.
We have generated a LDF containing only eukaryotic regulatory elements for
expression of foreign genes in Drosophila S2 cells flanking the MFSV-L gene
by means of PCR. Preliminary results suggest that the MFSV-L protein can be
produced in S2 cells, and experiments are underway to optimize the
expression of the L protein. In addition, expression of the T7 RNA
polymerase needed for synthesis of the MFSV genomic RNA in vitro was
tested. Our results indicated that the T7 polymerase was expressed in S2 cells
for at least 4 days when fused to a nuclear localization signal peptide (NLS),
but did not accumulate when lacking the NLS. These results are important in
order to optimize the conditions for the production of an infectious full-length
clone of MFSV in S2 cells.
Presence of airborne inoculum of Mycosphaerella graminicola and
occurrence of sexual reproduction during the growing season in Belgium
A. Clinckemaillie (1), G. Dedeurwaerder (1), M. Duvivier (2), J. Moreau (2),
A. LEGREVE (1)
(1) Université catholique de Louvain, Louvain-la-Neuve, BELGIUM; (2)
Centre wallon de Recherches agronomiques, Gembloux, BELGIUM
Phytopathology 100:S26
The impact of the sexual cycle of Mycosphaerella graminicola on the
evolution of the Belgian population was studied using two approaches. First,
the airborne inoculum of the fungus produced by sexual reproduction was
S26
PHYTOPATHOLOGY
Climate change and potato late blight suppression
J. P. Comstock (1), D. W. Wolfe (1), L. Joseph (1), A. T. Degaetano (1), W.
E. FRY (1)
(1) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S26
We used a mechanistic computer simulation model of potato late blight in
combination with historical weather and predictions of future weather to
assess possible effects of climate change on potato late blight and on the
amount of fungicide required to suppress the disease. We used historical and
projected weather data for Rochester NY. In general, weather was more
favorable for disease and there was greater variance in the disease severity and
amount of fungicide needed to suppress disease in 1977-2008 than in 19471966. The simulation model requires hourly temperature, relative humidity
and precipitation data. These variables were available from state-of-the-art
global climate model (GISS, GFDL, UKMO, NCAR and MIROC)
projections. In all cases the variables were statistically downscaled to achieve
the higher spatial and temporal resolution needed for the late blight
simulation. Climate change effects were estimated using a low emissions
scenario and a high emissions scenario. In both scenarios, average
temperatures increased. In the low emissions scenario, disease severity and
fungicide necessary to suppress disease in 2040-2067 remain similar to these
values in 1977-2008. In the high emissions scenario, fungicide needed to
suppress disease in 2040-2065 increased by 20–25% when compared to that
required in 1977-2008.
Candidate gene silencing in Glycine max using amiRNA to identify
soybean cyst nematode resistance gene(s) at the Rhg1 locus
D. E. COOK (1), S. Melito (2), T. Hughes (1), A. MacGuidwin (1), A. F. Bent (1)
(1) University of Wisconsin, Madison, WI, U.S.A.; (2) University of Sassari,
Sassari, ITALY
Phytopathology 100:S26
Soybean cyst nematode (SCN), Heterodera glycines, is the most economically
damaging soybean pathogen in the U.S. Once SCN is present in a field it
cannot be eradicated, and SCN is now endemic in major soybean-producing
counties across the Midwest. Two major loci, Rhg1 and Rhg4, have been
identified in multiple genetic mapping studies, accounting for a significant
portion of SCN disease resistance. It is important to identify the genes
underlying resistance at these loci to elucidate their mechanism of action and
to possibly broaden the scope of SCN resistance used today. We have
employed Agrobacterium rhizogenes mediated soybean transformation, a
unique amiRNA gene silencing vector, and a nematode demographic assay to
evaluate candidate resistance genes. Our recent efforts have focused on
identifying the Rhg1 locus gene(s) because it is the resistance source in a
majority of commercial cultivars. Recent research indicates that the proposed
LRR-kinase at Rhg1 is not responsible for controlling a significant portion of
the resistance seen in PI88788-derived sources. Results from testing other
candidate SCN resistance genes at the Rhg1 locus will be presented.
A bacterial pathogen uses distinct type III secretion systems to alternate
between host kingdoms
V. R. CORREA (1), D. R. Majerczak (2), E. Ammar (3), M. Merighi (2), R.
C. Pratt (1), M. G. Redinbaugh (4), D. L. Coplin (2), S. A. Hogenhout (5)
(1) Dept. of Horticulture and Crop Science, OARDC/The Ohio State
University, Wooster, OH, U.S.A.; (2) Dept. of Plant Pathology, The Ohio
State University, Columbus, OH, U.S.A.; (3) Dept. of Entomology,
OARDC/The Ohio State University, Wooster, OH, U.S.A.; (4) USDA, ARS
Corn and Soybean Research Unit / Dept. of Plant Pathology, OARDC / The
Ohio State University, Wooster, OH, U.S.A.; (5) Dept. of Disease and Stress
Biology / The John Innes Centre, Colney, Norwich, UNITED KINGDOM
Phytopathology 100:S27
Gram-negative bacterial pathogens of eukaryotes often secrete proteins
directly into host cells via a needle-like protein channel called a ‘type III
secretion system’ (T3SS). Bacteria that are adapted to either animal or plant
hosts use phylogenetically distinct T3SSs for secreting proteins. Here, we
report that Pantoea stewartii subsp. stewartii (Pnss), the causative agent of
Stewart’s wilt in maize, carries phylogenetically distinct T3SSs that enable it
to invade its insect and plant hosts. In addition to a Hrp-type T3SS, known to
be essential for maize pathogenesis, Pnss has a second T3SS (PSI-2) that is
required for persistence in its flea beetle vector, Chaetocnema pulicaria. PSI-2
belongs to the Inv-Mxi-Spa T3SS family typically found in animal pathogens.
Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for
building the structural components of T3SS and secretion of T3SS effectors,
greatly reduced both the persistence of Pnss in flea beetle guts and its
transmission to maize. Ectopic expression of the psaN gene complemented
these phenotypes. In addition, the relative expression level of the PSI-2 psaN
gene was higher in insects compared to maize tissues. When mechanically
inoculated, the Pnss psaN mutant was fully virulent on sweet maize,
indicating that PSI-2 is not required for plant pathogenicity. Our findings
demonstrate that the two T3SS in Pnss play different roles in the life cycle of
this bacterium as it alternates between insects and plants.
Exploring attraction of C. elegans to the Brown Garden Snail, Helix
aspersa
J. A. Cotton (1), K. R. SANCHEZ (1), E. P. Caswell-Chen (1)
(1) University of California, Davis, CA, U.S.A.
Phytopathology 100:S27
We isolated nematodes and bacteria associated with the Brown Garden Snail
and assessed the attractiveness of snail exudates (slime) and bacterial
associates to well-fed C. elegans (Ce N2) and food-deprived Ce. T-shaped
patterns in PDMS (polydimethylsiloxane) were placed on agar to serve as
bioassay arenas. Stimuli were placed at one end of the “T” cross-arm and
nematodes at the base of the vertical leg. Nematodes moved up the vertical leg
of the “T” and could move in two directions at the cross-arm - toward a
stimulus at the end of the cross arm or toward the blank control chamber
opposite. The stimuli were snail mucus, bacteria from snail mucus (P. putida,
S. kitahiroshimense, E. coli), and Lysobacter enzymogenes (C3). Ce was
attracted to mucus and to all of the bacteria included as stimuli, with the
attraction to bacteria being greater than to mucus. Well-fed Ce were attracted
to snail stimuli - 94% choosing C3 vs the control chamber, 98% choosing all
snail bacteria, and 78% choosing snail mucus. Food-deprived Ce were
attracted - 98% choosing snail bacteria vs the control chamber, and 67%
choosing snail mucus. L. enzymogenes was attractive and induced vivipary in
Ce. These results demonstrate possible roles of attraction in the association of
Ce with their snail hosts.
Status of dodine resistance and possibilities for renewed use against
Venturia inaequalis populations in the Northeastern U.S.
K. COX (1), S. Villani (1), G. Jacon (2)
(1) Department of Plant Pathology and Plant-Microbe Biology, Cornell
University, Geneva, NY, U.S.A.; (2) Groupe Agriphar, Ougree, BELGIUM
Phytopathology 100:S27
The development of site-specific fungicide resistance in Venturia inaequalis
populations in the Northeastern U.S. have left apple producers with few
options for managing apple scab. Producers now rely on calendar-based
applications of multi-site protectant fungicides to manage the disease. Decline
in the frequency of dodine resistant isolates within a population was
previously demonstrated for two orchards in the region. To investigate the
prevalence of declining dodine resistance, we surveyed 93 commercial, 6
baseline, and 18 research apple orchards from 2007–2009 for sensitivity to
dodine using microscopy-aided relative growth assays. Less than 27% of the
orchards surveyed had V. inaequalis populations with practical resistance to
dodine. Field trials were also conduced in an orchard formerly resistant to
dodine, but with a current population displaying reduced sensitivity. Dodine
programs were as effective or improved over standard programs of protectant
and site-specific fungicides for managing apple scab. Following applications
of dodine in the orchard, dodine sensitivity, expressed as population mean
percent relative growth, increased from 36.0 ± 3.0% in 2008 to 51.4 ± 6.0% in
2009. Although, the majority of the orchard populations in the survey were
composed of sensitive isolates or those with reduced sensitivity to dodine, it
remains to be seen whether dodine resistant V. inaequalis populations will reemerge following renewed use.
Real-time PCR detection of the Southern corn rust pathogen Puccinia
polysora
J. CROUCH (1), L. J. Szabo (1)
(1) Cereal Disease Laboratory, USDA-ARS, St. Paul, MN, U.S.A.
Phytopathology 100:S27
Southern rust, caused by Puccinia polysora, is an increasingly problematic
disease of corn (Zea mays) in the U.S. The fungus has been present in North
America since at least 1897, with epidemics occurring episodically throughout
the 20th century in Africa, China, Central and South America. Although
primarily a foliar disease, P. polysora may also infect sheaths and husk leaves,
causing severe and early senescence. Stem lodging may also occur as an
indirect result of photosynthate loss, and yield reductions may be
considerable. Southern rust may be distinguished from common corn rust
caused by Puccinia sorghi through expert examination of pustule color,
pustule location, and spore morphology, but the differences between the two
diseases and the causal organisms may be subtle or even impossible to detect,
especially in early stages of disease development. Therefore, to reliably
differentiate between these two pathogens, a real-time PCR assay based on the
nuclear ribosomal internal transcribed spacer region has been developed for P.
polysora and P. sorghi. This assay will be useful for monitoring and
evaluating the distribution and incidence of southern corn rust in the U.S.
Sequenced restriction-associated DNA (RAD) markers for SNP discovery
in the genus Colletotrichum
J. CROUCH (1), P. Oudemans (2), J. Polashock (3)
(1) USDA ARS, Cereal Disease Lab, St. Paul, MN, U.S.A.; (2) Rutgers
University, Chatsworth, NJ, U.S.A.; (3) USDA ARS, Chatsworth, NJ, U.S.A.
Phytopathology 100:S27
Colletotrichum species are among the most widespread and important plant
pathogens. Efforts are ongoing to better understand genetic variation within
this genus, but resources for the high-throughput discovery and development
of SNPs and other markers are currently very limited. In this study, we set out
to determine whether Illumina-sequenced RAD (restriction-associated DNA)
tags could be used to efficiently identify SNPs from Colletotrichum species,
with the C. graminicola genome sequence assembly serving as a reference.
28,699 RAD tags were sequenced from a sample of 59 Colletotrichum
isolated from grasses and cranberry, including C. graminicola, C. cereale, C.
acutatum and C. gloeosporioides. 39% of these sequences were mapped to the
C. graminicola genome, from which 4537 unique loci were identified. ~50%
of the mapped loci possessed two or more alleles (between 2-19 alleles/locus,
avg 3.9), with 1-6 SNPs present in each 49-bp sequence. For marker
development, the greatest number of polymorphic loci was identified from
grass-derived isolates of C. graminicola and its closest relatives (C. navitas,
C. nicholsonii), while far fewer alleles were observed from the wider
comparisons with the isolates from cranberry (C. acutatum and C.
gloeosporioides). Cluster analysis of the binary coded allelic dataset shows a
correspondence between the SNPs and known relationships previously
inferred through nucleotide sequence analysis and RFLP markers.
SM3: An intracellular paralog of the proteinaceous elicitor SM1 from
Trichoderma virens
F. K. CRUTCHER (1), L. J. Dangott (1), C. M. Kenerley (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S27
The biocontrol agent, Trichoderma virens, has the ability to protect plants
from pathogens by eliciting plant defense responses, involvement in
mycoparasitism, or secreting antagonistic secondary metabolites. SM1, an
Vol. 100, No. 6 (Supplement), 2010
S27
elicitor of induced systemic resistance, was found to have three paralogs
within the T. virens genome. The paralog sm3 is highly expressed in the
presence of plants and during mycoparasitism of Rhizoctonia solani.
Comparison of culture filtrate and tissue extracts by SDS-Page and Western
blots indicated that SM3, unlike SM1, is intracellular, indicating that SM3
may interact with other organisms only during direct contact with T. virens.
SM3 was purified using anion exchange and gel filtration chromatography and
sequenced for comparison to SM1. To determine the localization of the
protein within cells SM3 was tagged with red fluorescent protein (RFP). The
potential role of SM3 was assessed by testing the purified protein for its
ability to induce six defense related genes in maize in comparison with
induction by SM1. Gene deletion and over-expression mutants were generated
and compared with the wild type for induction of resistance in maize against
Colletotrichum graminicola. Understanding the roles of elicitor protein
families can greatly increase our understanding of plant-microbe interactions
and will give us new approaches to controlling plant diseases.
The role of calcium and other minerals on biofilm formation and
adhesion force in Xylella fastidiosa cells
L. CRUZ (1), L. De La Fuente (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S28
Xylella fastidiosa (XF) is known to infect a wide range of plant species, but its
mechanism of pathogenicity remains unclear. Plant susceptibility has been
mainly associated with water deficits from xylem vessel blockage caused by
plant-derived tyloses or gums and the formation of bacterial biofilm.
However, some of the symptoms are not fully explained by water stress.
Based on the similarity between symptoms of XF infection and plant
nutritional imbalances, we hypothesize that symptoms result from the capacity
of the bacteria to uptake minerals from the plant during pathogenesis.
Preliminary data suggests a positive correlation between increases in the
concentrations of calcium and iron and the formation of biofilm in vitro, while
a negative effect was found for copper and zinc. The addition of different
concentrations of chelators to PD2 complete media was used to further
corroborate these results. The specific Ca chelator ethylene glycol tetraacetic
acid (EGTA) produced the highest reduction in biofilm formation. The
specific Fe chelator deferoxamine (DFO) also produced a negative effect on
biofilm formation. The adhesion force of the cells under high concentrations
of Ca and EGTA was quantified using microfluidic chambers. The addition of
Ca to PD2 significantly increased the adhesion force of XF, while EGTA
significantly decreased adhesion to the substrate. These results are evidence of
a role of Ca in adhesion and biofilm formation of XF in vitro.
Evaluation of fluensulfone for root knot nematode on tobacco
A. CSINOS (1), J. Whitehead (2), L. L. Hickman (1), S. S. LaHue (1)
(1) University of Georgia, Tifton, GA, U.S.A.; (2) Makhteshim Agan of North
America, Oxford, MS, U.S.A.
Phytopathology 100:S28
Root knot nematodes are becoming an increasing problem on commercial
tobacco and management is exasperated with short supplies of nematicides,
high cost and lack of choice of materials. Makhteshim’s MCW-2
(fluensulfone) was evaluated on flue-cured tobacco for management of
Meloidogyne arenaria. 1, 2, 3 and 4 kg ai/ha of fluensulfone were applied to
the soil bed as separate treatments in a 30 cm band using a CO2 pressurized
back pack sprayer, and the material rototilled into the soil to a depth of 15 cm.
Aldicarb was applied to the bed at the rate of 3.36 kg/ha and rototilled into the
bed. Fenamiphos (3.36 kg/ha) was applied as described for MCW-2. The trial
was a RCB design with single row plots 11m long, 1.1m wide replicated 6
times. Soil was a sand loam, with a history of M. arenaria on peanuts. All
rates of MCW-2 had vigor ratings equal to aldicarb, except 4 kg/ha rate which
was higher. Root gall ratings at harvest and larval numbers for MCW-2 at 3
and 4 kg/ha were lower than aldicarb treated plots. All treatments except
fenamiphos had yields higher than the non-treated. Yields of MCW-2 were
not different from the aldicarb standard.
Impact of bispyribac-sodium on annual bluegrass control and brown
patch severity in tall fescue
M. A. CUTULLE (1), B. J. Horvath (2), J. Derr (3), A. Nichols (3), D. McCall
(4)
(1) Virginia Tech, Ellicott City, MD, U.S.A.; (2) University of Tennessee,
Knoxville, TN, U.S.A.; (3) Virginia Tech, Virginia Beach, VA, U.S.A.; (4)
Virginia Tech, Blacksburg, VA, U.S.A.
Phytopathology 100:S28
Tall Fescue is one of the most commonly utilized turfgrasses for home lawns
and other lower maintenance turf areas in the United States. Rhizoctonia
infects tall fescue stands during hot humid conditions when tall fescue is
under summer stress. The subsequent disease, brown patch is aesthetically
unpleasing and can thin out the turfgrass stand leading to the germination and
S28
PHYTOPATHOLOGY
encroachment of winter annual weeds such as annual bluegrass. A potential
postemergence herbicide for control of annual bluegrass in tall fescue is
bispyribac-sodium. However, preliminary reports indicate that applications of
bispyribac-sodium on tall fescue have increased its susceptibility to brown
patch, thus promoting the sequential increase of summer disease and fall weed
encroachment. Bispyrabac-sodium was applied at rates of 12 and 6 g ai ha-1
either April 22 and two weeks after or May 22 and two weeks after in 2009.
Applying the low rate of bispyribac-sodium on May 22 resulted in greater
than 60% brown patch in June, which was more than any other treatment.
More brown patch lesions were also recorded for later applications of the high
herbicide rate when compared to other treatment combinations. Overall, later
herbicide applications increased disease severity in July and August regardless
of herbicide rate. Early applications of the high herbicide rate resulted in less
annual bluegrass cover when compared to all other treatment combinations
with no objectionable phytotoxicity to tall fescue.
Interactions between lesion nematodes and Pythium ultimum on maize
seedlings
M. P. DA SILVA (1), G. Munkvold (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S28
Lesion nematodes (Pratylenchus penetrans) are known to interact with root
rot pathogens on a variety of host plants. The objectives of this research were
to measure the effects of P. penetrans infestation on seedling disease
symptoms caused by the fungal pathogen Pythium ultimum, and assess the
impacts of seed treatments on this interaction. Growth-chamber experiments
were conducted with 150-ml pots that were filled with an autoclaved sand-soil
mixture combined with fungal inoculum (colonized corn meal/sand mixture).
A suspension of 4000 P. penetrans (adults and juveniles) was added to the
pots at the time of planting. A factorial experimental design was used
including 8 seed treatments × 4 pathogen combinations × 4 replicates.
Experiments were harvested 30 days after planting. Shoot lengths, fresh and
dry shoot and root weights, and visual root health scores were determined.
Roots were scanned and image analysis conducted with WinRhizo software;
root length, volume, tips, branching, discoloration and diameter class
distribution were determined. The results indicate significant effects on root
health with interactions between fungal pathogens and nematodes. WinRhizo
color analyses indicate significant interactions between seed treatment and
nematodes affecting root health, length and volume. Root diameter class
distribution was significantly affected by nematode - fungus interactions that
resulted in a lower proportion of smaller diameter roots.
Identification of soybean lines resistant to Frog eye leaf spot at ultra-low
plant density
R. R. DABALA (1), W. D. Clark (1), S. K. Kantartzi (1)
(1) Southern Illinois University, Carbondale, IL, U.S.A.
Phytopathology 100:S28
Frog eye leaf spot caused by Cercospora sojina is an emerging problem in
Southern Illinois favored by hot and humid weather. The aim of this study was
to screen soybean (Glycine max (L.) Merr.) advanced breeding lines and to
identify the potential resistant ones. In total, 24 advanced breeding lines along
with seven checks including one resistant-‘Davis’ and one susceptible‘Blackhawk’ were tested through honeycomb selection, under the ultra-low
density of 1.2 plants/m2 in Carbondale IL in 2009. Data showed that there
were significant differences in disease severity among lines, indicating genetic
variability for FLS resistance. All the 24 lines subsequently tested for the
possible presence of previously reported QTLs using microsatellite markers
located on LG J. An integrated analysis of phenotypic and molecular data may
be useful in developing soybean cultivars with broad resistance to FLS and
adapted to Southern Illinois area.
Mating between Aspergillus flavus cryptic species I and II
K. E. DAMANN (1), C. DeRobertis (1), R. Sweany (1)
(1) Department of Plant Pathology & Crop Physiology, Louisiana State
University Agricultural Center, Baton Rouge, LA, U.S.A.
Phytopathology 100:S28
Aspergillus flavus was recently shown by others to be a sexually reproducing
Ascomycete and given the name Petromyces flavus for its teleomorphic stage.
Geiser, Pitt & Taylor (1998) reported phylogenetic divergence in a collection
of Australian isolates of A. flavus which they named cryptic species I and II.
We have determined the ability of 28 of their isolates to mate in vitro. Pairings
of Mat 1-1 and Mat 1-2 isolates within and between cryptic species revealed
successful matings in both circumstances. This is interesting because, as
originally reported, some cryptic species II isolates produce aflatoxins G1 and
G2, a trait which taxonomists have recently banished from the A. flavus
repertoire. The inheritance of aflatoxins B1, B2, G1, G2, cyclopiazonic acid,
sclerotial size, and the segregation of SSR haplotypes are reported.
Comparing ectomycorrhizal colonization on transgenic, hybrid, and
wildtype Castanea dentata
K. M. D’AMICO (1), T. R. Horton (1), C. A. Maynard (1), W. A. Powell (1)
(1) SUNY-ESF, Syracuse, NY, U.S.A.
Phytopathology 100:S29
The introduction of the fungal pathogen Cryphonectria parasitica (causal
agent of the chestnut blight) devastated populations of American chestnut
(Castanea dentata) across the eastern United States, effectively removing this
heritage tree species from the landscape. Efforts to develop an American
chestnut tree that is more resistant to blight include transformation with genes
to enhance the plant’s defense response. After a transgene has been introduced
into a plant, it is necessary to assess the non-target impacts that the gene
product might have on associated microbial populations. This study compares
the ectomycorrhizal colonization of American chestnut transformed with a
gene for oxalate oxidase to wildtype [American], Chinese chestnut (Castanea
mollissima), American x Chinese chestnut (C. dentata x C. mollissima), Red
Oak (Quercus rubra), and American Beech (Fagus grandifolia). Trees were
grown in soils collected in the field in a soil bioassay to bait for mycorrhizal
fungi. Mycorrhizal root tips were quantified and fungi were identified from
mycorrhizal root tips using RFLP and sequence analysis of the fungal ITS
region. The results of this study will increase our understanding of
ectomycorrhizal colonization in these Fagaceae species and inform the
deregulation process as part of a larger effort to restore American chestnut to
its natural range.
Physalis peruviana natural reservoir for Phytophthora infestans in the
field
G. DANIES (1), A. M. Vargas (1), C. A. Antolínez (1), G. Peña (1), A. J.
Bernal (1), S. Restrepo (1)
(1) Univ De Los Andes, Bogota, COLOMBIA
Phytopathology 100:S29
Phytophthora infestans is an hemibiotrophic plant pathogen that attacks a
great variety of crops belonging to the family Solanaceae, including Physalis
peruviana (cape gooseberry). Today Colombia is the leading producer of cape
gooseberry in the world. The aim of this study was to contribute to the
knowledge of the infection process of P. peruviana by P. infestans following
the development of the disease through an histological analysis and
quantitatively determining the expression of the biotrophic and necrotrophic
markers ipiO and npp1 respectively using qRT-PCR. Furthermore, we
compared the effect of infected cape gooseberries and potatoes as sources of
inoculum for cape gooseberries or potatoes in the field and in laboratory
conditions. Through the histological analysis it was possible to evidence
sporangia and zoospore germination. Sporulation and macroscopic symptoms were observed sporadically. The genes ipiO and npp1 showed
unexpected patterns of expression. Cape gooseberry plants ecotype
Colombia showed to be resistant while potatoes were susceptible to the P.
infestans inoculum circulating during the summer of 2009 in the northeast of
the United States. Our results suggest that different cape gooseberry ecotypes,
might play an important role in determining whether the plant is a host or a
non-host. Infected cape gooseberries may serve as inoculum for cape
gooseberries and potatoes, making this new host a possible source for resistant
genes.
Acholeplasmavirus P1 from Acholeplasma palmae, an ancestral relative of
plant pathogenic phytoplasmas
R. E. DAVIS (1), R. Jomantiene (2), Y. Zhao (1), I. Lee (1), E. L. Dally (1), J.
Shao (1)
(1) USDA-Agricultural Research Service, Beltsville, MD, U.S.A.; (2) Nature
Research Center, Vilnius, LITHUANIA
Phytopathology 100:S29
Phytoplasmas are cell wall-less prokaryotes that descended from an
acholeplasma-like ancestor and exist as transkingdom parasites of plant
phloem and phloem-feeding insects. Survey sequencing of the genome of
Acholeplasma palmae, a cell wall-less prokaryote isolated by others from
rotting tissues of a lethal yellowing phytoplasma-infected coconut tree,
revealed the presence of virus-related sequences. Cloning and results from
nucleotide sequence analysis indicated that A. palmae contained an
extrachromosomal, circular DNA molecule encoding putative proteins that
shared amino acid sequence similarities with proteins encoded by the
enveloped, double-stranded circular DNA acholeplasmavirus L2 from A.
laidlawii. Although the A. palmae virus shared similarities with
acholeplasmavirus L2, the A. palmae virus, termed acholeplasmavirus P1, was
distinct from L2. Results from comparative genomics revealed no homologous
potential protein coding regions (open reading frames, ORFs) in partially or
completely sequenced phytoplasma genomes. The findings are consistent with
the concept that acholeplasmaviruses L2 and P1 invaded Acholeplasma spp.
after evolutionary divergence of acholeplasmas from phytoplasmas.
Soil detection of crown and root rot of tomato caused by Fusarium in
Sonora and Baja California (Mexico) using soil phytopathometry
M. de Cara (1), D. Palmero (2), M. Vazquez-Mundo (3), F. Camacho (1), J.
TELLO MARQUINA (1)
(1) Univ of Almería, Almería, SPAIN; (2) Technical Univ of Madrid, Madrid,
SPAIN; (3) Almería, SPAIN
Phytopathology 100:S29
In 2008, crown and root rotten and dead tomato plants widely appeared in
several tomato fields in the states of Sonora and Baja California (Mexico). In
order to find the causal agent, 13 rhizosphere soil samples from three of these
fields were analyzed using the technique called “soil phytopathometry”a. This
technique consists on planting 10 germinated seeds of tomato (cv San Pedro,
susceptible to soil-borne pathogens) in a mixture of each rhizosphere sample
and sterile vermiculite (1:6 w/w) contained in 1-L plastic pots. Three pots
were used per sample. The plants were maintained for 60 days in a growth
chamber at 23 to 26°C with a 16-h photoperiod. Thirty days after sowing,
plants showed first symptoms, raising F. oxysporum f. sp. radicis-lycopersici: affected plants got wilted and exhibited crown and root rot. All
samples presented diseased plants, and Fusarium oxysporum was the
unique pathogen isolated from the rotten tissue when analyzed on malt extract
agar. Pathogenicity of eight isolates on tomato was confirmed, and no
pathogenicity was showed when these isolates were inoculated on sweetpepper and eggplant. To our knowledge this is the first report of F. oxysporum f. sp. radicis-lycopersici in Sonora and Baja California. The
usefulness of the technique was previously evaluated for F. oxysporum f. sp.
melonis (race 1) and Melon Necrotic Spot Virus (MNSV) and its vector
Olpidium bornovanus. aGeomicrobiology Journal, Volume 23, Issue 5 June
2006, pages 319-322
Influence of Xylella fastidiosa on mineral content of infected host plants
L. DE LA FUENTE (1), L. C. Cruz (1), J. K. Parker (1), P. A. Cobine (1)
(1) Auburn University, Auburn University, AL, U.S.A.
Phytopathology 100:S29
The bacterium Xylella fastidiosa (XF) infects several agronomically important
crops. The infection process involves the blockage of xylem vessels
responsible for the passage of water, but published work shows that water
deficit in grapes does not produce the same symptoms as XF infection. Based
on the similarities between mineral deficiency and XF infection symptoms,
we are studying the effects of the XF infection process on the mineral content
of host plants. A model system using greenhouse-cultivated tobacco
(Nicotiana tabacum cv. SR1) was established. Leaves of XF-inoculated and
buffer-inoculated control plants were collected before inoculation and
periodically over a 6–10 weeks period after infection. The mineral content of
the leaves was analyzed using inductively coupled plasma optical emission
spectrometry (ICP-OES) to look at the total mineral content of the leaf as well
as the distribution throughout the leaf. For the latter, small (1 cm2) sections
were excised from the leaves and analyzed individually. Data was
reconstructed following the leaf topography and plotted in contour maps.
Preliminary results indicate distribution of minerals in “hot spots” in infected
plants, while uninfected plants had an even distribution of the mineral content.
These studies are being complemented with in vitro experiments on the
influence of minerals on biofilm formation and attachment of XF. Our
preliminary data in both the host and the pathogen will be discussed.
Developing a taxonomic identification system based on microsatellites of
Phytophthora species
J. M. DEL CASTILLO (1), A. J. Bernal (1), S. Restrepo (1)
(1) Univ De Los Andes, Bogota, COLOMBIA
Phytopathology 100:S29
Phytophthora spp. is the most important genus of Oomycetes plant pathogens.
Actually there are 80 described species, and most of these are primary
invaders of plant tissues, and they are the causal agent of diseases in a wide
range of crops and natural plants. In order to develop control strategies against
Phytophthora spp., it is important to know the biology, mechanisms and
evolutionary processes of this important pathogen. The aim of this study was
to propose and validate a low cost identification system for Phytophthora
species based on a set of polymorphic microsatellite (SSRs) markers. For this,
30 isolates from P. infestans, P. andina, P. sojae, P. cryptogea, P. nicotianae,
P. capsici and P. cinnamomi were obtained, and 14 SSRs, potentially
transferable markers between these species were chosen. Amplification
conditions, including annealing temperature were standardized for several
markers. All of them were assayed on high-resolution agarose, and some on
polyacriyamide, and they specifically amplified in all species, showing
different alleles depending on the species. Also RFLP analysis of COX region
were performed, giving more tools to create an identification code to diagnose
and monitor this plant pathogen.
Vol. 100, No. 6 (Supplement), 2010
S29
First detection of Phakopsora pachyrhizi on Jicama in the United States
and on Florida beggarweed in Alabama
M. A. DELANEY (1), E. Sikora (2)
(1) Auburn University Extension, Auburn, AL, U.S.A.; (2) Auburn
University, Auburn, AL, U.S.A,
Phytopathology 100:S30
Soybean Rust, caused by Phakopsora pachyrhizi, was detected on Jicama
(Pachyrihius erosus) for the first time in the United States in November, 2009.
The pathogen was also detected on Florida beggarweed (Desmodium
tortuosum) for the first time in Alabama Phakopsora pachyrhizi was observed
on a potted Jicama plant grown outdoors in a residential area, as well as in a
demonstration plot at Auburn University. Symptoms appeared on the upper
leaf surface as chlorotic isolated areas near the leaf edge on leaves in the
lower plant canopy. Symptoms on the lower leaf surface exhibited brown
lesions and produced volcano-shaped pustules with urediniospores that were
characteristic of Phakopsora sp. Pustules and uredinospores were pale tan in
color. Leaves expressing disease symptoms of were analyzed using an
Envirologix monoclonal antibody test kit at the Auburn Plant Diagnostic
Laboratory. Symptomatic plant tissue was also sent to the USDA National
Identification Services Laboratory in Beltsville, MD for further confirmation.
The fungal structures present were confirmed to be Phakopsora sp. The
samples and forwarded to the USDA National Plant Germplasm and
Biotechnology Laboratory for DNA testing and confirmed as P. pachyrhizi.
Symptomatic tissue obtained from Florida beggarweed collected in Headland,
Alabama was submitted for identification in the same manner. To our
knowledge this is the first report of the disease on Jicama in the United States
and on Florida beggarweed in Alabama.
Effects of soybean cyst nematode infestation and resistance on Fusarium
root rot on soybeans
M. M. DIAZ ARIAS (1), G. Tylka (1), L. Leandro (1), G. Munkvold (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S30
Fusarium species are ubiquitous in soil and cause important soybean diseases
such as damping-off and root rot. At least 12 different Fusarium species have
been reported from soybean roots but their relative aggressiveness as root
pathogens is unknown. In some cases, root rots can be exacerbated by other
pathogens such as the soybean cyst nematode (SCN). To determine whether
SCN infestation enhances Fusarium root rot in soybean, greenhouse and field
trials were conducted using varieties that differ in genetic resistance to SCN.
Field plots were established in two Iowa locations (Story Co. and Hancock
Co.). Soil and plant samples were collected to test for soil SCN populations
and Fusarium root rot severity. In the field, the relationship between SCN
resistance and root rot was not consistent. However, some SCN-susceptible
cultivars such as 92M91 (Pioneer) had high root rot severity, and some SCN
resistant cultivars such as L2620RX (Latham) had lower disease severity. For
the Story location, regression analysis showed a poor relationship between
root rot severity and yield, but a positive relationship among nematode
population (0-3600 eggs/100cc soil) and disease severity. For the Hancock
location, percent root rot severity explained 42.1% of the variation in yield,
but there was a poor relationship between nematode populations and disease
severity. Selected isolates from eight different Fusarium species are being
used to test the interaction between SCN and Fusarium in greenhouse
conditions.
PCR detection of aflatoxin producing strains of Aspergillus spp. from
corn and red flour beetle
S. DOBHAL (1), D. Blazheva (2), M. Arif (1), P. Garrido (1), F. M. OchoaCorona (1), G. Opit (1), C. Garzon (1)
(1) Oklahoma State University, Department of Entomology and Plant
Pathology, Stillwater, OK, U.S.A.; (2) University of Food Technologies,
Department of Organic Chemistry and Microbiology, Plovdiv, BULGARIA
Phytopathology 100:S30
Aflatoxins are mycotoxins produced by some species of Aspergillus that
contaminate food and feed. This study describes the development of two
specific and sensitive primer pairs for PCR detection of aflatoxin-producing
Aspergillus spp. in contaminated corn grain and red flour beetles (Tribolium
castaneum), which have serious implications in food and agricultural
biosecurity. Primers were designed using the web interface software Primer3,
mFOLD and BLASTn with validated thermodynamic parameters. The primers
amplify 142 bp of the aflB-aflR intergenic spacer (aflatoxin Q) and 162 bp of
the conserved -tubulin gene (Aspergillus-specific). The two PCR assays can
detect down to 10–8 ng/µl of template DNA. To detect contamination of corn
seeds and T. castaneum with Aspergillus spp., DNA was extracted from both,
corn seeds and beetles. PCR products of the expected sizes were amplified,
purified, and confirmed by direct sequencing. These two PCR assays allow
the rapid detection of Aspergillus spp., and discriminates aflatoxin-producing
and nonproducing species or strains, and would assist decision making and
S30
PHYTOPATHOLOGY
assessment about stored grain contamination in conjunction with toxin
detection procedures.
Comparative analyses of the ‘Candidatus Liberibacter’ species reductive
genome features
H. DODDAPANENI (1), H. Lin (2), Y. Duan (3), V. Lou (4), C. Chen (4), C.
Vahling (3), Z. Lijuan (5), E. L. Civerolo (2)
(1) Carver Center for Genomics, Department of Biology, University of Iowa,
Iowa City, IA, U.S.A.; (2) USDA, ARS, SJVARC, Parlier, CA, U.S.A.; (3)
USDA, ARS, USHRL, Fort Pierce, FL, U.S.A.; (4) Guangxi Citrus Research
Institute, Guilin, Guangxi, China, Parlier, CA, U.S.A.; (5) University of
Florida, Indian River Research and Education Center, Fort Pierce, FL, U.S.A.
Phytopathology 100:S30
‘Candidatus Liberibacter’ species are gram-negative α-proteobacteria that are
associated with some destructive plant diseases such as citrus Huanglongbing
and potato ‘zebra chip’. These bacteria are transmitted by psyllids and are
classified into four species. Using whole genome amplification and 454
pyrosequencing, we have sequenced the genomes of ‘Ca. Liberibacter
asiaticus’ (Las) and ‘Ca. Liberibacter solanacearum’ (Lso). A total of 1,136
and 1,126 CDS were predicted in Las and Lso, respectively. Comparative
genomics and metabolic pathway analyses have revealed some details, such as
the presence of reductive oxidative phosphorylation, the reduction or complete
absence of metabolic enzymes and secretion systems in these genomes. There
are 867 conserved proteins, of which, 531 proteins shared ≥70% similarity
and may represent the core genome of the ‘Ca. Liberibacter’ species. The
remaining 336 proteins showed greater diversification and a significant
portion of those are associated with membrane and transport functions, and
that may help define their speciation. Similarly, Lso and Las genomes differ
by > 25% quantitatively among six functional COG categories. Five of these
categories are related to external interactions that are associated with a
pathogenic lifestyle. The reduced metabolic capabilities, which reflect their
fastidious nature, along with the presence of pseudogenes suggest ongoing
genome decay in this group of bacteria.
Response of late blight resistant tomato lines to Florida genotypes of
Phytophthora infestans
R. S. DONAHOO (1)
(1) University of Florida, Immokalee, FL, U.S.A.
Phytopathology 100:S30
Late blight of potato (Solanum tuberosum) and tomato (Solanum
lycopersicum) is caused by the Stramenopile Phytophthora infestans. Late
blight is common in south Florida during the winter months, where both crops
are produced, and environmental conditions are extraordinarily favorable for
disease development. Over the past five years, a shift in P. infestans
populations recovered from tomato has been observed. In an attempt to assess
late blight resistance to P. infestans isolates collected in Florida, seed from 13
cultivars were obtained from the Asian Vegetable Research and Development
Center. Six different late blight resistance genes (Ph+, Ph-1. Ph-2, Ph-3, Ph-4,
and Ph-6) have been introgressed into these ‘differential lines’. Using a
detached leaf assay, resistance to five Florida P. infestans genotypes are being
compared to that of the susceptible tomato cultivar ‘FL-47’. The results of the
detached leaf assays and the implications of P. infestans race structure in
Florida will be presented.
Effect of poultry litter on Heterodera glycines reproduction
P. DONALD (1), P. Allen (2), K. Sistani (3), D. Tyler (4), H. Tewolde (5)
(1) USDA ARS, Jackson, TN, U.S.A.; (2) Knoxville, TN, U.S.A.; (3) Bowling
Green, KY, U.S.A.; (4) Jackson, TN, U.S.A.; (5) Mississippi State, MS,
U.S.A.
Phytopathology 100:S30
Soybean cyst nematode (SCN), Heterodera glycines, management in soybean
production relies on use of incompletely resistant cultivars to reduce SCN
reproduction and associated potential risk of yield loss. A poultry litter study
was initiated to change soil biological composition and potentially reduce
SCN reproduction. Our objective was to use Normalized Difference
Vegetation Index (NDVI), soybean yield, plant height, leaf area index (LAI),
and SCN egg population density to quantify the impact of poultry litter
application on SCN reproduction and plant response. Data were collected for
three years as part of a field study with two rates of poultry litter applied
annually in the spring compared with conventional fertilizer application. Plots
receiving chicken litter had significantly higher yield in 2008 (P = 0.002) and
2009 (P = 0.03) than plots fertilized with conventional fertilizer. The 2007
growing season was especially dry and no treatment differences were
significant. NDVI and LAI were good predictors of plant height and soybean
yield for all years. Post-harvest SCN egg population density was inversely
correlated with yield (r = –0.47, P = 0.003) during 2007, but was positively
correlated with yield in 2008 (r = 0.61, P < 0.0001) and 2009 (r = 0.30, P =
0.06). Significant response of SCN egg population density to treatment may
have been masked by a strong anisotropic gradient present in the field.
Geostatistical analysis is being included to account for this.
Effect of light on germination, germ tube growth, and infection of daylily
by Puccinia hemerocallidis and of geranium by Puccinia pelargoniizonalis
W. DONG (1)
(1) University of Georgia, Griffin, GA, U.S.A.
Phytopathology 100:S31
The presence of rusts of daylily and geranium caused Puccinia hemerocallidis
and P. pelargonii-zonalis can result in reduced value of these ornamental
crops. Controlled environment experiments were conducted to determine the
effects of light on urediniospore germination, germ tube growth, and infection
of the two pathogens on detached leaves and plants. Exposure of non-hydrated
or hydrated spores of P. hemerocallidis and P. pelargonii-zonalis to cool
white fluorescent light (600µmol s–1m–2) or to sunlight (1200-1600 µmol s–
1m–2) for 4 h significantly reduced germination on detached leaves. Germ tube
growth of P. hemerocallidis was reduced by exposure to fluorescent light and
sunlight for 4 h on detached leaves of daylily. Germ tube growth of hydrated
spore of P. pelargonii-zonalis was reduced by a 4 h sunlight treatment,
however, exposure of both hydrated and non-hydrated spores to fluorescent
light or exposure non-hydrated spores to sunlight did not reduce the germ tube
length on detached leaves of geranium. The infection rate of P. hemerocallidis
on detached leaves of daylily decreased after 4 h treatment with fluorescent
light. Exposure of inoculated plants to sunlight for >2 h decreased the
infection rates of P. hemerocallidis and P. pelargonii-zonalis with a larger
effect observed on plants inoculated with hydrated compared to dry
urediniospores.
Effects of environment and cultivar on charcoal rot development in
soybeans
M. DOUBLEDEE (1), J. Rupe (1), C. Rothrock (1), S. Bajwa (1), A. Steger
(1), R. Holland (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S31
Charcoal rot, caused by Macrophomina phaseolina, is a soilborne disease
associated with hot, dry weather, however the above ground disease
symptoms are difficult to distinguish from those of drought. To separate the
affects of disease from drought, four soybean cultivars, DT97-4290, DPL
4546, R01-581F, and LS980358 were grown in microplots with soil that was
either infested or non-infested with M. phaseolina. Half of the plots were kept
well watered and the other half were allowed to water stress. Stomatal
conductance, canopy temperature, and spectral reflectance were measured
periodically though out the season. Yield, root/stem disease severity, plant
height stem discoloration and M. phaseolina colonization were determined at
harvest. On some dates, water stressed plants and infested plants had lower
stomatal conductance and higher canopy temperatures (based on infared
radiation expressed as Crop Water Stress Index) than well watered or noninfested plants. At flowering in 2008, plants in infested, non-irrigated plants
had lower stomatal conductance than those in infested irrigated or noninfested irrigated or non-irrigated plants. Spectral reflectance, disease
assessment, and yield data will also be presented. These results suggest that
infection with M. phaseolina may limit the water uptake in the plant, before
the onset of visible symptoms.
Distribution of Arabis mosaic virus on vineyards in northern provinces of
Iran
H. Doustseddigh (1), F. RAKHSHANDEHROO (1), M. Shamsbakhsh (2)
(1) Department of Plant Pathology, College of Agriculture and Natural
Resources, Science and Research Branch, Islamic Azad University, Tehran,
IRAN; (2) Plant Pathology Department, College of Agriculture, Tarbiat
Modarres University, Tehran, IRAN
Phytopathology 100:S31
Arabis mosaic virus (ArMV) is a Nepovirus with a very wide natural host
range. Because of a extra decrease of vineyard products for 5 recently years in
2 Iran provinces (Eastern and Western Azarbaijan) and observation of
doubtful symptoms to virus infection, a survey was conducted to determine
the incidence of Arabis mosaic virus in mentioned vineyards and also Tehran
province during the years 2008 to 2009. DAS-ELISA test was done with
ArMV specific polyclonal antiserum. According to the ELISA test results a
total number of 203 out of 453 tested leaf samples (44.8%) were infected with
ArMV with the infection rate of 61.3%, 12.5% and 9.5% for Eastern, Western
Azarbaijan and Tehran provinces respectively. Symptoms related to ArMV
infection were recorded as spotting (concentric rings), mottling, mosaic and
yellowing in surveid regions. Total RNA isolation performed with LiCl from
mechanically inoculated cucumber plants according to the published
protocols. Using specific primers for the coat protein of ArMV, extracted
RNA samples were detected by RT-PCR method. A DNA fragment of 519bp
was amplified for serological positive ArMV samples. The amplified
fragments of four isolates sequenced and then aligned with the corresponding
data available for other ArMV isolates in NCBI. Phylogenetic analysis
revealed that of all Iranian tested isolates together with some NCBI isolates
were categorized in one cluster while all other ArMV isolates from NCBI
were categorized in a separate cluster.
Evaluation of Thiazosulfene nematicide drip applications to manage rootknot nematode (Meloidogyne spp.) on yellow squash
J. G. DRIVER (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S31
Southern root-knot nematodes (Meloidogyne incognita) cause severe losses to
vegetable growers in the southeast U.S. Root-knot nematode damage results in
plant stunting, wilting, chlorosis and yield loss. Thiazosulfene is a new nonfumigant nematicide with potential for less crop phytotoxicity. Thiazosulfene
pre-plant drip applications were made in combination with thiazosulfene postplant drip applications at various rates, and were compared to oxamyl
(Vydate) and 1,3-dichloropropene (Telone II). Yellow squash (Cucurbita
pepo) were grown on LDPE white plastic mulch and root-knot galling and
yield was compared. The effect of thiazosulfene rates and application
sequence was not significant among the treated plots. However, the untreated
control had the highest root galling index and was significantly different
than all other treatments except thiazosulfene applied at 4.17 l/ha (P < 0.05).
The combination of a pre and post applications had no effect on root
galling. Plants tested with oxamyl had similar gall severity to thiazosulfene
and 1,3-dichloropropene was particularly effective at reducing root-knot
galling (P < 0.05). Thiazosulfene applied at 6.25 l/ha and oxamyl applied as a
pre and post plant treatment had similar results to 1,3-dichloropropene. Plants
grown with drip applied nematicides had intermediate vigor, with the
exception of plants grown with thiazosulfene at 4.17 l/ha rate that showed
poor vigor.
A broad host range tailocin from Burkholderia
I. DUARTE (1), G. Wang (1), J. J. Gill (1), R. F. Young (1), J. J. LiPuma (2),
C. F. Gonzalez (1)
(1) Texas A&M University, College Station, TX, U.S.A.; (2) University of
Michigan Medical School, Ann Harbor, MI, U.S.A.
Phytopathology 100:S31
The Burkholderia cepacia complex (Bcc) consists of at least 17
phenotypically similar but genotypically distinct species of non-fermenting,
Gram-negative bacteria that are found in a diverse set of niches. Members of
the Bcc can be involved in beneficial or pathogenic interactions with plants.
They are also considered opportunistic pathogens, especially for persons with
cystic fibrosis. Essentially all Bcc clinical isolates demonstrate broadspectrum antibiotic resistance in vitro. Combination antibiotic therapy
typically results in poor clearance of Bcc from infected individuals. There is a
substantial need to develop new strategies for antimicrobial therapy against
these pathogens. The use of phage-tail-like high molecular weight
bacteriocins, or “tailocins”, as a potential anti-bacterial agent against Bcc is
under investigation. We have identified a tailocin, designated Bcep0425,
which exhibits broad host range biocidal activity against members of the Bcc
and other bacterial genera. We have conducted genetic analysis of the tailocin
encoding genes and determined a high degree of similarity to defective phages
identified in sequenced Burkholderia genomes, except for the tail fiber
components which appear to be novel. Deletion analysis of tailocin structural
genes will be used to further characterize Bcep0425.
Pathogenic variation of Pectobacterium carotovorum isolates and the
effects of relative humidity on the severity of bacterial stem rot in
potato
J. K. Dung (1), B. K. SCHROEDER (1), D. A. Johnson (1)
(1) Washington State University, Pullman, WA, U.S.A.
Phytopathology 100:S31
Bacterial stem rot (BSR) of potato, usually caused by Pectobacterium
carotovorum (Pc), causes lesions and soft rot of aerial stems. The effects of
isolate and relative humidity (RH) were examined by syringe-inoculating
stems of “Umatilla Russet” potato plants with Pc cells (106 CFU) suspended
in sterile distilled H2O (sdH2O). Two Pc isolates obtained from BSR lesions
(O207.1 and V104.1), two Pc isolates collected from soft rot tubers (Ec101
and 1.1.2009) and a sdH2O control were used. Plants were subjected to 90–
100% (high) RH or 20–30% (low) RH for 24 hr post-inoculation (p.i.) and
placed in a greenhouse for evaluation. Areas under the lesion progress curve
(AULPC) were calculated from lesion length measurements taken at 2, 4 and
8 days p.i. Stems and progeny tubers were sampled and plated onto CVP to
detect pectolytic bacteria. Significant (P ≤ 0.0006) effects of isolate, RH and
isolate × RH interaction on AULPC values were observed. AULPC values
were significantly greater with isolates O207.1, V104.1 and 1.1.2009 than
Vol. 100, No. 6 (Supplement), 2010
S31
with the sdH2O control at both RH treatments and were significantly greater at
high RH than low RH. Isolate Ec101was not significantly different from the
sdH2O control at low RH but was significantly greater at high RH.
Pectolytic bacteria were recovered from ≥70% of inoculated stems but were
not isolated from progeny tubers. These results indicate that isolates of Pc
may vary in their capacity to cause BSR lesions and RH may affect symptom
severity.
rDNA IGS1 region. The size of the PCR products were 680 bp in AG-3
isolates and 650 bp in AG-4 isolate. To evaluate genetic diversity in
AG-3 group, thirteen restriction enzymes (seven 4-cuttrer and six 6-cutter)
were used while BamH I was just able to show the diversity among
isolates; consequently, AG-3 isolates were divided into two different groups.
No correlation was observed between genetic diversity and geographical
regions.
Management of Fusarium wilt of watermelon with fungicides
D. S. EGEL (1), K. Everts (2)
(1) Purdue University, Vincennes, IN, U.S.A.; (2) University of Maryland
College Park, Salisbury, MD, U.S.A.
Phytopathology 100:S32
Annular rings for enhancing photographs of perineal patterns of rootknot nematodes
J. D. EISENBACK (1)
(1) Virginia Tech, Blacksburg, VA, U.S.A.
Phytopathology 100:S32
Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. niveum (FON) is a
limiting factor in watermelon production across the eastern U.S. An increase
in FW has occurred with the phase out of methyl bromide as a soil fumigant,
the spread of race 2 of FON, and the increasing production of triploid
cultivars. Thus, the search for chemical management options has increased.
The U.S. national program IR-4 initiated trials of soil-applied chemicals to
manage FW. In 2008 in Indiana, Delaware and Maryland, fungicides were
applied once at transplanting. In Delaware, acibenzolar-S-methyl,
thiophanate-methyl, prothioconazole and ipconazole significantly reduced wilt
at 2 1/2 weeks. In Maryland, all treatments except azoxystrobin reduced wilt
incidence at 4 and 5 weeks. In Indiana, none of the treatments decreased wilt
as compared with the control, but propiconazole, and metconazole increased
vine vigor at 37 and 46 days. In 2009 at two sites, acibenzolar-S-methyl,
prothioconazole and thiophanate-methyl were applied alone and in
combination, through trickle irrigation immediately after transplanting and 2
and 4 weeks later. In Maryland, prothioconazole used alone, or with either
acibenzolar-S-methyl and/or thiophanate-methyl reduced wilt at 31 and 45
days. In Indiana, prothioconazole reduced wilt when used alone or in
combination with thiophanate-methyl whereas prothioconazole used in
combination with acibenzolar-S-methyl did not reduce wilt.
Morphology of the perineal pattern of female root-knot nematodes remains
useful for routine identification of Meloidogyne species. A new technique that
uses an annular ring in the stage condenser produces images with increased
resolution and enhanced surface morphology that look like images produced
with a scanning electron microscope. Annular rings are most often mounted in
the stage condenser of a light microscope for phase microscopy. They are
necessary for phase and have been shown to increase resolution. This increase
in resolution occurs from the coherent illumination (light waves that vibrate
with constant phase relationships) produced by the annular aperture that is
absent in the normal brightfield condenser. The initial picture appears similar
to a photographic negative but is inverted into a positive with image
processing software and a computer. This new technique of illumination may
increase the value of the observation of perineal patterns for identification of
Meloidogyne species by increasing resolution and enhancing surface
morphology.
Black leg of basil caused by Plectosporium tabacinum is reported in the
United States
D. S. Egel (1), G. S. RUHL (2), S. Hoke (1), B. Dicklow (3), R. Wick (3)
(1) Purdue University, Vincennes, IN, U.S.A.; (2) Purdue University, West
Lafayette, IN, U.S.A.; (3) University of Massachusetts, Amherst, MA, U.S.A.
Phytopathology 100:S32
Dark, irregular, black stem lesions of sweet basil (Ocimum basilicum
‘Genovese’) were observed in a hydroponic greenhouse in Indiana in 2007.
Diseased plant samples were sent to diagnostic clinics at Purdue University
and the University of Massachusetts. A fungus identified as Plectosporium
tabacinum was cultured from the basil stems. Inoculations were performed on
rooted basil plants in each of eight, 125-ml Erlenmeyer flasks. Four flasks
were filled with 100 ml of deionized water as negative controls and four were
filled with a 1 × 106 CFU/ml water suspension of P. tabacinum. After 24 h
incubation on a laboratory bench at 23C, the solutions in all flasks were
discarded and each flask and root system was rinsed three times with
deionized water and incubated an additional 7 days in deionized water. Dark
brown-to-black stem lesions similar to those described above developed at the
base of the hypocotyl at the water interface and extended to a mean of 22 mm
above the water interface on inoculated plants. Control plants remained
symptomless. P. tabacinum was recovered from symptomatic tissue of
inoculated plants to complete Koch’s postulates. These data indicate that P.
tabacinum was the causal agent of the symptoms observed on the hydroponic
basil. To our knowledge, this is the first report of P. tabacinum causing ‘black
leg’ and reduced growth on basil in the United States and the first report in the
world on hydroponic basil.
Genetic diversity of Anastomosis Group 3 of Rhizoctonia solani isolates
from potato in Iran by PCR-RFLP
F. EGHBALIAN (1), H. Zamanizadeh (1), B. Morid (2), S. Hajmansoor (3)
(1) Department of Plant Pathology, Science and Research Branch, Islamic
Azad University, Tehran, IRAN; (2) Department of Plant Protection, Takestan
Branch, Islamic Azad University, Tehran, IRAN; (3) Science and Research
Branch, Islamic Azad University, Tehran, IRAN
Phytopathology 100:S32
Rhizoctonia solani is one of the most important agents of potato losses in Iran
that causes stem canker and black scurf of tuber. Anastomosis Group 3 (AG3) is known as the major cause of the diseases. Recent researches have shown
that AG-3 isolates from potato and tobacco are distinct in genetic diversity. In
order to study genetic diversity in AG-3 population from potato, thirty one
isolates of Rhizoctonia solani were collected from different regions of Iran.
The anastomosis groups were determined by observation of hyphal
interactions. Thirty isolates were identified as AG-3 and one isolate as AG-4.
Molecular analysis was done based on PCR-RFLP by specific primers for
S32
PHYTOPATHOLOGY
Time spray strategies for Septoria leaf blotch disease progress on winter
wheat: The use of forecasting model
M. El Jarroudi (1), F. Giraud (2), P. Delfosse (3), L. Hoffmann (3), H. Maraite
(4), B. TYCHON (5)
(1) Univ of Liege, Arlon, BELGIUM; (2) Martillac, FRANCE; (3)
Belvaux, LUXEMBOURG; (4) Louvain-la-Neuve, BELGIUM; (5) Arlon,
BELGIUM
Phytopathology 100:S32
A mechanistic model, PROCULTURE, was used over the 2003–2009 period
to simulate septoria wheat leaf blotch progression in the canopy in fourreplicated field experiments located in three villages (Diekirch district:
Reuler; Grevenmacher district: Burmerange and Christnach), representative of
the different agroclimatological zones of the Grand-Duchy of Luxembourg.
This model has been developed in order to find the optimum time of fungicide
spray in fields. On the basis of simulated disease progression on the upper
leaves. A weekly PROCULTURE recalibration is routinely done using actual
disease levels observed on site. The results indicated that the relationship
between disease control by fungicides and yield loss varies from site-to-site
and from season-to-season. On average, only one application of fungicide is
required to control efficiently the Septoria leaf blotch disease.
PROCULTURE forecasts have been shown to be correct in about 85% of all
cases. The fungicide treatment determined by the simulation model over
2003–2009 period resulted in a better return on investment (80%) than the
other single treatments tested and as important as the double fungicide
application (GS31 and GS 59) for Everlange, Christnach and Burmerange. At
Reuler, between 2003 and 2009, treatments based on the Septoria risk
simulation model were recommended only once, in 2007. The climatic
conditions of this last site tend to favour organic farming in this region where
foliar disease pressure is very weak.
Assessment of the night weather parameters and their use in forecasting
model of leaf rust
M. El Jarroudi (1), F. Giraud (2), P. Delfosse (3), L. Hoffmann (3), H. Maraite
(4), B. TYCHON (5)
(1) Univ of Liege, Arlon, BELGIUM; (2) Martillac, FRANCE; (3) Belvaux,
LUXEMBOURG; (4) Louvain-la-Neuve, BELGIUM; (5) Arlon, BELGIUM
Phytopathology 100:S32
A stochastic model was developed to predict the wheat leaf rust (Puccinia
triticina Eriks.) severity (percentage of leaf area with symptoms showing
uredinia) in four-replicated field experiments located in three villages
(Diekirch district: Reuler; Grevenmacher district: Burmerange and
Christnach), representative of the different agroclimatological zones of
Luxembourg. The model was elaborated by the analysis of the night weather
and leaf rust incidence. Statistical validation using regression analysis reports
a strong correlation between the number of hours with specific meteorological
conditions and the percentage leaf area covered by brown rust lesions for the
two upper and youngest leaves, which are mostly responsible for
photosynthesis activity and assimilates production filling the grains. The
development of the brown rust requires a period of at least twelve consecutive
hours with temperatures between 8 and 16°C and a relative humidity (RH)
greater than 60%, with optimal values lying between 12 and 16°C and RH
greater than 80%. During the 2004 to 2009 period, at four sites, the linear
regression between simulated and observed values for Puccinia triticina was
highly significant (P < 0.01) and R2 (coefficient of determination) explained
80 to 85% of the variability. Efforts are now being developed to better define
thresholds for fungicide applications and to spatialize the outputs of the model
over the entire Luxembourg territory.
Effect of climate change on plant-pathogen-beneficial microorganism
interactions
Y. ELAD (1), O. Agra (2), H. Ben Kalifa (2), D. Rav David (2), M.
Borenshtein (2)
(1) ARO, The Volcani Center, Bet Dagan, ISRAEL; (2) Volcani Center, Bet
Dagan, ISRAEL
Phytopathology 100:S33
The interactions of tomato plants, yeast or bacterial potential biocontrol agents
(BCAs) and either humidity-promoted diseases (late blight and gray mold) or
a disease that is also active under less humid conditions (Oidium
neolycopersici powdery mildew) were studied, in order to later model the
effects of climate change on plant diseases and suggest adaptive measures.
The effects of wetness duration, level of RH and temperature change on the
pathogens, the survival of the BCAs and disease suppression were studied.
Under high-temperature and low-RH conditions, the examined bacterium’s
(Pseudomonas sp.) survival was poorer than that of the examined yeast
(Rhodotorula sp.). The microorganisms survived well at 10–15°C under
high RH conditions. The bacterium survived better under high RH conditions than under lower RH conditions in a net house, and also survived better
on leaves with powdery mildew than on symptomless leaves. The yeast was
less affected by microclimatic conditions and survived for 14 days.
Establishment of the two humidity-promoted diseases was lower and
suppression by BCAs was better when wet conditions persisted for 8 h than at
24-h wet period, no disease control was observed. Thus, environmental
conditions affect disease intensity, BCA survival and the efficacy of the
introduced microorganisms. Furthermore, it is clear that such effects will
occur and that adaptive measures need to be developed in order to respond to
these expected changes.
Influence of time, host plant and location on diversity of aster yellows
phytoplasma strains
S. Y. ELATEEK (1), M. L. Ivey (1), A. P. Michel (1), P. A. Paul (1), S. A.
Miller (1)
(1) Ohio State University, Wooster, OH, U.S.A.
Phytopathology 100:S33
Surveys of aster leafhoppers (Macrosteles quadrilineatus) were conducted in
the summer of 2008 and 2009 to assess the distribution of strains of aster
yellows phytoplasma in two major vegetable production areas (Celeryville
and Hartville) in northern Ohio. Aster leafhopper adults were collected
every 2 weeks from mid-June until mid-September from red leaf, green
leaf and romaine lettuce, cilantro and parsley production fields. A total of
3,330 leafhoppers were collected and tested by nested and multiplex PCR to
identify phytoplasma strains. The percentage of AYP-positive aster
leafhoppers peaked in August in both years and locations, and was higher
in Celeryville than Hartville. Four AYP strains were identified: AY-WB
(aster yellows phytoplasma subgroup 16SrI-A) and AY-BW, AY-BD2 and
AY-S (16SrI-B). Some leafhoppers (13.4%) carried unknown AYP strains.
AY-WB was only detected in leafhoppers collected in green leaf and
romaine lettuce and parsley; 16SrI-B strains were found in leafhoppers from
all crops tested. Strains AY-BW and AY-BD2 were detected in similar
numbers and were predominant in 2008 in both locations, while in 2009, AYBW was predominant in Hartville and AY-WB was most abundant in
Celeryville. The distribution and/or abundance of aster yellows phytoplasma
strains appear to be influenced by time, host plant and location of production
fields.
Impact of gaseous ozone on postharvest fungal decays of tomato fruits
M. ELKAHKY (1), J. Bartz (2), M. El-Sheshtawi (1), S. Elafifi (1), M.
Elmazaty (1)
(1) Mansoura University / Faculty of Agriculture, Mansoura, EGYPT; (2)
University of Florida / IFAS, Gainesville, FL, U.S.A.
Phytopathology 100:S33
Postharvest fungal decays of tomato caused by Alternaria alternata, Botrytis
cinerea, Geotrichum candidum and Rhizopus stolonifer result in significant
economic losses during different stages from harvesting from farm to fork.
The efficacy of ozone gas for controlling fungal postharvest fungal decays of
tomato was evaluated. In the laboratory tests, Ozone 0.1 ppm in the
atmosphere above inoculated PDA didn’t affect radial growth, fresh weight or
dry weight of the previously mentioned fungi, but, the development of aerial
mycelium over the cultures appeared to be blocked. On the other hand, ozone
significantly decreased the spore germination of the most tested fungi. Ozone
also, decreased the density of fungal spores in the air of a storage room when
it was applied for 24 hours. In fruit tests, ozone 0.15 ppm at 10°C and RH
99% prevented the development of lesions on wound-inoculated tomato fruits
for 7 days. After 10 days, progressive lesions were observed but were smaller
than those on control fruit. In contrast, when the treatment was applied at
22°C, lesions developed similarly to those on control fruit but sporulation was
inhibited. There was no evidence of phytotoxicity associated with these ozone
treatments.
Identifying resistance to Pythium irregulare and Fusarium graminearum
in soybean
M. L. ELLIS (1), P. A. Paul (1), A. E. Dorrance (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S33
Pythium irregulare and Fusarium graminearum have emerged as important
soybean seedling pathogens in Ohio. The objective of this research was to
assess and characterize resistance to these two pathogens. A greenhouse assay
was used to evaluate 105 soybean genotypes for potential resistance to two
isolates of P. irregulare. Data for seed germination, total weight, root weight,
and a root rot score using an ordinal scale were collected. Twenty of these
genotypes were then evaluated for resistance to F. graminearum, using a
rolled towel method. Data for seedling disease severity was collected. Based
on the results for the greenhouse assay, the isolate × genotype interaction for
root weight was not significant; however there was a significant difference
between the two isolates and among genotypes (P < 0.0001). There was also a
significant difference among genotypes (P < 0.0001) inoculated with F.
graminearum. A number of potential candidates with high levels of resistance
to both pathogens were identified.
Description of two putative new species of Pythium isolated from soybean
and corn in Ohio
M. L. ELLIS (1), K. D. Broders (2), P. A. Paul (1), A. E. Dorrance (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.; (2) University of
Guelph, Guelph, ON, CANADA
Phytopathology 100:S33
During a survey of agronomic soils from 88 locations in Ohio, a distinct
morphological group of Pythium species, designated as group 7 (G7), was
recovered from 30% of the locations and was pathogenic on both corn and
soybean. The objective of this study was to use both morphological and
sequence data to characterize this group. Sequence analysis of the ITS1-5.8SITS2 region of the ribosomal DNA for 21 isolates separated them into two
distinct groups within the E1 clade of the genus Pythium. The first group
consisted of eleven isolates that were 99% similar to P. acrogynum and P.
hypogynum. The second group consisted of ten isolates that were 97% similar
to P. longandrum and P. longisporangium. Therefore these two subgroups,
previously designated as G7, are proposed as two new species based on
morphological and sequence analysis. The frequency that these new species
were isolated from agronomic production fields makes them an important
component to characterize for future management of the Pythium complex
affecting corn and soybeans in Ohio.
Sick Plants and a Hungry World: An online course for Master Gardeners
S. D. ELLIS (1), P. J. Bennett (2), M. J. Boehm (1)
(1) The Ohio State University Department of Plant Pathology, Columbus, OH,
U.S.A.; (2) Ohio State University Extension State Master Gardener Program,
Springfield, OH, U.S.A.
Phytopathology 100:S33
What was the future in teaching and learning is now the reality. Online
courses can now be found on any topic and are not just for those seeking
college degrees, but also for those dedicated to lifelong learning. One group
who falls under the latter category are the Master Gardeners, who are
individuals from all over the United States that specialize in horticultural
topics and are required to earn continuing education credits each year. With
this in mind, a collaborative effort between The Department of Plant
Pathology and the Ohio Master Gardener Volunteers took shape. The
department took an online course currently offered asynchronously to Ohio
State students and transformed it into an online course specific to Master
Gardener Volunteers. The non-credit course entitled Sick Plants and a Hungry
World is offered through the free course management system Moodle and
covers topics ranging from the history of plant diseases to global issues
in plant pathology. Ten modules make up the content of the course where
little involvement from the instructor is needed. Students register through
the Office of Continuing Education at Ohio State and have ten weeks to
complete the self-paced course. Self-assessments allow students to test
themselves on the material. Cost of the course is $35. Over 200 individuals
from 16 states have registered for the course since its launch in March 2009.
Vol. 100, No. 6 (Supplement), 2010
S33
Those completing the course receive a certificate of completion from the
department.
Biological control of bacterial spot of tomato and capsicum caused by
Xanthomonas campestris and Pseudomonas solanacearum by bacteriophages in the UAE
K. A. EL-TARABILY (1), F. McKenna (2)
(1) United Arab Emirates Univ, Al-Ain, UAE; (2) Natural Science Center
Inc., Steele, AL, U.S.A.
Phytopathology 100:S34
Five different highly virulent and polyvalent phages were isolated from
tomato and capsicum rhizosphere soil in the United Arab Emirates (UAE)
using selective enrichment technique. These phages were screened for their
abilities to lyse in vitro Xanthomonas campestris and Pseudomonas
solanacearum, the causal agents of bacterial spot disease of tomato and
capsicum in the UAE. The presence of these pathogenic bacteria in the UAE
vegetable fields affects dramatically plant production and leads also to
environmental pollution due to the excessive bactericide application by the
commercial farmers. The phage suspension (x 109 pfu ml–1) was prepared in
especially designed mini bioreactor. These five phages were subsequently
tested in the greenhouse, individually or as a mixture, for their ability to
suppress the incidence of the disease. Antagonistic Streptomyces sp. which
was found to be resistant to each individual phage was used with the mixtures
of the five phages in order to improve the efficiency of the phages to reduce
the incidence of the disease. The treatment which included all five phages
combined with Streptomyces sp. was significantly superior to all other
treatments in suppressing the disease severity more than the application of
each phage alone or with the mixture of phages alone. Results showed that
there is a potential to use a mixture of phages combined with Streptomyces sp.
for the field management of tomato and capsicum bacterial spot disease in the
UAE.
Biological control of bean broomrape (Orobanche crenata) and hemp
broomrape (Orobanche ramosa) by Fusarium isolates
K. A. EL-TARABILY (1), M. Abouzeid (2)
(1) United Arab Emirates Univ, Al-Ain, UAE; (2) Ain Shams University,
Cairo, EGYPT
Phytopathology 100:S34
Thirty-nine Fusarium isolates were obtained from infected bean broomrape
(Orobanche crenata) and hemp broomrape (Orobanche ramosa) collected
from infested fields in Egypt. The effect of Fusarium culture filtrates on
germination of seeds of the two Orobanche species was tested in vitro. The
culture filtrates of Fusarium species isolated from O. crenata were found to
be more toxic to the seeds of both Orobanche species than those obtained
from O. ramosa. Seeds of O. crenata were shown to be more resistant to
Fusarium culture filtrates compared to those of O. ramosa. The highest
inhibition values in germination of Orobanche seeds were recorded for six
Fusarium isolates, one identified as F. oxysporum, one as F. equiseti and four
as F. compactum. Aqueous mixtures of mycelia and conidia of all Fusarium
isolates were directly sprayed on O. ramosa tubercles formed on roots of
tomato plants grown in transparent plastic bags and were used also to infest
soil in pots seeded with both faba bean and O. crenata. Two of the four F.
compactum isolates were significantly more pathogenic against O. crenata
and O. ramosa, respectively, compared to other Fusarium isolates tested in the
pots and plastic bags. This study clearly shows the potential to use biocontrol
agents originating from one Orobanche sp. (e.g. O. crenata) to control another
(e.g. O. ramosa) as many Fusarium isolates originating from O. crenata were
found to be more pathogenic to O. ramosa seeds than isolates originating from
O. ramosa.
Isolates of Phytophthora capsici differ in their ability to cause disease on
cucurbit fruits
T. B. ENZENBACHER (1), M. K. Hausbeck (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S34
Studies were undertaken to elucidate the differences in disease response
among eight types of cucurbit fruits and differences in virulence among five
unique isolates of Phytophthora capsici. Isolates differed in mating type,
mefenoxam sensitivity, and host origin. Unwounded, summer squash types
and cucumbers were inoculated with a 5-mm plug of mycelia and sporangia
from a 5-to 7-day-old culture and were incubated at room temperature and
high relative humidity under laboratory conditions. Hard squash types were
wounded with a sterile probe 3 to 5 mm below the fruit surface before being
inoculated and exposed to high humidity in a greenhouse environment. Fruits
were measured for lesion and pathogen sporulation diameter (cm) and
evaluated for sporulation density using a visual scale. All P. capsici isolates
used incited disease on cucurbit fruit with significant differences observed
S34
PHYTOPATHOLOGY
among the isolates and fruit type tested. This study suggests that multiple
isolates should be utilized in future cucurbit germplasm screenings for P.
capsici fruit resistance.
Resting spores for long-term storage of Synchytrium solstitiale, a
candidate for biological control of yellow starthistle
F. M. ESKANDARI (1), W. L. Bruckart (1), T. L. Widmer (1)
(1) USDA ARS FDWSRU, Fort Detrick, MD, U.S.A.
Phytopathology 100:S34
An isolate of Synchytrium solstitiale from France has been evaluated recently
for biological control of yellow starthistle (YST, Centaurea solstitialis).
Protocol was needed for long-term storage of S. solstitiale for research and
archival purposes. In greenhouse studies, germination of S. solstitiale resulted
with mature resting spores in dried YST leaves. Leaves were surface
sterilized, and resting spores were removed by scraping or grinding leaf tissue.
Spores were placed on 2% water agar in Petri dishes that were wrapped with
Parafilm and aluminum foil and incubated at 10/15°C (night/day
temperatures). One vesicle per resting spore, each with a single sporangium (=
sorus), developed in 7–20 days. Zoospores were released from sori in sterile
distilled water with 100 ppm Streptomycin. Plants also inoculated with sori
from resting spores were incubated in moist plastic bags at 10/15°C (night/day
temperatures) and an 8-hour photoperiod. Plants were removed from the
growth chamber after 10 days, placed in a 20°C greenhouse, and observed for
symptom development. Successful germination and plant infection occurred
from inoculation by resting spores following this protocol. A test set up to
measure viability and virulence of resting spores after 2, 3, or 4 years of
storage resulted in successful germination and infection of YST plants using
the protocol described. Thus, long-term storage and maintenance protocol for
S. solstitiale has been achieved.
Effect of foliar fungicides on hail damaged corn in Wisconsin in 2009
P. D. ESKER (1), N. C. Koval (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S34
On 24 July 2009, two trials located at Lancaster, WI were impacted by 0.6 to
1.3 cm-sized hail. In both trials, plants were at the tasseling into silking
growth stages. The first trial was established to study the effect of fungicide
timing (V5-V6, R1, or combinations) on corn disease development (all
diseases) and yield and the second to study the effect of corn hybrid (n = 6)
and fungicide on corn anthracnose and grain yield. Both trials had four
replications. Trials were sprayed on 29 July at R1. In the first trial, fungicides
included pyraclostrobin, azoxystrobin+propiconazole, and propiconazole+trifloxystrobin. Only pyraclostrobin was applied in trial two. Late season
disease measures included early evidence of ear rot, top dieback, lodging due
to anthracnose, and common smut. Corn plants (n = 5) were also destructively
sampled for stalk assessments (0–5 rating scale). Yield measures were
moisture, test weight, and grain yield (adjusted to 15.5% moisture). In trial
one, there was no evidence of a difference among treatments (P > 0.05) for
late season diseases or yield. Grain yield ranged from 93 to 141 bushels per
acre (CV = 25.1%) and the increased variability was attributed to hail. In trial
two, there were differences among treatments (P < 0.05), but these were
primarily a function of corn hybrid and no differences in yield were observed.
These results suggest that hybrid selection is still the primary factor to
consider for corn disease management.
Citrus greening in commercial orchards in Puerto Rico
C. ESTEVEZ DE JENSEN (1), A. Vitoreli (2), F. Roman (3)
(1) University of Puerto Rico, Juana Diaz, U.S.A.; (2) Plant Disease
Clinic/University of Florida, Gainesville, FL, U.S.A.; (3) University of Puerto
Rico, Mayaguez, U.S.A.
Phytopathology 100:S34
Citrus greening (CG) associated with Candidatus Liberibacter asiaticus, was
recently identified in Puerto Rico. Symptomatic and asymptomatic leaves of
sweet oranges (cv. Valencia and Washington Navel), Lime (cv. Tahiti),
grapefruit and mandarin were sampled in Castañer, Juana Diaz, Yahuecas and
Isabela Municipalities. Standard Polymerase Chain Reaction (PCR) was
performed on DNA extractions using primers OI1 and OI2. The 16S rDNA
fragments with molecular weight of 1160 bp were amplified in an agarose gel
at 1.5% and corresponded to the bacterium Candidatus Liberibacter asiaticus.
Sequencing of the PCR products from Isabela confirmed amplification of Ca.
L. asiaticus DNA. Sweet oranges and mandarins were severely affected at
Isabela where tree decline was observed. In Castañer in a 600 acre orchard
two out of 25 samples were positive for the disease. In a three year old Thaiti
lime orchard in Juana Diaz, symptoms developed from mottled areas and
yellowed shoots to stem and limb dieback within five months in 81 out of 352
trees. Ten samples out of 25 samples were positive for C. L. asiaticus. A
survey of citrus greening and implementation of IPM practices to prevent the
spreading of the disease are needed.
Fungal and oomycete pathogens associated with crown and root diseases
of strawberry in Western Australia
X. L. FANG (1), D. Phillips (2), H. Li (1), K. Sivasithamparam (3), M.
Barbetti (4)
(1) University of Western Australia, Perth, AUSTRALIA; (2) Department of
Agriculture and Food Western Australia, Midland, AUSTRALIA; (3)
University of Western Australia, Nedlands, AUSTRALIA; (4) University of
Western Australia, Crawley, AUSTRALIA
Phytopathology 100:S35
Strawberries are a high-value export crop in Western Australia (WA),
constituting more than 70% of Australia’s strawberry exports. Crown and root
diseases pose an increasing challenge to strawberry production in WA, with
more than 1 m plants p.a. dying from such diseases. Field surveys undertaken
in 2008 showed that the percentage plant decline indices (%DI) across all sites
ranged from 3 to 40. The mean level of plant decline across all sites rose
sharply from a %DI of 13 in August to 39 in October. Based on
morphological and molecular identification, the potential fungal and oomycete
pathogens associated with crown and root diseases were Fusarium oxysporum,
Rhizoctonia (R. solani, Ceratobasidium AG-A, AG-C, AG-I and other taxa of
Ceratobasidium), Cylindrocarpon destructans, Phoma exigua, Gnomonia
fructicola, Phytophthora cactorum, Pythium ultimum and Macrophomina
phaseolina. F. oxysporum was most frequently isolated from crowns, at a
frequency of 41%, and its incidence was strongly correlated with the severity
of crown disease. Rhizoctonia and C. destructans were most frequently
isolated from roots, at a frequency of 12% for each. There was a poor
relationship between the incidence/severity of crown disease and root disease.
This work not only demonstrates that strawberry production in WA is severely
compromised by crown and root diseases, but implicates Fusarium wilt in
particular as the major disease associated with the extensive plant deaths
occurring in WA.
Identification and characterization of resistance to Meloidogyne incognita
in wild species of Cucurbitaceae
T. FASKE (1)
(1) Tarleton State University, Stephenville, TX, U.S.A.
Phytopathology 100:S35
Resistance of four wild species of Cucurbitaceae to Meloidogyne incognita
was evaluated in two greenhouse experiments. The objectives were to find
new sources of resistance and determine the mechanism of resistance among
resistant species. Wild species included Cucumis melo var. dudaium, C. melo
var. texanus, C. dipsaceus, and Cucurbita fastidissima, which are commonly
found in the southern U.S. Each entry was inoculated at the first true leaf stage
with 1,000 nematodes per 500 cm3 of soil. Only C. melo var. dudaium, C.
melo var. texanus supported less (P = 0.05) reproduction than the susceptible
control, C. sativus ‘Straight eight’. Resistance in these species was as great as
C. metuliferus, a resistant species. The mechanism of resistance in these
species was attributed to fewer (P = 0.05) juveniles penetrating root tips and
delayed (P = 0.05) maturity of juveniles into mature females, than the
susceptible control. A similar mechanism of resistance was observed in C.
metuliferus. Cucumis melo var. dudaium and C. melo var. texanus may be
useful sources of resistance to M. incognita in honeydew and muskmelon.
Screening of organically certifiable fungicides and natural compounds to
control anthracnose caused by Colletotrichum orbiculare in cantaloupe
M. FELICIANO-RIVERA (1)
(1) University of Kentucky, Lexington, KY, U.S.A.
Phytopathology 100:S35
Control options for management of cucurbit anthracnose in organic
production are poorly developed. Thus, the objective of this research was to
investigate potential disease control obtained with natural, organically
certifiable spray materials against Colletotrichum orbiculare in vitro and in
vivo. Materials tested included: essential oils, bicarbonate salts, commercial
products and chitosan. Antifungal activity was evaluated in vitro with 96-well
plates using absorbance (450 nm) to measure mycelial growth, and/or by
observing spore germination. In vivo experiments were performed under
greenhouse conditions. Treated plants were inoculated, and disease severity
was recorded using APS Assess Software 2.0. In vitro, bicarbonate salts
(KHCO3, NaHCO3 and NH4HCO3) provided >50% inhibition at 0.25 molarity, while Bordeaux®, Kocide 2000® and SoilGard 12G® inhibited mycelial
growth by >70% at concentrations ≥50 µg/ml. Horticultural lime sulfur
completely inhibited spore germination at 2.5 µg/ml. Over 65% inhibition was
obtained using chitosan at concentrations ≥100 µg/ml. In vivo, NH4HCO3,
Serenade Max®, Bordeaux®, Kocide 2000®, SoilGard 12G®, Horticultural
lime sulfur and chitosan provided >85% disease control and were statistically
significant different from the non-treated plants (P < 0.05). None of the
essential oils provided a significant reduction (P > 0.05) in disease. These
results suggest the potential for use of these organically certifiable fungicides
and natural compounds to control anthracnose in cucurbit.
A spinach BAC library for marker development, gene discovery, and
functional genomics
C. FENG (1), J. C. Correll (1), B. H. Bluhm (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S35
Spinach production has increased dramatically during the past two decades in
the U.S. Downy mildew remains the most economically important disease of
spinach and improving genetic resistance to downy mildew is a high priority
in all spinach breeding programs. A bacterial artificial chromosome (BAC)
library was constructed from a spinach near-isogenic line harboring the downy
mildew resistance locus RPF1. The library contains 73,728 clones with an
average insert size 183 Mb, thus providing approximately 13X coverage of the
989 Mb spinach genome. An initial examination of 3535 BAC-end sequences
identified gene sequences encoding conserved proteins such as kinases,
endonucleases, dehydrogenases, ATPases, cellulose synthases, droughtinduced proteins, low-temperature inducible proteins, permeases, germination
proteins, zinc-finger proteins and other transcription factors. Over 120 BACend sequences contained simple sequence repeats (SSRs), including the di[(AT)n, (AG)n, and (AC)n], tri- [(AAC)n, (GAT)n, and (CTT)n], tetra[(TTTA)n, (GTTG)n, (ACAT)n], and penta- (TAGAC)n nucleotide repeats. Diand tri-nucleotide repeats were most prevalent. Polymorphic repeats were
detected among selected cultivars, and heterozygosity of some loci was
detected. This BAC library will assist with gene discovery, the development
of markers linked to important traits such as disease resistance, and the
integration of genetic and physical maps with genomic sequence.
The CYP51C gene, a novel marker for phylogenetic analysis of Fusarium
species
D. Fernandez-Ortuno (1), E. Loza-Reyes (1), S. L. Rogers (1), B. A.
FRAAIJE (1)
(1) Rothamsted Research, Harpenden, UNITED KINGDOM
Phytopathology 100:S35
The CYP51 gene encodes sterol 14α-demethylase, a key enzyme in the
pathway leading to ergosterol, phytosterol and cholesterol biosynthesis in
fungi, plants and mammals, respectively, and the target of triazole fungicides.
Common resistance mechanisms towards triazoles are CYP51 mutation and
over-expression. Some filamentous ascomycetes, including Aspergillus
fumigatus and Rhynchosporium secalis, have two CYP51 genes, CYP51A and
CYP51B. However, a third copy, CYP51C, has only been identified in
Fusarium species and appears to be unique to this genus. In this study, we
investigated if the Fusarium-specific CYP51C gene can be used as an
informative marker to differentiate Fusarium species present in cereals.
Molecular and phylogenetic analyses based on 46 CYP51C sequences of 18
Fusarium species revealed sufficient sequence variability to differentiate
between them. The species-dependent separation showed clear correlation
between major phylogenetic lineages and abilities to synthesise particular
classes of toxins. The interspecific divergence was used to design speciesspecific primers. The resulting PCR assays differentiated F. asiaticum/F.
vorosii, F. cerealis, F. equiseti and F. poae from other members of the
Fusarium head blight complex. Early detection and control of different
Fusarium spp. is crucial to prevent toxins entering the food chain and a useful
tool in disease management practices.
Bacterial communities associated to Eucalyptus plants infected by
Ceratocystis fimbriata
A. FERREIRA (1), E. R. Gonzáles (2), F. D. Andreote (3), J. Azevedo (1), W.
L. Araújo (4)
(1) Department of Genetics, Escola Superior de Agricultura “Luiz de
Queiroz”, University of São Paulo, Piracicaba, SP, BRAZIL; (2) Suzano
Bahia Sul Papel e Celulose S/A, Suzano, SP, BRAZIL; (3) Embrapa Meio
Ambiente, Jaguariúna, SP, BRAZIL; (4) Laboratory of Molecular Biology and
Microbial Ecology, NIB, University of Mogi das Cruzes, SP, Mogi das
Cruzes, SP, BRAZIL
Phytopathology 100:S35
Brazil is one of the largest producers of Eucalyptus in the world, and the
demand for this wood has increasing in the last few years. The expansion of
the cultivation area results in changes of factors influencing eucalyptus growth
and incidence of some pathogens such as Ceratocystis fimbriata. The
pathogen-plant interaction may be affected by host ecology and associated
microbial communities. Therefore, we used C. fimbriata infected plants with
four infestation symptoms to assess the effects of this infestation on
rhizoplane and endophytic bacterial communities. The culturable bacterial
diversity was assessed by isolation and 16S rRNA gene characterization by
ARDRA and sequencing. Additionally, total bacterial diversity was assessed
by the culture-independent approach DGGE. Results showed that healthy
plants presented a higher bacterial density in the rhizoplane, while the
endophytic community was higher in infected plants. Thirteen and eight
ARDRA ribotypes were observed in the cultured bacteria isolated from
Vol. 100, No. 6 (Supplement), 2010
S35
rhizoplane and endosphere of roots, respectively. The most frequent species
was Bacillus cereus. However, only the occurrence of species Pseudomonas
fluorescens, P. veronii and Rahnella aquatilis were correlated with less
disease occurrence. The DGGE analysis showed that the pathogen infestation
interferes in the composition of the assessed bacterial communities. Principal
Components Analysis (PCA) on the basis of DGGE band patterns separated
samples in the four stages of disease infection. The results obtained in the
present work provide information about the pathogenic-associated
microorganisms and show important features related to C. fimbriata infection.
ITS sequence analysis of Brazilian Stenocarpella macrospora and
Stenocarpella maydis isolates
A. FESSEHAIE (1), G. Munkvold (2), E. Alves (3)
(1) Seed Science Center, Iowa State University, Ames, IA, U.S.A.; (2) Dept.
of Plant Pathology, Iowa State University, Ames, IA, U.S.A.; (3) Pioneer
Sementes Ltda., Rodovia GO 210 km 04, BRAZIL
Phytopathology 100:S36
Stenocarpella macrospora and S. maydis cause ear rot, stalk rot, and leaf
diseases in maize. These diseases can cause considerable yield losses in maize
in warm, humid environments. The objective of this work is to assess the
occurrence and distribution of Stenocarpella species in maize producing
regions of Brazil and evaluate variability among isolates based on geographic
origin. Stenocarpella species were isolated from maize kernels and leaf tissues
obtained from representative locations, with two independent isolations made
from each kernel and leaf sample. Pure cultures were identified based on
morphological characteristics and the analyses of nucleotide sequence of the
rDNA ITS regions. Stenocarpella species were isolated from 100% (12 of 12)
and 37.5% (5 of 16) of the kernel and leaf samples, respectively. S.
macrospora occurred in nine of the twelve kernel samples as a sole
contaminant. Two samples were infested by both species and only one sample
was infested by S. maydis alone. From maize leaf samples, only S.
macrospora was recovered. Phylogenetic analyses based on the rDNA ITS
sequence data revealed that S. macrospora isolates formed a homogenous
cluster regardless of geographic origin. S. maydis isolates grouped in a
separate distinct cluster. S. macrospora was more prevalent as a pathogen of
maize in Brazil. S. maydis occurred only in samples from southern Brazil.
Real-time PCR to measure head smut infection in maize seedlings
A. FESSEHAIE (1), G. Munkvold (2)
(1) Seed Science Center, Iowa State University, Ames, IA, U.S.A.; (2) Dept.
of Plant Pathology, Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S36
Head smut caused by Sporisorium holci-sorghi can be a devastating disease
on every continent where maize is grown. The disease can cause severe yield
loss by replacing the reproductive parts of the plant with fungal sori.
Evaluating control methods or genetic resistance for maize head smut is
difficult because of the need for field trials that are slow, costly, and highly
variable. The main purpose of this study was to develop a real-time PCR assay
to evaluate maize seedlings for S. holci-sorghi infection that occurs during
germination and emergence. We report a novel real-time PCR assay for the
detection and quantification of S. holci-sorghi in maize seedlings. Specificity
of the forward primer used in this study was reported previously. Sensitivity
of the assay was determined using serial dilutions of genomic DNA extracted
from pure teliospores, maize leaf tissue spiked with spores and naturally
infected maize plant tissue. The detection limit of the assay for purified DNA
from teliospores was 250 fg per 25 µl PCR reaction mixture. PCR detected
target DNA extracted from tissues spiked with teliospores and from naturally
infected maize tissue at minimum quantities of 2.5 pg and 25 pg, respectively.
The new real-time PCR assay is sensitive and simple; it could be useful for
more rapid evaluation of fungicide seed treatments or genetic resistance for
management of S. holci-sorghi in maize.
Leaf blotch disease complex in Norwegian wheat
A. FICKE (1), O. Elen (1), U. Abrahamsen (1), G. Brodal (1)
(1) Bioforsk Norwegian Inst of Agric & Env Res, Aas, NORWAY
Phytopathology 100:S36
The leaf blotch disease complex (LBD) frequently reduces yield of wheat in
Norway. In visual assessments field symptoms can be difficult to attribute
definitively to specific causal agents, and may be caused by any or all of the
following three pathogens: Stagonospora nodorum (teleomorph
Phaeosphaeria nodorum) causing Stagonospora Nodorum or glume blotch
(SNB), Septoria tritici (teleomorph: Mycosphaerella graminicola) causing
Septoria tritici or speckled leaf blotch (STB), and Drechslera tritici-repentis
(teleomorph Pyrenophora tritici-repentis causing tan spot (DTR). There is no
broad resistance to all three pathogens in commercially relevant wheat
varieties. We analyzed 9 years of historical data on severity of LBD in the
field and 36 years of historical data on post-harvest SNB infection of wheat
kernels. Overall, correlation between leaf severity and seed severity over years
S36
PHYTOPATHOLOGY
was low (r = 0.5). However, during the last 4 years correlation between SNB
seed infection and severity of LBD increased (r = .825). LBD severity varied
significantly with geographic location and increased exponentially on the last
3 leaves between BBCH stage 70 and the last assessment at BBCH stage 89.
An improved understanding of environmental and host developmental factors
as they affect each member of the LBD complex in the field will be essential
to screening for quantitative and durable resistance to LBD.
AvrGf1 from Xanthomonas citri subsp. citri strain Aw targets the
chloroplast in grapefruit leaves
J. FIGUEIREDO (1), N. Potnis (1), P. Romer (2), T. Lahaye (2), J. Jones (1)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) Martinsried,
GERMANY
Phytopathology 100:S36
A related strain of Xanthomonas citri subsp. citri strain A that causes Asiatic
citrus canker, X. citri subsp. citri strain Aw (Xcc-Aw) isolated in Florida, is
pathogenic to Key lime but not grapefruit or orange. Infiltration of Xcc-Aw in
grapefruit leaves triggers a hypersensitive response (HR). This defense
response results from the recognition of a type III effector, AvrGf1, by an
unknown “resistance” gene in grapefruit. Analysis of the AvrGf1 amino acid
sequence using neural-network based predictors PCLR, ChloroP and LOCtree
to determine the subcellular localization, predicted a chloroplast transit signal
at the N-terminus within the first 87 amino acids. To test this prediction, we
constructed a T-DNA binary vector expressing AvrGf1 driven by the 35S
constitutive promoter and C-terminally tagged GFP. Chloroplast localization
was visualized in leaves six days after infiltration using confocal laser
scanning microscopy. AvrGf1-GFP is visibly concentrated in stomatal guard
cells in which chloroplasts are abundant. Two mutants carrying deletions at
the N-terminus disrupting the putative chloroplast signal was designed,
Gf1ΔN116, which 116 amino acids were deleted, abolished GFP expression in
grapefruit leaves and HR induction. However, the mutant Gf1ΔN13, with 13
amino acids deleted, resulted in HR elicitation but not GFP expression. Thus,
we suggest that disruption of the putative chloroplast signal, even in the few
first amino acids, is essential for the subcellular localization of AvrGf1
protein.
Screening of St. Augustinegrass (Stenotaphrum secundatum) germplasm
for brown patch and large patch resistance
N. C. FLOR (1), P. F. Harmon (1), L. E. Datnoff (2), R. N. Raid (3)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) Department of Plant
Pathology & Crop Physiology, Louisiana State University, Baton Rouge, LA,
U.S.A.; (3) University of Florida, Belle Glade, FL, U.S.A.
Phytopathology 100:S36
St. Augustinegrass (SAG) is a warm season grass commonly used in the
United States. Large patch disease, caused by Rhizoctonia solani AG-2-2 LP,
is considered one of the most important fungal diseases of SAG. While brown
patch disease, caused by other anastomosis groups of R. solani primarily
affects cool season grasses, but does occasionally occur on warm season
grasses as well. Differences in large patch resistance in SAG cultivars has
been observed but never quantified. One BP and one LP isolate from St.
Augustinegrass were chosen to screen twenty genotypes of SAG. Pots of SAG
plants were inoculated with mycelial plugs and maintained in an incubator at
23 to 26°C. Disease progress on each genotype was assessed daily and the
intrinsic rates of infection were calculated. AUDPC also was calculated for
each genotype. Trials were replicated, randomized, and repeated. Means were
separated after ANOVA with a Waller-Duncan k-ratio t-test. Results indicated
that some genotypes were more resistant to large patch and brown patch than
others, but results did not correlate between isolates for all genotypes. In
general, dwarf genotypes of SAG developed more disease than standard
height genotypes. Ploidy level within genotype did not significantly impact
resistance. Future screening efforts should focus on large patch isolates of R.
solani. Additional work is needed to further characterize brown patch isolates
from warm season turfgrass and to determine the potential impact of this
disease.
Hormetic effect of cyazofamid on the radial growth of Pythium
aphanidermatum
F. FLORES (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S36
Hormesis is an adaptive biphasic response of an organism, in which low doses
of an inhibitory agent stimulate, while higher doses suppress a determined
endpoint. Scarce studies have focused on the occurrence of hormesis in plant
pathogens. The hormetic effects of low doses of pesticides may be relevant to
disease management if partial application results in stimulatory dosages at
some spatial scales. The aim of this research was to assess the effect of small
doses of the pesticide cyazofamid on the radial growth of Pythium
aphanidermatum in vitro. Petri dishes containing solid growing media
amended with different doses of the pesticide were inoculated with a 5 mm
diameter plug colonized by actively growing mycelium. Seven of the nine
doses tested were at subinhibitory concentrations. Non-amended control plates
were also inoculated. The radial growth of mycelial colonies was measured
after 24 h incubation at 28°C. Five replicates for each treatment were
performed with five repetitions over time. The dose response curve, inferred
EC50 and the non observable adverse effect limit (NOAEL) were analyzed
using a modified log-logistic model. Radial growth stimulation up to 13%
occurred when P. aphanidermatum was exposed to concentrations between
0.07 and 0.13 ppb cyazofamid. These observations provide the groundwork
for future studies confirming the occurrence of such responses in plant
production systems.
Luna fungicides for the control of diseases of horticultural crops
L. FOUGHT (1), G. H. Musson (2), H. Young (2)
(1) Bayer CropScience, Fresno, CA, U.S.A.; (2) Bayer CropScience, Res
Triangle Park, NC, U.S.A.
Phytopathology 100:S37
Luna fungicides are a family of horticultural fungicide products in
development by Bayer CropScience. Luna products are based on the new
active ingredient fluopyram. Four mixture products have also been submitted
to the U.S. EPA as combinations with other active ingredients: trifloxystrobin,
pyrimethanil, tebuconazole, and prothioconazole. The mixtures have
demonstrated excellent crop safety and outstanding control of a broad range of
major foliar and fruit diseases such as powdery mildew, brown rot blossom
blight, early blight, gray mold, and scab. Multiyear trial results will be
presented.
Evaluation of the expression of genes associated with resistance to
Aspergillus flavus colonization and aflatoxin production in different
maize lines
J. C. Fountain (1), Z. Chen (2), B. Scully (1), R. C. Kemerait (3), R. L. Dewey
(4), B. GUO (1)
(1) USDA-ARS, Crop Protection and Management Research Unit, Tifton,
GA, U.S.A.; (2) Department of Plant Pathology and Crop Physiology,
Louisiana State University, Baton Rouge, LA, U.S.A.; (3) Department of
Plant Pathology, the University of Georgia, Tifton, GA, U.S.A.; (4) Department of Crop and Soil Sciences, the University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S37
Aflatoxins are carcinogenic toxic compounds produced by Aspergillus flavus
during infection of crops including maize (Zea mays L.). Contamination of
maize with aflatoxin is exacerbated by late season drought stress. Previous
studies have implicated numerous resistance-associated proteins (RAPs) that
may be responsible for resistance to A. flavus colonization and aflatoxin
accumulation. This study examined the expression of three genes encoding
RAPs, ZmPR-10 (PR-10), glyoxalase I (GLX-I), and a 14-kDa trypsin
inhibitor (TI-14), in different maize genotypes under drought stressed and
irrigated conditions to determine their potential utility as molecular markers
for germplasm screening and evaluation utilizing quantitative real-time PCR.
Results suggested that drought stress during kernel development affected gene
expressions differently in different genotypes. Results were generally consistent with expectations in that RAP-coding gene expressions correlated well to
known resistant traits of the examined genotypes. However, more genotypes
should be studied in order to apply these genes’ expression as selection markers.
Characterization of canker resistance in citrus plants created by ‘somatic
cybridization’
M. I. FRANCIS (1), A. Pena (1), J. W. Grosser (1), J. H. Graham (1)
(1) University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S37
Cybrids are an asymmetric hybrid that contain the nucleus of one parent in
combination with the mitochondrial and/or chloroplast genome of a second
parent. Mitochondria and chloroplasts have a central regulatory role in
integrating stress and/or programmed cell death signaling. The model cybrid
(RL+MK) for evaluation was a hybrid between susceptible ‘Rough’ lemon
(RL, Citrus jambhiri) and resistant ‘Meiwa’ kumquat (MK, Fortunella
crassifolia.) Resistant MK developed a hypersensitive response (HR) with
necrotic lesions and low population of Xanthomonas citri subsp. citri (Xcc) in
detached leaf and in attached leaf assays. Early expression of genes related to
programmed cell death was identified in MK. RL+MK cybrid produced an
intermediate reaction and Xcc population response between susceptible and
resistant parents at low Xcc inoculum (105 cfu/ml) and necrosis indicative of a
resistant reaction at high Xcc inoculum (108 cfu/ml). To validate the
inheritance of resistance from MK, 22 cybrids of ‘Ruby red’ grapefruit (RG)
with MK were compared to both parents using detached leaf assays. RG+MK
cybrids had significantly lower lesion number per inoculation site (20 to
88%), compared to the RG parent. Xcc populations in RG+MK cybrids varied
from 7 to 2.2 log units. Thus, resistance in cybrids may be inherited at
different levels depending which sets of genes contained in the mitochondrion
or chloroplast genomes are transferred to RG in the cybridization process.
Mutations in the target protein of succinate-dehydrogenase inhibitors
(SDHI) conferring changes in fungicide sensitivity
M. FRANK (1), A. Glaettli (1), S. Schlehuber (1), G. Stammler (1)
(1) BASF S.E., Limburgerhof, GERMANY
Phytopathology 100:S37
Fungicide resistance of fungal plant pathogens is mostly based on point
mutations in the target proteins. Monitoring of the sensitivity of different
target pathogens of the SDH Inhibitor Boscalid was carried out in the recent
years. While in most species the sensitivity of all isolates were within the normal
or baseline range, cases of resistance were found e.g. in B. cinerea (Stammler
et al., 2007), Corynespora cassiicola (Ishii, 2008) or Alternaria alternata
(Avenot et al., 2008). Target gene analysis of such isolates showed mutations
in the subunits of the SDH B, C and D. The current status of SDHI sensitivity
in the field will be reported. Moreover, point mutations identified in field
isolates of various fungi are reviewed and their structural implications and effect
on fungicide binding is discussed based on three-dimensional protein models.
Gene expression during appressorium formation by Phakopsora pachyrhizi
R. D. FREDERICK (1), C. L. Stone (1)
(1) USDA ARS FDWSRU, Fort Detrick, MD, U.S.A.
Phytopathology 100:S37
Phakopsora pachyrhizi, which causes Asian soybean rust (ASR), has spread
from southern Asia and Australia to Africa and South America, and more
recently to North America. At present, U.S. commercial soybean cultivars do
not have any resistance to ASR. To develop novel methods for controlling this
disease, it is important to understand the molecular processes that occur
throughout the infection cycle. This study examined gene expression during
appressorium formation, which is required for the pathogen to breach the leaf
surface. An appressorium-enriched cDNA library was constructed with
mRNA extracted from appressoria produced by germinating urediniospores on
polystyrene plates and subtracting with mRNA extracted from urediniospores
germinated on water. A total of 1152 cDNA clones were sequenced and
compared to P. pachyrhizi germinating urediniospore ESTs, and 31 clones
were found only in the appressorium-enriched cDNA library. BlastX analysis
revealed sequence similarity to known proteins for 20 clones, and identified
three clones as hypothetical proteins. Eight clones showed no significant
similarity to protein sequences in GenBank. Genes identified in this study fell
into functional categories of metabolism, cell cycle and DNA processing,
protein fate, cellular transport, cellular communication and signal transduction, and cell rescue.
Alteration in lignin biosynthesis restricts growth of Fusarium species in
brown midrib sorghum
D. L. FUNNELL-HARRIS (1), J. F. Pedersen (1), S. E. Sattler (1)
(1) USDA ARS, Lincoln, NE, U.S.A.
Phytopathology 100:S37
To improve sorghum for bioenergy and forage uses, brown midrib6 (bmr6)
and bmr12 near-isogenic genotypes were developed in different sorghum
backgrounds. bmr6 and bmr12 grain had significantly reduced colonization by
members of the Gibberella fujikuroi species complex, compared with wildtype, as detected on two semi-selective media. Fusarium species were identified using sequence analysis of a portion of the translation elongation factor 1α gene (TEF). The pathogens Fusarium thapsinum, Fusarium proliferatum
and Fusarium verticillioides, G. fujikuroi members, were commonly recovered. Other frequently isolated Fusarium species likely colonize sorghum
asymptomatically. Chi-square analyses showed that the ratios of Fusarium
species colonizing bmr12 grain were significantly different from wild-type,
indicating that bmr12 affects colonization by Fusarium spp. One Fusarium
incarnatum/equiseti species complex (FIESC) genotype, commonly isolated
from wild-type and bmr6 grain, was not detected in bmr12 grain. Phylogenetic
analysis suggested that this FIESC genotype represents a previously
unreported TEF haplotype. When peduncles of wild-type and near-isogenic
bmr plants were inoculated with F. thapsinum F. verticillioides, or Alternaria
alternata, the resulting mean lesion lengths were significantly reduced relative
to wild-type in one or both bmr mutants. This indicates that impairing lignin
biosynthesis results in reduced colonization by Fusarium spp. and A. alternata.
Soil and rhizosphere populations of fluorescent Pseudomonas spp.
associated with field-grown plants are affected by sorghum genotype
D. L. FUNNELL-HARRIS (1), J. F. Pedersen (1), S. E. Sattler (1)
(1) USDA ARS, Lincoln, NE, U.S.A.
Phytopathology 100:S37
Sorghum [Sorghum bicolor (L.) Moench] is valued for bioenergy, feed and
food. Potential of sorghum genotypes to support differing populations of rootVol. 100, No. 6 (Supplement), 2010
S37
and soil-associated fluorescent Pseudomonas spp. or Fusarium spp., in two
soils, was assessed. Pseudomonads and Fusarium spp. were assessed from
roots and soil of field-grown sorghum genotypes Redlan and RTx433, along
with biological control traits including hydrogen cyanide (HCN) and 2,4diacetylphlorogluconol (phl) production. In dryland field conditions, RTx433
roots had greater numbers of pseudomonads than Redlan before anthesis but
similar numbers after. There were no differences in numbers of
pseudomonads from dryland soil or roots or soil of irrigated plants.
Percentages of HCN-producing root isolates and phl soil isolates declined on
irrigated Redlan plants, but percentages of HCN-producers increased in
dryland conditions. Redlan roots had greater percentages of Fusarium isolates
in the Gibberella fujikuroi species complex. Results indicated that sorghum
genotype affected rhizosphere populations of fluorescent Pseudomonas spp.
and Fusarium spp. across soil environments.
inhibit the growth of several bacteria, fungi and oomycetes. Activity was
detected against the taxonomically related Clavibacter michiganensis subsp.
nebraskensis, a corn pathogen causing Goss’ wilt and blight, as well as
Pantoea stewartii subsp. stewartii (Pss) strains, fungi including Rhizoctonia
solani, Sclerotinia sclerotium, Bipolaris sorokiniana and Fusarium graminearium, and an oomycete (Phythium ultimum). Inhibition zones were detected
after two days at 28°C, and varied in size and appearance. Thus, the endophytes
produced both bacteriocin(s) and one or more other antimicrobial compounds.
Density dependent latency in the grapevine powdery mildew (Erysiphe
necator)
D. M. GADOURY (1), M. M. Moyer (2), R. C. Seem (2), W. F. Wilcox (2),
L. M. Wakefield (2), P. A. Magarey (3)
(1) Cornell University, Geneva, NY, U.S.A.; (2) Cornell University NYSAES,
Geneva, NY, U.S.A.; (3) Magarey Plant Pathology, Loxton, SA 5333,
AUSTRALIA
Phytopathology 100:S38
Cyst nematodes, major pathogens of soybean, potatoes, sugar beets, and other
crops, are sedentary root feeders that limit the size of ingested molecules by
use of an organelle-like feeding tube believed to act as a “molecular sieve”
between the nematode stylet and the surrounding syncytial plant feeding site.
The feeding tube constrains the delivery of proteins and nucleic acids from the
plant to the nematode. The size cut-off for molecular uptake into the soybean
cyst nematode (SCN), Heterodera glycines, has never been determined and
work on other cyst nematode species provides conflicting values between 20
and 40 kDa. We have therefore studied the uptake of variously sized
fluorescent proteins expressed in transgenic roots into H. glycines in
comparison to another sedentary endoparasite, root knot nematode
(Meloidogyne incognita) and the migratory lesion nematode (Pratylenchus
scribneri). We provide the first evidence for uptake of plant-expressed
fluorescent proteins into the SCN intestine and describe this uptake by
developmental stage. We show how the likelihood of uptake into the intestine
decreases with molecular weight establishing a molecular size cut-off for
ingestion. The results obtained from this study clarify and expand
understanding of SCN host-plant feeding and provide guidance for the
development of biotechnology-based strategies for nematode control.
Duration of the latent period in E. necator has been estimated in previous
studies by depositing relatively large numbers or dense aggregations of
conidia by various means upon relatively small areas of host tissue. In natural
epidemics, inoculum is more commonly dispersed on air currents and arrives
at the infection court as a single conidium or ascospore. Vitis vinifera
‘Chardonnay’ was inoculated using 5 mml droplets of a conidial suspension
containing 1 to 250 germinable conidia. Under the most favorable
environmental conditions and upon the most susceptible leaves, the latent
period was relatively constant above 10 spores per inoculation point, but as
density approach 1 spore per point the latent period increased by more than
50%. Unrealistically dense inoculation will therefore yield unrealistically
short latent periods. Under field conditions, the longer latent periods
interacted with ontogenic resistance of leaves, whose aging during latency
further delayed sporulation. Mortality of hyphae in nascent colonies due to
environmental factors or fungicide effects could also delay sporulation by
reducing colony density below a critical level. Conversely, dense colonization
typically observed on shoots emerging from dormant infected buds (flag
shoots) could minimize the latent period. Thus there may be qualitative as
well as quantitative differences between epidemics developing from
ascosporic inoculum compared to flag shoots.
Broad spectrum virus resistance using oligoadenylate (OAS) system
L. C. GALVEZ (1), Z. Zhang (2), A. Mitra (1)
(1) University of Nebraska Lincoln, Lincoln, NE, U.S.A.; (2) University of
Missouri, Columbia, MO, U.S.A.
Phytopathology 100:S38
Tobacco SR1 and Benthamiana plants expressing mammalian oligoadenylate
system (OAS) system exhibit resistance to tobacco etch virus (TEV),
cucumber mosaic virus strain D (CMV-D) and cucumber mosaic virus strain y
(CMV-y). The system is composed of two enzymes, a 2,5A synthetase that
produces 5’-phosphorylated, 2’,5’-linked oligoadenylates (2-5A) upon
activation by viral dsRNA, and a 2-5A-dependent RNase L. Transformed
plants individually carrying the two enzymes were crossed and the progenies
were tested against TMV, TEV and CMV. Detached leaf assays of plants
carrying the two enzymes showed necrotic lesion formation which is a
manifestation of hypersensitive response (HR) against the virus infection. On
the other hand, plants with RL construct alone showed a typical systemic
infection. It was interesting that SR1 plants carrying 2,5A alone also
manifested hypersensitive response against TEV. Transgenic soybean carrying
the OAS system was also evaluated for resistance against soybean mosaic
virus (SMV) and bean pod mosaic virus (BPMV). Transformed soybean leaf
protoplasts inoculated with SMV were ELISA negative whereas protoplasts
from control soybean showed normal viral replication. The OAS system has
the potential to provide broad spectrum resistance against all + RNA and
dsRNA viruses.
Production of antimicrobial compounds by endophytic bacteria
L. C. GALVEZ (1), A. K. Vidaver (1)
(1) University of Nebraska Lincoln, Lincoln, NE, U.S.A.
Phytopathology 100:S38
The in vitro inhibition assay for testing endophyte strains (Vidaver, 1972),
was used to screen for production of antimicrobial compounds. The capability
of endophytic bacteria to produce antimicrobial compounds is a major
mechanism by which they qualify as candidates biocontrol agents. This study
showed that antimicrobial compounds produced by Gram-positive endophytes
S38
PHYTOPATHOLOGY
Uptake and exclusion of plant-expressed fluorescent proteins by the
soybean cyst nematode Heterodera glycines
B. GAO (1), J. Bradley (1), M. Hresko (1), A. Caruano-Yzermans (1), J.
Williams (1)
(1) Divergence Inc., St. Louis, MO, U.S.A.
Phytopathology 100:S38
Characterization of EDS1 as a component of Vitis defense responses to
Erysiphe necator
F. GAO (1), X. Shu (2), M. B. Ali (2), S. Howard (2), N. Li (2), P.
Winterhagen (2), W. Qiu (2), W. Gassmann (1)
(1) University of Missouri, Columbia, MO, U.S.A.; (2) Missouri State
University, Mountain Grove, MO, U.S.A.
Phytopathology 100:S38
Vitis vinifera is the most economically important deciduous fruit crop, but
cultivated grapevine varieties lack adequate innate immunity to a range of
diseases. To identify defense pathways in grapevine, we focus on orthologs of
the central Arabidopsis defense regulator ENHANCED DISEASE
SUSCEPTIBILITY1 (EDS1). The family of EDS1-like genes is expanded in
grapevine, and members of this family were previously found to be
constitutively upregulated in the resistant variety ‘Norton’ of the North
American grapevine species Vitis aestivalis, while they were induced by
Erysiphe necator, the causal agent of grapevine powdery mildew (PM) in the
susceptible V. vinifera variety ‘Cabernet Sauvignon’. We determined the
responsiveness of Vitis EDS1-like genes to PM and salicylic acid, and find
that EDS1-like paralogs are differentially regulated in ‘Cabernet Sauvignon’,
while two are constitutively upregulated in ‘Norton’. Sequencing Vitis EDS1like cDNA and genomic clones with highest sequence similarity to AtEDS1
revealed high conservation in the protein encoding sequence and some
divergence of the promoter sequence in the two grapevine varieties. By
complementing an Arabidopsis eds1-1 mutant, we showed that Vitis EDS1
from either grapevine variety is a functional ortholog of AtEDS1. Together,
our analyses show that differential susceptibility to PM is correlated with
differences in EDS1 expression, not differences in EDS1 function, between
resistant ‘Norton’ and susceptible ‘Cabernet Sauvignon’.
A role for WRKY proteins in the low 18:1-derived defense signaling
pathway
Q. GAO (1), D. Navarre (2), A. Kachroo (1)
(1) University of Kentucky, Lexington, KY, U.S.A.; (2) USDA-ARS,
Washington State University, Prosser, WA, U.S.A.
Phytopathology 100:S38
A reduction in oleic acid (18:1) levels simultaneously upregulates salicylic
acid (SA)-mediated responses and inhibits jasmonic acid (JA)-inducible
responses in plants. Plants containing low 18:1 exhibit enhanced resistance to
biotrophic pathogens but are hypersusceptible to necrotrophic pathogens.
Replenishing 18:1 levels restores both Sa- and JA-related defense phenotypes.
We recently reported that SA and the signaling component, enhanced disease
susceptibility 1 (EDS1) function redundantly in this low 18:1-induced
pathway to upregulate SA-derived responses, including resistance to
biotrophs. Here, we show that WRKY proteins mediate the low 18:1-derived
repression of JA signaling, and accumulation of SA. Knockout mutations in
two WRKY genes lower SA levels in the low 18:1-containing Arabidopsis
mutant, ssi2, but not to basal levels. The double mutant plants are not restored
in SA-mediated signaling, and continue to express pathogenesis-related genes
constitutively and exhibit enhanced resistance to Pseudomonas syringae. In
contrast, both JA-inducible PDF1.2 expression, and basal resistance to
Botrytis cinerea are restored in these plants. Thus, two WRKY proteins
positively regulate SA accumulation and negatively regulate JA-derived
resistance signaling in the low 18:1-induced defense pathway.
Expression and homology modeling of Dihydroorotate dehydrogenase
from the phytopathogenic Oomycete Phytophthora infestans
M. F. GARAVITO (1), L. Garcia (1), G. L. Lozano (1), A. J. Bernal (1), B. H.
Zimmerman (1), S. Restrepo (1)
(1) Univ De Los Andes, Bogota, COLOMBIA
Phytopathology 100:S39
The oomycete Phytophthora infestans (Mont.) de Bary, the causal agent of the
tomato and potato late blight, causes tremendous crop and economic losses
worldwide. In Colombia this pathogen is currently a devastating risk for
highlands dedicated to production of its potato host Solanum tuberosum.
Current fungicide based control strategies are far from being adequate and
new ones are urgently needed. As P. infestans seems to be more related to the
apicomplexan parasites than to true fungi, we believe that inhibiting the
metabolic processes used to control human parasites might work to control
this plant pathogen. In this study we investigated the dihydroorotase
dehydrogenase DHODase, one of the key enzymes of the de novo pyrimidine
biosynthetic pathway as a potential drugable target to develop novel control
strategies. In silico, our preliminary molecular docking assays by MOLEGRO
using three-dimensional (3D) homology modeled structures suggest that 6 of 7
parasite DHODase inhibitory compounds exert as well an inhibitory activity
against the P. infestans enzyme. A counter selection strategy using the S.
tuberosum model revealed that two promising inhibitory compounds seem to
be species-selective and exert low or no inhibition over the hosts DHODase.
Using the P. infestans N-terminally truncated DHODase expressed and
purified recombinant protein that complemented a DHODase-deficient
bacterial host we expect the to validate our in silico hypotheses.
Phylogenetic relations within Aspergillus parasiticus imply host adaptation and global transport of aflatoxin-producing fungi
N. GARBER (1), L. C. Grubisha (2), A. Ortega-Beltran (1), C. Probst (1), P.
J. Cotty (2)
(1) University of Arizona, Tucson, AZ, U.S.A.; (2) USDA-ARS, University of
Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S39
Aflatoxin contamination of food and feed is an economic and health concern
worldwide. In the U.S., active enforcement of maximum allowable aflatoxin
levels for consumer protection can eliminate profit from crops including
maize and cotton if aflatoxin is detected at concentrations of 20 parts per
billion or greater. In countries with no aflatoxin-monitoring, human
consumption of aflatoxins can result in aflatoxicosis with symptoms that range
from immune system suppression to death. Two species in Aspergillus section
Flavi are responsible for most aflatoxin contamination events: A. flavus and A.
parasiticus. A. flavus communities span large geographic areas and associate
with multiple crops, while A. parasiticus communities are discrete and
commonly associated with few crops. In order to relate community structure
to geography, host, and aflatoxin contamination risk, microsatellite loci and
portions of multiple genes were amplified from A. parasiticus associated with
peanut, maize, sugarcane and mixed cropping systems in North America, Asia
and Africa. Phylogenetic analyses yielded multiple concordant gene trees
whose topologies identify A. parasiticus lineages both associated with maize
and peanut cropping worldwide, and a putative new species with an ancient,
global and almost exclusive association with sugarcane (Saccharum cultivars).
Population structure will be discussed as it relates to host preference, genetic
diversity and geographic dispersal of A. parasiticus.
Comparative proteomic analysis of sugarcane response to infection by
Xanthomonas albilineans, the causal agent of leaf scald
F. GARCES (1), J. W. Hoy (1), Z. Chen (1)
(1) Louisiana State University, Plant Pathology and Crop Phisiology
Department, Baton Rouge, LA, U.S.A.
Phytopathology 100:S39
Leaf scald is a systemic, vascular disease of sugarcane that can cause severe
yield reduction and the elimination of potential varieties. Although the use of
resistant cultivars is the best control method, the molecular mechanisms of
resistance in sugarcane are unknown. A comparative proteomic analysis to
study differentially expressed proteins could provide a better understanding of
the sugarcane resistance response during infection by X. albilineans. Single
susceptible and resistant cultivars were selected according to disease severity,
vascular infection and bacterial quantification by qPCR. After X. albilineans
inoculation, leaf samples were collected during a time-course encompassing
the response to initial and systemic infection. Total proteins were extracted
and separated by two-dimensional electrophoresis. A relatively low number of
proteins were differentially regulated during the initial infection of inoculated
leaves in both susceptible and resistant cultivars. The number of up- and
down-regulated proteins in the resistant cultivar increased during the systemic
phase of infection 8 weeks after inoculation. To identify and determine
possible functions of the proteins associated with the resistance response, they
are being recovered for peptide sequencing. The preliminary proteomic
analysis, vascular infection, and bacterial population evidence suggest that
resistance is accomplished by mechanisms that limit systemic infection.
Control of Fusarium vascular wilt on carnation using soil biodisinfection
A. García (1), D. Palmero (2), M. de Cara (3), M. Santos (3), J. TELLO
MARQUINA (4)
(1) Instituto Andaluz de Investigación y Formación Agraria, Pesquera,
Alimentaria y de la Producción Ecológica (IFAPA), Cadiz, SPAIN; (2)
Technical University of Madrid, Madrid, SPAIN; (3) Almería, SPAIN; (4)
Univ of Almeria, Almeria, SPAIN
Phytopathology 100:S39
The most important area for fresh cut flowers production in Spain is located in
Cadiz province, attending 553 ha within a total of 1,164 ha in the whole
country. Fusarium wilt (causal agent Fusarium oxysporum f. sp. dianthi) is the
most important disease in carnation crops, meaning a limiting factor for
production. Chemical soil fumigation together with the use of resistant
cultivars against fungal infection used in the area did not provide a level of
efficiency that makes carnation crop profitable. Efficacy of soil
biodisinfection treatments using organic materials (C/N ~36) with and without
polyethylene cover were evaluated for two consecutive years. Results showed
that soil biodisinfection using compost of carnation and chrysanthemum
(5kg·m-2) + hen manure (5kg·m-2) + solarization confers a suitable protection
against the Fusarium vascular wilt during two years. Production and flower
quality were significantly higher than any of the chemical fumigants tested
(methyl bromide, 1,3 dichloropropene (1,3-D) + chloropicrin, metam-sodium)
with and without polyethylene cover. Results also suggest that hen manure
and standard high-density polyethylene film (HDPE) were both the key factor
to the success of the biodisinfection. The proposed method using crops
residues allows, not only the disease control, but also the reutilization of crop
debris by using healthy and even diseased plants.
Population study on the Western Australian strains of Sclerotinia
sclerotiorum
X. GE (1), K. Sivasithamparam (1), H. Li (1), M. Barbetti (1)
(1) The University of Western Australia, Crawley, AUSTRALIA
Phytopathology 100:S39
Sclerotinia disease, caused by the fungus Sclerotinia sclerotiorum, is one of
the most important yield-limiting and certainly the least controllable of
diseases threatening oilseed and vegetable Brassica production world-wide,
including Australia. In Western Australia, the disease is severe but the status
of the pathogen population is largely unknown. Research is now being
undertaken to 1) distinguish the variability within the pathogen population
with respect to morphological characteristics, including cultural and
physiological differences; 2) develop a set of host differentials to delineate
pathotypes/strains of the pathogen using the inoculation and assessment
methods developed by our team, 3) undertake molecular/ phylogenetic studies
to compare strainal variation in Western Australia and elsewhere. One
hundred and seventy isolates from oilseed and vegetable Brassicas and from
some other broad-acre crop species from 17 sites in Western Australia were
isolated and studied. Morphological studies indicated significant cultural and
physiological differences among isolates, even within isolates sampled from
the same site but from different individual host stems. A set of differential
resistant response hosts was screened from ninety three genotypes of B. napus
and B. juncea from China and Australia under semi-field conditions.
Molecular and phylogenetic analyses are underway to determine the extent of
genetic variation within the pathogen population.
Survey of soybean diseases in the Ohio River Valley Region of Ohio
during 2009
K. GEARHART (1), D. Dugan (2), J. Grimes (3), L. Farley (4), J. Fisher (5),
C. Burskey (4), A. E. Dorrance (1)
(1) Dept. of Plant Pathology, The Ohio State University, OARDC, Wooster,
OH, U.S.A.; (2) Ohio State University Extension, Georgetown, OH, U.S.A.;
(3) Ohio State University Extension, Hillsboro, OH, U.S.A.; (4) Ohio State
University Extension, Owensville, OH, U.S.A.; (5) Ohio State University
Extension, Waverly, OH, U.S.A.
Phytopathology 100:S39
Vol. 100, No. 6 (Supplement), 2010
S39
Soybean yields in the Ohio River Valley Region of Ohio have been
substantially less than other soybean producing regions of the state. The
objective of this study was to determine what the production limitations are in
the seven counties during 2009. A total of 132 plant and soil samples were
collected at crop growth stages VE to V2 and R5 to R7 from 6 and 4 fields,
respectively. Fields were sampled based on crop rotation practices and/or a
history of low yields. Plant samples were transported back to the laboratory
and symptomatic root tissue was plated onto selective media for both fungi
and oomycete pathogens. Soil samples were processed for soybean cyst
nematode (SCN) and soil analysis. Pythium, Phytophthora, Fusarium,
Macrophomina, Diaporthe, Sclerotinia, and Rhizoctonia spp. were isolated
from plant material. SCN was identified in 17 of the 38 locations and counts
ranged from 0 to 3300 eggs/100cc soil. This is the first reported find of SCN
for Pike Co. The next step is to further characterize the disease complexes that
may be present, evaluate management strategies, and educate producers in this
region.
Risk factors and modeling of powdery mildew occurrence on hop cones
D. H. GENT (1), J. L. Farnsworth (2), M. E. Nelson (3), G. G. Grove (3)
(1) USDA ARS NFSPRC, Corvallis, OR, U.S.A.; (2) Oregon State
University, Corvallis, OR, U.S.A.; (3) Washington State University, Prosser,
WA, U.S.A.
Phytopathology 100:S40
Powdery mildew of hop (caused by Podosphaera macularis) can substantially
reduce crop cone yield and quality. Risk factors associated with the incidence
(proportion) of diseased cones were identified and formalized in a linear
mixed model based on published data from 12 fungicide efficacy trials
conducted during 2000 to 2008. Models that included risk factors of disease
incidence on leaves, rain, temperature, and fungicide timing during critical
cone developmental stages explained 87 to 91% of the variability in observed
incidence of diseased cones. Predictions of disease levels in 2009 with data
sets collected independently of those used for model developed (validation
data sets) were imprecise (R2 = 0.55), in part because fungicide effects were
not represented accurately. When the fungicide effect variable was revised,
the model explained 74% of the variability in the observed incidence of
diseased cones in the validation data sets. Sensitivity analyses indicated that
the effect of fungicide application timing is substantial, and suggests
appropriate timing of efficacious treatments is critical for minimizing levels of
powdery mildew at harvest. Future research is planned to link the disease risk
model to a crop damage function to inform and optimize late season
management decisions for powdery mildew.
Identification and characterization of cucurbit powdery mildew in
Florida
A. J. GEVENS (1), G. S. Maia (2)
(1) University of Wisconsin, Madison, WI, U.S.A.; (2) University of Florida,
Gainesville, FL, U.S.A.
Phytopathology 100:S40
Cucurbit powdery mildew (PM) is a common and economically important
foliar disease in vegetable production. Although cucurbit PM can be caused
by two obligate pathogens, Podosphaera xanthii and Golovinomyces
cichoracearum, P. xanthii has been most commonly identified. Recently, there
has been an increase in occurrence and severity of cucurbit PM in FL, raising
concern of a shift in the population. In this study, we identified and
characterized single colony isolates of cucurbit PM from multiple sites, dates,
and hosts. Two butternut squash fields (Live Oak and Citra, FL) were sampled
during spring 2009 and the Citra site was again sampled in fall 2009. For
comparison, additional cucurbit isolates were collected from SW and NE FL.
For all 264 PM isolates from butternut squash from Live Oak and Citra,
conidia were hyaline, ellipsoid-ovoid, in single chains per conidiophore, and
had dimensions of 32-44 X 18-20 µm (N = 50) and footcells of 47-61 X 11-13
µm (N = 25). All isolates exhibited fibrosin bodies in immature conidia and
conidia edges were crenate. Isolates from butternut squash and additional
cucurbit hosts from varied locations and dates were subjected to multiplex
polymerase chain reactions with species-specific primers for P. xanthii
(S1/S2) and G. cichoracearum (G1/G2). With S1/S2, a specific PCR product
(454 bp) was amplified from genomic DNA of all isolates. All FL 2009 PM
cucurbit isolates were identified as P. xanthii.
Enhanced quality, value, yield, carbon capture and sustainability of
switchgrass biomass by the improvement of root, microbe and soil
interactions
S. GHIMIRE (1), K. Craven (1), S. Chaluvadi (2), J. Bennetzen (2), E. Worley
(1), J. Yang (1), M. Udvardi (1), M. Keller (3)
(1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.; (2) The
University of Georgia, Athens, GA, U.S.A.; (3) BioEnergy Science Center,
Oak Ridge, TN, U.S.A.
Phytopathology 100:S40
S40
PHYTOPATHOLOGY
Sustainable production of switchgrass (Panicum virgatum L.) for biofuel will
depend, in part, on maximizing nutrient acquisition and assimilation
throughout the growing season as well as minimizing nutrient loss at harvest.
Nutrient acquisition and uptake by plants can be enhanced by beneficial soil
microbes as well as those existing endophytically within the roots of the
switchgrass host. We have undertaken a comprehensive characterization of the
microbes associated with the rhizosphere of planted switchgrass cultivars
and those found within the healthy, surface-sterilized root systems of plants
from natural habitat. High levels of microbial biodiversity were detected
for both fungi and bacteria, and several strains have been isolated for
evaluation of fitness effects on elite switchgrass cultivars. Dramatic
differences in rhizosphere and endophyte microbial populations have been
found to be a function of host genetics by analysis of different switchgrass cultivars, and mapping studies are now initiated to identify the host
genes that determine microbial composition in and around the root. The
evaluation of 31 switchgrass accessions for natural variations in nutrient-use
and remobilization efficiencies of 20 different elements in shoots of fieldgrown plants at maturity and after senescence revealed that accessions
differed in elemental composition in these two developmental stages.
Implication of these findings in sustainability of switchgrass production will
be presented.
Powdery scab effect on potato Solanum tuberosum and S. phureja
E. GILCHRIST (1), J. Soler (2), J. G. Morales (1), S. Reynaldi (1)
(1) Universidad Nacional de Colombia, Departamento Ciencias Agronómicas,
Medellin, COLOMBIA; (2) Universidad Nacional de Colombia, Medellin,
COLOMBIA
Phytopathology 100:S40
Spongospora subterranea f. sp. subterranea is the causal agent of the potato
powdery scab, this microorganism infects young tissues. There is nor effective
control method available for the disease neither resistance varieties, it was
evaluated the influence of the cycle culture duration on the powdery scab
effects over two potato species. The potato Solanum tuberosum variety Diacol
Capiro is six months cycle culture duration and Solanum phureja variety
Criolla Colombia is four months. These were planted in free and infect soil
whit S. subterranea. Both species had galls at 100% of the roots in infected
soil, those galls covered the 5% of the root surface in S. tuberosum and the 8%
in S. phureja. There was no quantitative relation between the affected root
surface with the length decrease, foliar dry weight and production. There were
similar disease effects in both potato species, suggesting that there is not a
susceptibility window related with the cycle culture duration. Next researches
should focus in determinate if species or varieties with different development
speed have any differential susceptibility to the disease, it should evaluate the
powdery scab effects at the same sampling time.
Trichoderma asperellum T109 effect over Spongospora subterranea in
potato field
E. GILCHRIST (1), J. Pérez (2), H. A. Vargas (2), S. Reynaldi (1)
(1) Universidad Nacional de Colombia, Departamento Ciencias Agronómicas,
Medellin, Antioquia, COLOMBIA; (2) Universidad Nacional de Colombia,
Medellin, COLOMBIA
Phytopathology 100:S40
The aim of this research was to determinate the T. asperellum T109 effects
over S. subterranea cystosori and over the powdery scab in potato Solanum
tuberosum variety Diacol Capiro in field evaluating three doses. The removal
of cystosori seems to be regulated by the concentration of the inocula, both for
fault and for excess. It was determinate the effect of the T. asperellum T109
application on S. tuberosum plants without (control) and with 1082 ± 187
cysotosory/g soil of S. subterranean. In contaminated soil the plants were
26% less big and its production was 30% less, the T. asperellum T109
application reduce in 64% and in 54% the powdery scab effects in the plant
growth and production, respectably. Future researches will have to
determinate what factors limit the T. asperellum T109 activity in field.
Evaluation of fungicides on white mold of peanuts
S. GLUCKSMAN (1), C. R. Semer (1)
(1) University of Florida, Gainesville, FL, U.S.A.
Phytopathology 100:S40
Peanuts, found in foods such as candy bars, peanut butter, peanut brittle, and
peanut flour, are also found in cosmetics, nitroglycerine, plastics, dyes, and
paints. Peanut oil is also a commodity derived from this legume, and is
responsible for approximately 4% of the global vegetable oil production at
4.93 million metric tons. Florida produced approximately 10% of the 5.15
billion pounds of peanuts produced in the United States in 2008. White mold
causes an estimated 7% loss from disease on peanuts annually valued at $7
million. The purpose of this experiment was to evaluate the efficacy of several
fungicides for the control of white mold (Sclerotium rolfsii) on peanuts. An
alternating application of Bravo Weatherstik® 1.25pt/A (AFG) and Provost®
10.7oz/A (BCDE), treatment 10, was found to produce the highest yield at
4543.31lbs/Acre. Besides the control, the lowest yield was treatment 12
(Bravoweatherstik® 1.25pt/A (ABCDEFG)) at only 3645.97 lbs/Acre.
SSR-based genetic analysis of Candidatus Liberibacter asiaticus isolates
from multiple continents
J. M. GLYNN (1), H. Lin (1), C. Chen (2), Y. Duan (3), L. Zhou (4)
(1) United States Department of Agriculture, Parlier, CA, U.S.A.; (2) Guangxi
Citrus Research Institute, Guangxi, PRC PEOPLES REP OF CHINA; (3)
United States Department of Agriculture, Ft. Pierce, FL, U.S.A.; (4)
University of Florida, Ft. Pierce, FL, U.S.A.
Phytopathology 100:S41
Huanglongbing (HLB) is one of the most devastating citrus diseases that
threaten citrus production worldwide. The causal agents are believed to be
Candidatus Liberibacter spp. Although substantial efforts have been made
toward detection of the presumed pathogens, information regarding to the
pathogen’s genetic variation, population structure, and epidemiological
relationships is limited-- such data might provide useful insights into the
evolutionary adaptations and host selection mechanisms utilized by this group
of bacteria. The objective of this study is to identify the phylogenetic
relationship between Candidatus Liberibacter asiaticus (Las) strains isolated
from Florida, Brazil, India, and China. We used a panel of at least 8 simple
sequence repeat (SSR) markers to infer phylogenetic relationships amongst
HLB-associated Las strains from these four geographically- and ecologicallydiverse regions. Our results indicate that Las strains from Florida share
features with strains from three other regions of the world and provide insights
into regions of the Las genome that may be associated with altered levels of
pathogenicity.
Analysis of induction and establishment of dwarf bunt of wheat under
marginal climatic conditions
B. J. GOATES (1), G. L. Peterson (2), R. L. Bowden (3), L. D. Maddux (4)
(1) USDA ARS, Aberdeen, ID, U.S.A.; (2) USDA ARS, Frederick, MD,
U.S.A.; (3) USDA ARS, Manhattan, KS, U.S.A.; (4) Kansas State University,
Topeka, KS, U.S.A.
Phytopathology 100:S41
Dwarf bunt caused by Tilletia contraversa has limited distribution due to
essential climatic requirements; primarily persistent snow cover. The
pathogen is a quarantine organism in several countries outside of the U.S.A.,
some of which may have marginal climate for the disease, including the
People’s Republic of China. To evaluate the risk of disease introduction,
experiments were conducted in Kansas which is a climatic analog to the
northern winter wheat areas of China. Four replicate 27 m2 plots, planted with
a susceptible cultivar, were inoculated at six rates ranging from 0.88 to 88,840
teliospores/cm2. Three separate nursery sites were inoculated once, each site
in a separate season, followed by replanting and examination for disease for 4
to 6 years afterward. Any diseased spikes produced were crushed and returned
to the plots. Bunt was induced at trace levels at the three highest inoculation
rates in two of the three nurseries. One nursery had no disease during all of six
seasons. Disease carryover occurred during one year in one nursery at the
highest inoculation rate, but no disease occurred in three subsequent seasons.
In all nurseries, the disease eventually disappeared. A duplicate nursery
planted in a disease conducive area showed the highest inoculum rate caused
almost 100% infection. This research substantiates the critical importance of
climatic conditions for establishment of this pathogen and contributes to pest
risk assessment efforts.
Genetic diversity of the vegetable pathogen Phytophthora capsici in
Argentina
D. J. GOBENA (1), J. Roig (2), J. Hulvey (1), K. Lamour (1)
(1) University of Tennessee, Knoxville, TN, U.S.A.; (2) INTA EEA La
Consulta, Mendoza, ARGENTINA
Phytopathology 100:S41
Phytophthora capsici is an oomycete plant pathogen that attacks solanaceous
and cucurbit hosts worldwide. In the U.S., populations from single fields often
exhibit extensive genotypic diversity. However, in Peru a single A2 mating
type clonal lineage dominates over a wide area. Here we present data on 43
isolates of P. capsici recovered from pepper, paprika and pumpkin hosts in
2006, 2007, 2008 and 2009 from different regions in Argentina. All of the
isolates were the A1 mating type. The isolates were assayed for population
specific single nucleotide polymorphism (SNP) and AFLP profiles. The
SNP’s were identified by re-sequencing single copy genes in two isolates
from the Argentina populations and were assessed for all isolates using high
resolution DNA melting analysis. Our preliminary data indicates four unique
genotypes with one clonal lineage comprising 86% of the population and two
other clonal lineages with only two isolates. An overview of the marker
development, application and resultant findings will be presented.
Cell cycle regulator MoCDC15 is required for conidiation and
pathogenicity in Magnaporthe oryzae
J. GOH (1), K. Kim (1), Y. Lee (1)
(1) Department of Agricultural Biotechnology, Center for Fungal Genetic
Resources, and Center for Fungal Pathogenesis, Seoul National University,
Seoul, SOUTH KOREA
Phytopathology 100:S41
Rice blast, caused by Magnaporthe ozyzae, is a serious constraint to rice
production, and has emerged as an important pathosystem to uncover
molecular mechanisms in plant-fungus interactions. Like other fungal
pathogens, M. oryzae produces a massive amount of conidia, via conidiation
for dissemination and exacerbation of the disease. However, relatively little is
known about molecular mechanism of conidiation in M. oryzae. To better
understand conidiation mechanism, we identified a conidiation-defective
mutant, ATMT0225B6 (MoCDC15T-DNA), from a T-DNA insertional mutant
library. Molecular and bioinformatics analyses revealed that T-DNA was
inserted in a gene encoding a Pombe CDC15 Homology (PCH) ortholog
(MoCDC15) in ATMT0225B6. To unveil functional roles of MoCDC15, the
same allele mutant (ΔMoCDC15T-DNA) was generated by a single step gene
replacement strategy. Both mutants exhibited the same phenotype defects in
conidiation, conidial germination, appressorium formation, and pathogenicity.
Conidia of these mutants were in abnormal shapes with reduced adhesion
ability to hydrophobic surface. All these phenotypic defects were recovered in
the complemented strains (Mocdc15c). These results indicated that MoCDC15
is essential for conidiation, infection-related development, and pathogenicity
in M. oryzae.
DNA pyrosequencing to determine the influence of fallow period on soil
microbial communities in the Bolivian highlands
L. GOMEZ (1), A. Jumpponen (1), M. A. Gonzales (2), J. Cusicanqui (3), C.
Valdivia (4), P. Motavalli (4), M. Herman (1), K. A. Garrett (1)
(1) Kansas State University, Manhattan, KS, U.S.A.; (2) Fundacion
PROINPA, La Paz, BOLIVIA; (3) Universidad Mayor de San Andres, La Paz,
BOLIVIA; (4) University of Missouri, Columbia, MO, U.S.A.
Phytopathology 100:S41
In the Bolivian highlands (Altiplano; approx. 4000 masl), traditional fallow
periods are being shortened in an effort to increase short-term crop yields,
which may be at the expense of soil quality and plant health. Using 454pyrosequencing and DNA-tagging, we characterized the response of the
microbial community to (1) the length of fallow period and (2) the presence of
Parasthrephia sp. and Baccharis sp. (both locally known as ‘Thola’),
considered beneficial to the maintenance of soil health in fallow fields in this
region. The two study regions, Umala and Ancoraimes, differ in their soil
characteristics, which may be a fundamental reason for the inherent
differences in regional management practices. Soils in Ancoraimes have
higher levels of organic matter, nitrogen and other macronutrients, which
supported more diverse fungal communities (P < 0.001). The presence of
Thola after ten years of fallow had a positive effect on soil fungal diversity.
Unexpectedly, the longer fallow periods were associated with lower fungal
richness and diversity, perhaps because some fields with longer fallow periods
were perceived by managers to have lower quality soils. Analyses of bacterial
communities and fungal community composition are underway. Our results
suggest that the drivers of microbial richness may be more complex than
predicted by fallow period alone, and that plant cover may be important in
conserving microbial communities.
Relationship between Citrus Variegated Chlorosis intensity and yield
reduction
F. P. GONÇALVES (1), E. S. Stuchi (2), S. A. Lourenço (1), L. Amorim (1)
(1) Escola Superior de Agricultura Luiz de Queiroz / USP, Piracicaba,
BRAZIL; (2) Embrapa Mandioca e Fruticultura / Estação Experimental de
Citricultura de Bebedouro, Bebedouro, BRAZIL
Phytopathology 100:S41
Citrus Variegated Chlorosis (CVC), caused by Xylella fastidiosa, causes
losses of around 120 million dollars/year in Brazil. CVC is present in Brazil
since 1987, even though, the relationship between disease intensity and yield
losses has never been established. The objective of this study was to
determine this relationship. The incidence of branches with symptoms of CVC
and the asymptomatic fruit yield (asymptomatic fruit production) were
evaluated during three years (2006 to 2008), in orchard located at São Paulo
State. The experiment design was in factorial randomized blocks (3 × 2). The
treatments were: no irrigation, irrigated with 50% and 100% of crop
evapotranspiration (ETc), combined with natural and artificial inoculation of
X. fastidiosa. Each plot consisted of 6 plants, with 4 replication, total 144 trees
(10-yr-old Natal sweet orange). The single plant method was used to quantify
disease damage. For all treatments, the decrease of yield (asymptomatic fruit
production) was associated with the increased of CVC incidence, described by
the negative exponential model. The reductions of yield rates were similar for
Vol. 100, No. 6 (Supplement), 2010
S41
all treatments (b = 0.014 to 0.019). Because of this, one single model can be
used to describe the yield-CVC intensity relationship: y = (114.
07)*exp(0.017) *x), where y is yield (Kg of fruit asymptomatic) and x is
disease incidence, p < 0.01 with R2 = 0.45. According to the model, 10% of
symptomatic branches lead to a reduction of 18.3 kg/plant. Supported by
CNPq and Fundecitrus.
Morphological and molecular characterization of Olpidium virulentus, the
fungal vector of the Macana virus disease in Colombia
C. GONZALEZ (1), D. L. Osorio (1), M. C. Cepero de García (1), L.
Sastoque (1), C. Beltrán (1), A. M. Cotes (1)
(1) CORPOICA, Mosquera, Cundinamarca, COLOMBIA
Phytopathology 100:S42
Olpidium spp. is a fastidious root-infecting plant parasite whose eradication is
difficult because of its condition as obligated root plant parasite as well as it
having resistant structures. It is considered as transmission vector of virus
such as Dianthovirus from the family of the Tombusviridae, which is a causal
virus of the ‘Macana’ or necrotic streak disease of fique (Furcraea spp.). The
Macana disease symptoms are characterized by necrotic stripes which appear
on the infected plant leaves, followed by growth reduction and finally, total
death. In order to to identify the Olpidium specie involved in the Macana
disease in Colombia, extensive field surveys in three municipalities in the
Cauca department, and microscopic studies of non-motile reproductive stage
and motile zoospore was conducted using a contrast phase microscope. Fungal
isolate was directly obtained by cultivation of motile zoospore from infected
roots on lettuce plant. Fungal DNA was obtained from both zoospore and root
plants, and a multiplex PCR was carried out using reported species-specific
primers of three Olpidium spp. The microscopic analysis and PCR data
indicated that fungal specie obtained from fique corresponded to Olpidium
virulentus.
Genotypic diversity of Phytophthora ramorum in Canada
E. M. GOSS (1), M. Larsen (1), A. Vercauteren (2), S. Werres (3), K.
Heungens (2), N. J. Grunwald (1)
(1) USDA ARS, Corvallis, OR, U.S.A.; (2) Institute for Agricultural and
Fisheries Research, Merelbeke, BELGIUM; (3) Julius Kuehn Institute Federal Research Centre for Cultivated Plants, Braunschweig, GERMANY
Phytopathology 100:S42
Characterization of the genetic structure and diversity of the sudden oak death
pathogen, Phythophthora ramorum, in ornamental nurseries in the United
States has shown that all three known clonal lineages of the pathogen are
present. The most common clonal lineage in U.S. nurseries has been the NA1
clonal lineage, which has the wider distribution in the United States as a result
of interstate shipments of infected nursery stock. British Columbia (BC),
Canada is also known to have nursery infestations of P. ramorum, and
shipments of infected plants between the United States and BC have occurred.
We investigated the genotypic diversity of P. ramorum in BC nurseries and
compared this population to U.S. and European nursery populations. All three
of the P. ramorum clonal lineages were found among Canadian nursery
isolates, but the most common was the NA2 lineage. The NA1 clonal lineage
was found infrequently in comparison to the United States. The EU1 lineage
was observed almost every year and shared multilocus genotypes with isolates
from Europe and the United States. Appropriate markers for the
characterization of the NA2 lineage are needed.
Fungal gene expression patterns during infection of field pea roots by F.
graminearum and F. avenaceum
R. S. GOSWAMI (1), K. Chittem (1)
(1) North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S42
Fusarium root rot of field peas has been a growing concern in the Midwestern
production areas. Surveys conducted over the past three years demonstrated
that species such as F. avenaceum and F. graminearum, previously associated
with cereals in the U.S., were infecting roots of this major rotational crop.
Information regarding the infection of legumes by these species is limited and
our goal is to elucidate the mechanism of disease development through
genomic analysis and pathogenicity studies. Fungal gene expression during
infection of field pea roots by these two pathogens was evaluated as a part of
this study. Over eleven thousand F. graminearum genes were detected, 6.57%
of which are considered to be associated with cell defense, rescue and
virulence. Initial comparisons suggest that 1606 F. avenaceum genes
identified are similar to those expressed by F. graminearum during pea root
infection. Genes common between crown rot and head blight in wheat and
those associated specifically with crown rot were identified in this study in
addition to genes involved in the trichothecene biosynthetic pathway. Analysis
of fungal gene expression patterns, the ability of F. graminearum to produce
mycotoxins on peas and their potential role in infection of this host will be
presented.
S42
PHYTOPATHOLOGY
Spatial and temporal analyses of Plum pox virus survey data
A. GOUGHERTY (1), R. Welliver (2), N. Richwine (2), F. W. Nutter (1)
(1) Iowa State University, Ames, IA, U.S.A.; (2) Pennsylvania Department of
Agriculture, Harrisburg, PA, U.S.A.
Phytopathology 100:S42
Plum pox virus (PPV) was recently declared to be successfully eradicated in
Pennsylvania, following the implementation of a statewide eradication
program initiated in 2000. There were approximately 400 PPV-positive trees
detected in 2000, however, the number of PPV-positive trees decreased
exponentially during the following years until 2007, when no new PPVpositive trees were detected. The success of the eradication program can be
attributed to an extensive annual state-wide survey and the removal of all
susceptible hosts within 500 m of a PPV-positive tree. Using 10 years of
survey data, a number of spatial analyses were performed at the block and
homeowner scale to quantify the spatial and temporal dynamics of the
pandemic. In 2000, positive blocks were found to be clustered for distances
between 0.7 and 4.9 km. In later years, the distance to 50% of new PPVpositive blocks/homeowners (D50) increased from 1.3 km in 2000 to 17.2 km
in 2005. In 2006, the last year any PPV-positive blocks/homeowners were
detected, the D50 decreased to 6.3 km. An approximate two year periodicity
was found to exist arising from previous positive locations, suggesting that the
location of a PPV-positive block/homeowner was having an impact on the
health status of other Prunus blocks at least 2 years after being removed. This
was further supported by a nearest neighbor analysis that revealed the time
since removal of the nearest positive block/homeowner averaged to be 1.8
years.
Development of primers and probes for detection of Citrus Candidatus
Liberibacter species by real-time PCR
A. GOVINDARAJULU (1), N. Choudhary (1), J. S. Hartung (2), A. L. Stone
(3), V. D. Damsteegt (3), R. H. Brlansky (1)
(1) University of Florida, Lake Alfred, FL, U.S.A.; (2) USDA-ARS,
Beltsville, MD, U.S.A.; (3) USDA-ARS, Fort Detrick, MD, U.S.A.
Phytopathology 100:S42
Citrus huanglongbing (HLB) or citrus greening is the most threatening
bacterial disease of Citrus spp. Three uncultured Candidatus Liberibacter spp.
are usually detected by conventional PCR or real-time PCR using species
specific primers and probes based on the 16S rRNA gene. Recent molecular
analyses suggest that the rpoB gene (encoding the -subunit of RNA
polymerase), is useful for bacterial identification. We report the design of a
pair of degenerate primers and probe in the rpoB region and its application for
the detection of the three Liberibacter species (Ca. Liberibacter asiaticus, Ca.
L. africanus and Ca. L. americanus). In addition, a multiplex format was
standardized and applied to detect all three species. This one step real-time
PCR diagnostic method is reliable for the detection of Liberibacter infection
regardless of species. This method also may be useful to detect unidentified
Liberibacter species in citrus.
Soil application of neo-nicotinoid insecticides and acibenzolar-S-methyl
for induction of SAR to control citrus canker on young citrus trees
J. H. GRAHAM (1), M. Myers (1)
(1) University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S42
Soil application of systemic imidacloprid produces season-long control of
citrus canker caused by Xanthomonas citri subsp. citri. This neo-nicotinoid
insecticide induces systemic acquired resistance (SAR) measured as an
increase in salicylic acid levels and expression of PR-2 gene in citrus leaves.
Soil drenches of imidacloprid (Admire, Bayer Crop Science), thiamethoxam
(Platinum, Syngenta Crop Protection), and the commercial SAR inducer
acibenzolar-S-methyl (Actigard, Syngenta Crop Protection) were compared
with copper hydroxide (CH, Kocide 3000, Dupont Crop Protection) applied as
a spray at 21 da interval for canker control on foliage of 4-yr old ‘Ray Ruby’
grapefruit trees. Canker on each set of foliar flushes was assessed as the
percentage of the leaves with lesions. All treatments significantly reduced
incidence of foliar canker compared to the untreated check. Platinum and
Actigard as soil drenches were as effective as previously tested drenches of
Admire for sustained control of canker on young trees under epidemic conditions. Although the level of control did not match that of 11 contact sprays of
Kocide 3000, SAR activity persisted season long. For both the 2008 and 2009
seasons, canker control with SAR inducers was highest for four applications
of Actigard at 60 da interval. This treatment demonstrates the efficacy of
repeated soil applications for maintaining SAR throughout the season.
Genetic variation and adaptation of M. perniciosa isolates in Bahia, Brazil
K. P. GRAMACHO (1), J. Pires (2), R. Moreira (3), U. V. Lopes (2)
(1) CEPLAC CEPEC SEFIT, Itabuna, Bahia, BRAZIL; (2) CEPLAC CEPEC
SEGEN, Itabuna, BRAZIL; (3) UFR, Cruz das Almas, BRAZIL
Phytopathology 100:S42
The incidence of witches’ broom disease of cacao (Moniliophthora
perniciosa) in Bahia, Brazil has increased towards the main source of
resistance: the Scavinas. It is hypothesized that this alterations is due to
pathogen variability. Cross-infectivity of isolates of M. perniciosa from the
Scavina’s descendent was evaluated. The experiment was arranged as a
randomized complete block design with 4 replicates of treatments that
included four isolates of M. perniciosa derived from the Scavina’s descendent
(Sca6, THS1188, TSH565, TSH516) and two from other resistant (CCN10)
and susceptible (SIC2) genotypes. Clones of Sca6, TSH1188, TSH565,
CCN10, and SIC 23 (susceptible) were inoculated with 20-µL droplet of 2 ×
105 basidiospores/mL per shoot. Sixty days after the inoculation day, plants
were evaluated considering the variables SINT (disease incidence) and ID
(disease index). There were significant differences among the M. perniciosa
isolates. Most Scavina’s descendent isolates were significantly more virulent
than the isolate derived from the susceptible genotype. Moreover, based on
molecular markers (RAPD), fungal isolates from resistant genotypes differed
(p < 0.05) from the susceptible ones. Considering the increase in susceptibility
in Scavina descendants, associated with the distinction of isolates from
susceptible/resistant genotypes, it can be concluded that the increase in
susceptibility is the result of the increase in frequency of strains capable of
overcoming the resistance of Scavina.
The effects of temperature, humidity, and wounding on development of
Phytophthora rot of cucumber
L. L. GRANKE (1), M. K. Hausbeck (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S43
The effects of temperature (10, 15, 20, 25, 30, 35°C at >97% relative
humidity) and relative humidity (<45%, ~60%, ~70%, ~80%, ~90%, and
>97% at 25°C) on development of Phytophthora fruit rot of pickling
cucumber were investigated in controlled growth chamber studies. In addition,
the effect of wounding on size related resistance of pickling cucumber to P.
capsici was characterized for three fruit sizes: 2.0 to 2.5 × 8 to 9 cm (small),
3.0 to 4.0 × 12.0 to 13.0 cm (medium), and >4.5 cm × >14 cm (large). No
lesions developed on cucumbers incubated at 10°C, but lesions were observed
on cucumbers incubated at all other temperatures tested. Lesion development
was delayed for cucumbers incubated at 15°C. Lesion diameter and
sporulation were greatest on cucumbers incubated at 25°C at 4 days post
inoculation (dpi). Lesion development was greatest on cucumbers incubated at
≥80% relative humidity, but lesions formed on cucumbers incubated at all
levels of relative humidity tested. Wounding was found to lessen size-related
resistance in pickling cucumber. Lesion size was similar for all wounded
cucumbers at 4 dpi regardless of fruit size. The smallest lesions were observed
on unwounded large cucumbers. Sporangial production was greatest on
wounded small and medium fruits. Fewer sporangia were produced on the
large unwounded fruits than on any of the other cucumbers tested. Similar
numbers of sporangia were produced on wounded large fruits and unwounded
small and medium cucumbers.
Influence of environment on periodicity and concentration of airborne
Pseudoperonospora cubensis sporangia in commercial cucurbit fields
L. GRANKE (1), M. Hausbeck (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S43
Airborne concentrations of Pseudoperonospora cubensis sporangia were
monitored from 2006 to 2009 in commercial cucurbit fields in four Michigan
counties. An additional two counties were monitored for one or two growing
seasons. Environmental conditions (temperature, relative humidity, leaf
wetness, and rainfall) were recorded at select sites during the 2008 and 2009
growing seasons. The concentrations of airborne sporangia showed a marked
diurnal periodicity with the highest concentrations between 600 and 1300 h.
Airborne sporangial concentrations were positively related to average
temperature and negatively correlated with both average relative humidity and
leaf wetness at all of the sites monitored in 2008 and 2009. The results of this
study may be used to improve the regional disease warning system, which
already uses sporangial counts to guide fungicide applications.
Detecting Sugarcane yellow leaf virus in asymptomatic sugarcane leaves
with hyperspectral remote sensing and associated leaf pigment changes
M. P. GRISHAM (1), R. M. Johnson (1), P. V. Zimba (2)
(1) USDA ARS, Houma, LA, U.S.A.; (2) Texas A&M - Corpus Christi,
Corpus Christi, TX, U.S.A.
Phytopathology 100:S43
Sugarcane yellow leaf caused by Sugarcane yellow leaf virus (SCYLV) does
not produce visual symptoms in most susceptible sugarcane plants until late in
the growing season. High-resolution, hyperspectral reflectance data from
SCYLV-infected and non-infected leaves of two cultivars, LCP 85-384 and
Ho 95-988, were measured and analyzed on 13 July, 12 October, and 4
November 2005. Infection was determined by reverse transcriptasepolymerase chain reaction (RT-PCR) analysis. Results from discriminant
analysis showed that leaf reflectance was effective at predicting SCYLV
infection in 73% of the cases in both cultivars using resubstitution and 63%
and 62% in LCP 85-384 and Ho 95-988, respectively, using cross validation.
Leaf pigments were extracted from leaf samples collected on 12 October and
analyzed for chlorophylls and carotenoids concentrations. SCYLV infection
influenced the concentration of several of the plant pigments including
violaxanthin and b-carotene. Pigment data was effective at predicting SCYLV
infection in 80% of the samples in the combined data set using the derived
discriminant function with resubstitution, and 71% with cross validation.
Developing technology to remotely detect SCYLV infections without a
laboratory-based diagnostic technique would provide an efficient method to
insure that seed cane is free of the SCYLV.
Evaluation of multiple management strategies for FHB barley
P. L. GROSS (1), S. M. Neate (2), R. Brueggeman (1)
(1) North Dakota State University, Fargo, ND, U.S.A.; (2) Toowoomba,
AUSTRALIA
Phytopathology 100:S43
Fusarium head blight (FHB) has been a devastating disease for U.S. barley for
over a decade. The disease causes kernel discoloration and the production of
deoxynivalenol (DON). Genetic resistance, cultural practices, and fungicides
have reduced the disease and toxin levels, but often not to levels required by
the malting and brewing industry. We investigated the effects of resistant
lines, fungicide treatment at heading, and crop rotations on FHB severity and
DON accumulation in barley at Fargo, ND in 2009. Four six-row and four
two-row barley lines with differential reactions to FHB were grown on ground
planted the previous year to either wheat or soybean. Additionally, these plots
had a fungicide treatment (Prosaro applied at 0.48 L/ha) at heading or were
left untreated. FHB incidence, severity DON levels, yield and test weight were
evaluated. Disease levels were very low in this study. Planting barley into
soybean ground significantly increased yield and test weight for both barley
types, across all lines, but disease parameters generally were not impacted by
crop rotation. Fungicide treatment significantly reduced DON levels across
the six-row lines, but not the two-row. Several-two row and one six-row line
had significantly lower DON levels than more susceptible lines, across
rotation and fungicide treatments. No significant differences were observed
between interactions in six-row or two-row barley lines. This study will be
repeated again in 2010.
Morphological and cytological modifications of Gibberella zeae germlings
induced by mating pheromones and affinity-selected peptides
N. W. GROSS (1), J. E. Scholez (1), F. J. Schmidt (2), J. T. English (1)
(1) Division of Plant Sciences, University of Missouri, Columbia, MO,
U.S.A.; (2) Department of Biochemistry, University of Missouri, Columbia,
MO, U.S.A.
Phytopathology 100:S43
Head blight of wheat is initiated in the spring by the homothallic ascomycete
Gibberella zeae. This floral disease reduces grain yield but also results in the
accumulation of various mycotoxins, the most notable among them being
deoxynivalenol (DON). To reduce the need for fungicide treatment, wheat
with robust resistance is necessary. However, in the absence of a dominant
resistance gene, only defined types of partial resistance have been available to
producers. Small peptides with specific fungistatic activity towards G. zeae
are being developed as an alternative management strategy. These peptides,
derived from combinatorial phage-display libraries and also from native G.
zeae mating pheromone peptides, inhibit ascospore germination and induce
abnormal germling phenotypes. Abnormalities induced by these mating
pheromone peptides include extended periods of isotropic growth, apolar
germtube emergence, and increased hyphal branching. After one day of
peptide exposure, as many as 95% of ascospores exhibit abnormal
morphological characteristics and these effects were observed over a wide
range of peptide concentrations. Cytological analyses of ascospore and
germling structure are in progress to better understand the basis of these
morphological abnormalities. These data will advance our understanding of
small-peptide inhibition of fungal spores which can be potentially exploited as
a means of blight control.
Evolutionary relationships among Aspergillus flavus
compatibility groups
L. C. GRUBISHA (1), P. J. Cotty (1)
(1) USDA-ARS, University of Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S43
vegetative
Aspergillus flavus is a fungal plant pathogen of many diverse crops including
cotton, peanuts, maize, almond, and pistachio. During infection by A. flavus,
crops are frequently contaminated with highly carcinogenic aflatoxins. A.
flavus populations are composed of numerous vegetative compatibility groups
Vol. 100, No. 6 (Supplement), 2010
S43
(VCGs), however not all VCGs produce aflatoxin. Identifying A. flavus
isolates to VCG is a laborious and costly enterprise. We genotyped isolates
using 19 microsatellite loci and the mating type locus, MAT, to elucidate
evolutionary relationships among 20 VCGs. In addition, we assessed the
utility of these molecular markers for initial VCG grouping. For the first
objective, isolates from 20 VCGs were obtained from A. flavus populations
associated with cottonseed in Texas and Arizona. For objective 2, 80 A. flavus
isolates from maize in Texas were independently and blindly grouped into
VCGs by both auxotroph complementation and microsatellite haplotype
similarity. Results from these studies demonstrate that the same microsatallite
markers can be used on A. flavus populations from both different geographic
regions and different plant hosts. Relationships among isolates and VCGs will
be discussed.
Characterization of the occ gene cluster required for the production of
antifungal compound occidiofungin in Burkholderia contaminans strain
MS14
G. GU (1), K. Chen (1), L. Smith (1), S. Lu (1)
(1) Mississippi State University, Mississippi State, MS, U.S.A.
Phytopathology 100:S44
plants. The infected plant showed leaf mottling, chlorotic blotch and
interveinal yellowing symptoms. The causal agent of this disease was named
Calibrachoa mottle virus (CbMV). Based on the particle morphology, dsRNA
profile and genome organization, CbMV was suggested to be a member of
genus Carmovirus, family Tombusviridae. CbMV has a single stranded,
positive-sense RNA genome of 3,919 nucleotides with five open reading
frames (ORFs). To facilitate study of carmovirus proteins at molecular level,
an infectious full-length cDNA clone of CbMV was constructed. RNA was
extracted from purified virions and used for cDNA synthesis. Full length
cDNA was constructed by using two overlapping fragments covering the
entire CbMV genome. The T7 RNA polymerase promoter sequence was
added upstream of 5’-UTR using PCR. Whole genome cDNA with fused T7
promoter sequence was cloned into pUC18 vector. Uncapped and capped in
vitro RNA transcripts derived from the full-length cDNA clone of CbMV
were infectious causing symptoms indistinguishable from those of the wildtype isolate on Chenopodium quinoa. The presence and validity of the
progeny virus were verified by ELISA, RT-PCR and nucleotide sequencing.
Characterization of the HrpG and HrpX regulons of Xanthomonas
axonopodis pv. citri
Y. GUO (1), N. Wang (2)
(1) University of Florida, Lake Alfred, FL, U.S.A.; (2) Citrus Research and
Education Center, University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S44
Occidiofungin produced by Burkholderia contaminans strain MS14 is an
octapeptide dedicated to a broad range of antifungal activities of the
bacterium. The 58-kb genomic fragment containing 18 open-reading frames
(ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin
production. Putative proteins encoded by five nonribosomal peptide
synthetase genes (occA – occE) of the gene cluster were predicted to contain
the catalytic modules responsible for the biosynthesis of occidiofungin.
Transcription of all the ORFs identified in the region except ORF1 and
ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory
genes located at the left border of the cluster. Sequence analysis revealed that
the occ gene cluster, excluding ORF1 and ORF16, shared high similarity
(99% nucleotide coverage and 91% identity) to an uncharacterized DNA
region of B. ambifaria strain AMMD. The gene cluster was not found in other
Burkholderia strains available in GenBank (nucleotide coverage < 24%).
Analysis of G+C composition and prediction using “IslandPick” indicate that
the occ gene cluster has possibly been horizontally transferred between
bacteria. More importantly, the absence of the gene cluster in clinical strains
of Burkholderia indicates that occidiofungin is not required for potential
human pathogenesis. The findings have provided insights into the
development of antifungal medicines or agricultural fungicides based on
occidiofungin.
Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus canker
and has a wide host range. The type III secretion system and effectors, which
are essential for the pathogenicity of Xac, is controlled by two transcription
regulators, HrpG and HrpX. We have been aiming at identification of the
effectors of Xac. We postulate that effector genes will be control by HrpG
and/or HrpX. In this work, we designed and conducted oligomicroarray
analysis to characterize the regulon of HrpG and HrpX. Our analyses revealed
that the expression of nearly 300 genes and 350 genes was significantly
influenced by the mutation of the two transcription regulators HrpG and
HrpX, respectively. The differentially expressed genes encode proteins
belonging to 17 functional categories including potential effectors. Besides the
known activities such as regulation of the expression of hrp and effector
genes, microarray analyses also showed that HrpG and HrpX also influence
flagellum synthesis, extracellular enzymes. Our results suggest that HrpG and
HrpX not only regulate the expression of the type III secretion system and
effector genes, but also influence genes in other functional categories during
infection.
The role of fungal endophytes in the production of natural products in
Echinacea purpurea
R. J. GUALANDI (1), K. D. Gwinn (1), B. H. Ownley (1), R. M. Auge (1)
(1) University of Tennessee, Knoxville, TN, U.S.A.
Phytopathology 100:S44
The gene FvNoxR of Fusarium verticillioides is required for its female
fertility, normal hyphal ROS localization and full virulence on maize
L. GUO (1), G. A. Kuldau (1)
(1) Pennsylvania State University, University Park, PA, U.S.A.
Phytopathology 100:S44
Echinacea purpurea is a native herbaceous perennial with substantial
economic value for its medicinal and ornamental qualities. Arbuscular
mycorrhizae (AM) are symbiotic fungi that form relationships with plant roots
and are known to enhance growth in the host. AM and other fungal
endophytes often affect stress resistance and secondary metabolism in the host
as well as the ecology of other endophytes in the plant. A newly emerging
paradigm in sustainable biotechnology is the targeted use of fungal
endophytes to enhance growth and secondary metabolism in crops. Many of
the therapeutic compounds in E. purpurea could be affected by fungal
colonization. This research tests the effects of intentional inoculation, of E.
purpurea, with the AM fungi Glomus intraradices and Gigaspora margarita
and the entomopathogenic fungal endophyte Beauveria bassiana (Bb). A
series of greenhouse experiments tested endophyte colonization and changes
in plant growth and phytochemistry. AM and Bb effectively colonized E.
purpurea with some significant interactive effects on colonization. Consistent,
substantial and significant increases in all growth parameters were observed in
AM treatments. Substantial increases in P and N fertilization were necessary
to produce AM and non-mycorrhizal samples of similar size. Bb had minor
and inconsistent effects on some growth parameters and did exhibit some
significant interactive effects with AM. Results of phytochemical analyses
will be discussed.
Fusarium verticillioides is a filamentous Ascomycete growing as a corn
endophyte and causing corn ear rot, stalk rot and seedling blight. Fumonisin
mycotoxins produced by this fungus pose a threat to animal health by causing
fatal animal diseases such as equine leukoencephalomalacia and are
associated with esophageal cancer of humans. Despite much understanding
about fumonisin production and regulation it remains largely unknown how F.
verticillioides causes disease and grows as an endophyte. Using genomic
resources, we are studying the function of several F. verticillioides genes
during fungal growth and disease development. An in silico search of the F.
verticillioides genome using the Epichloë festucae NADPH oxidase regulator
(NoxR) sequence as the probe revealed a hypothesized protein (FvNoxR)
sharing 80% sequence similarity with NoxR. Deletion of the putative FvNoxR
gene has shown pleiotropic effects. ΔFvNoxR exhibits little aerial hyphae,
reduced conidiation and female sterility, although the mutant radial growth
rate is similar to the wild type. Reactive oxygen species (ROS) level in
ΔFvNoxR is comparable to the wild type in vitro but its localization differs.
The mutant produces multiple hyphal branches on conidial germ tubes, a
possible sign of lost hyphal polarity. Importantly, ΔFvNoxR has attenuated
virulence on maize seedlings, ears and stalks. Efforts are underway to
characterize the mutant in planta fungal growth and ROS level.
Infectious RNA transcripts derived from cloned cDNA of Calibrachoa
mottle virus (CbMV)
A. GULATI-SAKHUJA (1), H. Liu (1)
(1) USDA-ARS, Salinas, CA, U.S.A.
Phytopathology 100:S44
Calibrachoa is an important new horticultural plant both in Europe and the
United States. Commercial reproduction of Calibrachoa plants and
maintenance of genetic mother stock are done by means of vegetative
propagation. A virus with spherical particles was isolated from Calibrachoa
S44
PHYTOPATHOLOGY
Fungicidal control of stem rust (Puccinia graminis f. sp. tritici) of wheat
V. GUPTA (1)
(1) South Dakota State University, Brookings, SD, U.S.A.
Phytopathology 100:S44
Stem Rust, caused by Puccinia graminis f. sp. tritici (Pgt), is one of most
devastating diseases of wheat throughout the world. The management of stem
rust with fungicides is of particular interest due to the development of highly
virulent races of Pgt in eastern Africa (e.g. Ug99). Herein we present results
from studies evaluating fungicidal activity against Pgt. First, inhibition of
urediniospore germination was evaluated in vitro for most of the
commercially available wheat fungicides as single active ingredient dilution
series. EC50 values were calculated and it was found that strobilurins were
best at inhibiting spore germination followed by the newer triazoles. Second,
application timing was evaluated in the greenhouse with fungicides applied 24
hr, 48 hr, and 96 hr before or after inoculation. All of the pre-inoculation
treatments inhibited symptom development, however the triazoles were
significantly better when applied after infection. Field experiments were
conducted in 2008 and 2009 using commercial products. In 2008, all
treatments had lower stem rust severity and higher yields in comparison to
untreated control. Tebuconazole and prothioconazole were most effective in
controlling stem rust. In 2009, no significant differences were measured,
however symptom development was limited on the untreated plots due to
below-average temperatures.
Analysis of Mycosphaerella graminicola populations from California,
Indiana, Kansas and North Dakota with mating type and SSR markers
S. GURUNG (1), S. B. Goodwin (2), M. Kabbage (3), W. W. Bockus (4), T.
B. Adhikari (1)
(1) North Dakota State University, Fargo, ND, U.S.A.; (2) USDA-ARS, Crop
Production and Pest Control Research Unit, Purdue University, West
Lafayette, IN, U.S.A.; (3) Texas A & M University, College Station, TX,
U.S.A.; (4) Kansas State University, Manhattan, KS, U.S.A.
Phytopathology 100:S45
Septoria tritici blotch, caused by Mycosphaerella graminicola, is one of the
most important foliar diseases of wheat. Genetic diversity of 333 isolates of
M. graminicola collected from spring (California, North Dakota) and winter
wheat (Indiana, Kansas) was analyzed for mating type and 17 SSR markers.
The Indiana, Kansas, and North Dakota populations were further subdivided
into two, six, and two subpopulations, respectively according to sampling sites
and years of collection. Both mating types were equally distributed in clonecorrected populations from Kansas, Indiana and North Dakota, while mating
type frequency deviated from a 1:1 ratio in the California population. Gene
diversity values for the California, Indiana, Kansas, and North Dakota
populations were 0.44, 0.53, 0.40, and 0.57, respectively. No evidence of
linkage disequilibrium was observed in all populations or subpopulations
analyzed. High gene flow was observed between the California and Kansas
(Nm = 15.914) and Indiana and Kansas (Nm = 16.89) populations. Analysis of
molecular variance showed that most genetic variation (> 82%) was within
populations and subpopulations; less than 18% occurred between the
populations or subpopulations. These results indicate that frequent sexual
recombination occurs in M. graminicola populations in spring and winter
wheat. Furthermore, all geographically separated populations of M.
graminicola are genetically similar, suggesting a panmictic structure.
Genome wide association mapping of resistance to tan spot and spot
blotch in spring wheat
S. GURUNG (1), J. M. Bonman (2), M. Acevedo (2), T. B. Adhikari (1)
(1) North Dakota State University, Fargo, ND, U.S.A.; (2) USDA-ARS, Small
Grains and Potato Germplasm Research Unit, Aberdeen, ID, U.S.A.
Phytopathology 100:S45
Tan spot, caused by Pyrenophora tritici-repentis, and spot blotch, caused by
Cochliobolus sativus, are destructive diseases of wheat worldwide. Studying
the inheritance of resistance to these diseases using bi-parental mapping
populations is time consuming and, in addition, the complex inheritance and
partial effects of the resistance makes conventional breeding difficult.
Molecular markers linked to resistance genes could facilitate resistance
breeding. The main objective of this study was to utilize association mapping
to identify molecular markers associated with tan spot and spot blotch
resistance. A total of 582 accessions of spring wheat landraces of diverse
origin from the USDA National Small Grain Collection were assessed for
resistance to tan spot (race 1 and race 5) and spot blotch at seedling stage
during 2009 and 2010 in a greenhouse at NDSU. Diversity Arrays Technology
(DArT) marker-based linkage analysis for quantitative trait analysis (QTL) of
disease resistance loci is in progress. Whether the newly identified
genes/alleles are novel or identical to those previously reported in wheat
genome will be discussed.
Changes in flavonoid biosynthetic pathway genes and anthocyanins due
to virus infection in grapevine (Vitis vinifera L.) leaves
L. R. GUTHA (1), L. F. Casassa (1), J. F. Harbertson (1), R. A. Naidu (1)
(1) Washington State University, Prosser, WA, U.S.A.
Phytopathology 100:S45
Symptoms of grapevine leafroll disease in red-fruited wine grape (Vitis
vinifera L.) cultivars consist of green veins and red and reddish-purple
discoloration of inter-veinal areas of leaves. In this study, reversetranscription quantitative PCR was used to compare the expression of
flavonoid biosynthetic pathway genes between symptomatic leaves infected
with Grapevine leafroll-associated virus-3 and uninfected green leaves in cv.
Merlot. Using two constitutively expressed reference genes as the most
invariant internal controls, we normalized the expression levels of candidate
genes in virus-infected and uninfected leaves. The expression levels of
different genes ranged from two- to fifty-fold increase in virus-infected leaves
when compared to uninfected leaves, with CHS3, F3’5’H, F3H1, LDOX,
LAR1 and MybA1 showing greater than 10-fold increase. HPLC profiling of
anthocyanins extracted from infected and uninfected leaves indicated the
presence of cyanidin 3-glucoside and malvidin 3-glucoside only in virusinfected, symptomatic leaves. Our results also showed significantly higher
levels of two flavonols in virus- infected leaves than in uninfected leaves, with
quercetin followed by myricetin being the predominant compounds. These
results suggested modulation of anthocyanin pathway genes towards de novo
synthesis of certain classes of end -products contributing to phenotypic
expression of disease symptoms.
Novel detection of Ralstonia solanacearum race 3 biovar 2 using magnetic
capture hybridization and real time-PCR
Y. HA (1), T. P. Denny (1), M. A. Schell (1)
(1) University of Georgia, Athens, GA, U.S.A.
Phytopathology 100:S45
Ralstonia solanacearum race 3 biovar 2 (R3bv2), a quarantined pathogen not
present in the U.S., is a soil-borne bacterium that causes lethal wilting of
potato, tomato, geranium and many weeds. Rapid, robust and sensitive
detection methods are needed to maintain exclusion of R3bv2. Standard and
real time-PCR (RT-PCR) can be used for specific identification of cultured
R3bv2 cells, but compounds in soil and plants inhibit amplification when
sampling directly from these sources, resulting in false negatives. To
circumvent this problem, we developed paramagnetic beads coated with
biotinylated single-stranded DNA ‘capture probes’ that anneal to R3bv2specific target sequences. Magnetic capture hybridization (MCH) using these
beads was then used to purify and enrich R3bv2 DNA for subsequently
detection by RT-PCR. Control experiments showed that the RT-PCR
detection threshold was 10-fold higher before MCH than after MCH both for
R3bv2 genomic DNA (10 fg/µl vs. 1 fg/µl) or R3bv2 bacteria suspended in
water (103 cells/ml vs. 102 cells/ml). More importantly, when R3bv2 cells
were suspended in 1% soil solution RT-PCR failed, whereas MCH followed
by RT-PCR detected as few as 103 cells/ml. Similarly, amplification failed
when R3bv2 cells were suspended in 6% potato extract, but use of MCH prior
to RT-PCR detected down to 50 R3bv2 cells/ml. Therefore, by removing
inhibitory compounds present in soil and plant samples, MCH should greatly
enhance the utility of RT-PCR for detecting R3bv2.
Non-host resistance response at the nucleosome level
L. HADWIGER (1)
(1) Washington State University, Pullman, WA, U.S.A.
Phytopathology 100:S45
In non-host resistance (innate immunity) the activation of an assortment of
pathogenesis- related (PR) genes is responsible for slowing the growth of the
invading pathogen. In the interaction, between pea endocarp and Fusarium
solani f. sp. phaseoli, total resistance develops within 6 h and is associated
with PR gene activations being initiated within 2 h. The mechanisms involved
mirror those recently observed in animal systems and may result from
alterations or remodeling of chromatin. The PR gene DRR206 activation
occurs as there is an ubiquitination/reduction of histones H2A/H2B and a
reduction in the architectural transcription factor, HMG A. The pea RNA
polymerase and these nuclear proteins can be located within the region of the
DRR206 promoter DNA and subsequently within the open reading frame at 2
and 4 h pi with chromatin immunoprecipitation (ChIP) analyses. Also there is
a simultaneous reduction in the HMG A in this region. The RNA polymerase
complex appears to be in place but “paused” at the promoter and subsequent
movement through nucleosomes is likely reinitiated by the transient
disassembly of histones H2A/H2B from DNA and in its wake a reformation of
these and other nuclear components -- all occurring within sensitive regions of
the pea chromosome.
Fine scale genetic structure of flowering dogwood in the Great Smoky
Mountains National Park
D. HADZIABDIC (1), B. M. Fitzpatrick (1), X. Wang (2), P. A. Wadl (1), T.
A. Rinehart (3), B. H. Ownley (1)
(1) University of Tennessee, Knoxville, TN, U.S.A.; (2) Texas A&M - Texas
AgriLife Research and Extension Urban Solutions Center, Dallas, TX, U.S.A.;
(3) USDA-ARS, Southern Horticultural Laboratory, Poplarville, MS, U.S.A.
Phytopathology 100:S45
Flowering dogwood (Cornus florida L.) populations have experienced severe
declines caused by dogwood anthracnose in the past three decades. Mortality
has ranged from 48 to 98%, raising the concern that genetic diversity of this
native tree has been reduced significantly. However, the response of each
species to habitat disturbance may differ greatly depending on their biological
Vol. 100, No. 6 (Supplement), 2010
S45
attributes, particularly pollen and seed dispersal ability. Nineteen microsatellite loci were used to evaluate the level and distribution of genetic
variation throughout Great Smoky Mountains National Park (GSMNP).
Significant genetic structure at both landscape and local levels were found.
We infer that two genetic clusters exist within the park, mostly separated by
the main dividing ridge of the Great Smoky Mountains. The differentiation is
statistically significant, but subtle, with gene flow evident through lowelevation corridors. Even accounting for this structure, we observed heterozygote deficiency across all loci, implying nonrandom mating at a finer scale.
This pattern of variation implies that pollination occurs primarily between
related individuals despite wide dispersal of seeds.
Application rate and interval impact the efficacy of Heritage 50WDG
fungicide for the control of Alternaria leaf spot on marigold
A. K. HAGAN (1), J. R. Akridge (2)
(1) Auburn University, Auburn, AL, U.S.A.; (2) Brewton Agricultural
Research Unit, Brewton, AL, U.S.A.
Phytopathology 100:S46
Heritage 50WDG at 0.04, 0.08, and 0.16 g ai/l when applied at 2-, 3-, and 4wk intervals was compared with Daconil Weather Stik 6F and Eagle 40W for
control of Alternaria leaf spot (Alternaria tagetica) in field-grown marigold in
Alabama. While the French Dwarf marigold (Tagetes patula) ‘Little Hero’
was used in 2001, the American marigold (T. erecta) ‘Discovery Yellow’ was
planted in the remaining study years. Fungicides were applied to run-off with
a CO2-pressurized sprayer from June 6 to September 1, 2001; April 30 to
August 2, 2002; April 30 to September 17, 2003; and May 14 to September
10, 2004. When applied at 2-wk intervals, all rates of Heritage 50WDG gave
better disease control in all years than Daconil Weather Stik 6F or Eagle 40W,
which showed no activity against this disease. In two of three years, Daconil
Weather Stik 6F applied weekly decreased disease severity compared with the
non-fungicide treated control, but when applied at 2-wk intervals, disease
levels were similar to the non-fungicide treated control in two of four years.
Alternaria leaf spot intensified when Heritage 50WDG rates declined from
0.16 to 0.04 g ai/l and intervals increased from 2- to 4-wk. At 0.04 g ai/l,
disease control with Heritage 50WDG was better at the 2- than at the 3- and
particularly 4-wk schedule. Regardless of the level of disease, Heritage
50WDG at 0.08 and 0.16 g ai/l when applied every 3-wk gave effective
control of Alternaria leaf spot on marigold.
Yield response and control of crown rust on oats with fungicides
A. K. HAGAN (1), M. B. Pegues (2)
(1) Auburn University, Auburn, AL, U.S.A.; (2) Gulf Coast Resarch and
Extension Center, Fairhope, AL, U.S.A.
Phytopathology 100:S46
Efficacy of selected fungicides for the control of crown rust was assessed on
the rust susceptible oat variety ‘Coker 227’ in 2008 and 2009. The
experimental design was a randomized complete block with four replications.
Fungicides were applied with CO2-pressurized back pack sprayer at full flag
leaf extension (GS 9) and/or head extension (GS 10.5). Crown rust was rated
on a 0 to 10 scale where 0 = no disease, 1 = 1 to 10% leaf area symptomatic to
10 = flag leaf dead. In both years, all fungicide treatments had lower rust
ratings than the control. In 2008, best rust control were obtained with two
applications of Tilt 3.6E and Stratego 2.08E, while single applications of 3.1
and 6.2 fl oz/A Quadris 2.08SC were less ineffective. Highest yield gains
were obtained with Tilt 3.6F, Stratego 2.08E, Headline 2.09E, and Quilt
1.67E. For 2009, single applications of Tilt 3.6E, Stratego 2.08E, Headline
2.09E and Quadris 2.08SC at GS 10.5 but not GS 9 gave the same level of rust
control as two the two application programs with the same fungicides. Despite
differences in rust severity, yields with the two applications of Tilt 3.6E,
Stratego 2.08EC, Headline 2.09E at 6 fl oz/A, and Quadris 2.08SC were
similar to that with a single GS 9 or GS 10.5 application of the same
fungicide. Yield gains up to 46 and 33 bu/A were seen with fungicides in
2008 and 2009, respectively, for a net income increase of up to $145 to $203
per acre for seed oats valued at $5.30/bu.
Genetic diversity of Iranian Fusarium oxysporum f. sp. ciceri by random
amplified polymorphic DNA or vegetative compatibility groups
S. HAGHIGHI (1)
(1) Islamic Azad University, Damghan Branch, Tehran, IRAN
Phytopathology 100:S46
Fusarium wilt of chickpea is a devastating disease in chickpeas growing in
different regions of Iran. This fungal disease is caused by Fusarium
oxysporum f. sp. ciceri and this pathogen can reduce yield by 15%. In order to
study genetic diversity of this pathogen, thirty isolates of Fusarium
oxysporum f. sp. ciceri were selected from different origins of Iran. In vitro
pathogenicity tests of the isolates were performed using a root-dip assay and
isolates were classified into three categories of highly, moderate and weakly
virulent groups. Genetic diversity was analysed based on RAPD-PCR using
S46
PHYTOPATHOLOGY
ten random primers. Cluster analysis of DNA fragments were performed using
MVSP software and a UPGMA method with jaccard coefficient. Results of
RAPD-PCR showed a high genetic diversity among the isolates and clustered
them into 17 groups. There was a correlation between RAPD groups and
geographical localization of the isolates. The basis for VCG assignment was a
complementation reaction between nit1 or nit3 and nitM mutants on minimal
medium. Pairs of isolates which exhibited vigorous site of the two nit mutantmycelia- indicating the formation of heterokaryon-were determined as
vegetatively compatible and by using this method; the isolates were classified
into eight groups. In conclusion, the results of isolate classification by RAPD
and VCG are similar but, the RAPD technique is more efficient than VCG
method and a population genetic analysis can be performed with greater
precision.
Spread of huanglongbing through citrus and citrus relatives in retail
nurseries and garden centers
S. E. Halbert (1), K. L. Manjunath (2), C. Ramadugu (3), R. F. LEE (2)
(1) Florida Division of Plant Industry, Gainesville, FL, U.S.A.; (2) USDA
ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside,
CA, U.S.A.; (3) Department of Botany and Plant Sciences, University of
California, Riverside, Riverside, CA, U.S.A.
Phytopathology 100:S46
Huanglongbing (HLB or greening) was first found in Florida in 2005, has
spread to all the citrus growing counties in the state. Infected nursery trees and
psyllid vectors carrying the HLB associated bacteria need to be avoided for
successful management of the disease. In the present study, over 1200 psyllid
(Diaphorina citri) samples were collected from plants for sale in retail centers
and garden centers in Florida over a period of four years and tested for the
presence of HLB associated “Candidatus Liberibacter asiaticus” (LAS) by
real time PCR. 5–12% of psyllid samples tested in different years were
positive for LAS. About 8% of psyllid samples collected from citrus, 10%
from Murraya paniculata and 6% from other plants were positive for LAS. M.
paniculata is a preferred host of D. citri and is used widely as an ornamental
plant in Florida. Presence of LAS positive samples in counties having no
commercial citrus provides additional evidence for the role of retail nurseries
and garden centers in spreading HLB throughout the state of Florida. The
importance of regulation to prevent both the psyllids and the HLB associated
Liberibacters in retail nurseries in regions where the disease has not yet
established will be discussed.
Assessment of agreement between apple scab and fire blight forecasts
based on SkyBit predictions and on-site weather records
N. O. Halbrendt (1), J. W. Travis (1), H. K. NGUGI (1), J. Russo (2)
(1) Penn State University, Biglerville, PA, U.S.A.; (2) ZEDX, Inc., Bellefonte,
PA, U.S.A.
Phytopathology 100:S46
SkyBit is a site-specific electronic weather service that delivers daily weather
and pest management information to fruit growers in the mid-Atlantic region.
The goal of this project was to assess the quality and reliability of the disease
forecast information delivered to growers via SkyBit. We evaluated weather
data and disease infection predictions delivered via SkyBit in comparison with
data collected on-site at the Penn State Fruit Research & Extension Center,
Biglerville, Pennsylvania. Overall, forecasts of scab infection periods based
on SkyBit remote-sensed data showed significant agreement with predictions
from a Spectrum Technologies model using on-site data (χ2 = 2.0; P = 0.157,
based on McNemar’s test), and weakly agreed with forecasts based on the
Mills Table scab model (χ2 = 3.6; P = 0.059). Fire blight infection predictions
based on SkyBit (with ‘++’ counted as an infection period) did not agree with
MaryBlyt predictions (χ2 = 6.4; P < 0.011). However, when the high risk of
infection events (H) from the MaryBlyt model were counted as infection
periods, predictions based on the SkyBit fire blight model agreed with
MaryBlyt forecasts (χ2 = 0.333; P = 564). Estimates of daily rainfall from
SkyBit closely agreed (r = 0.882; P < 0.001) with on-site records but SkyBit
under-estimated the actual amounts. We conclude that SkyBit delivers reliable
data for predicting scab infection periods and daily rainfall, but it tends to
overestimate fire blight infection periods.
Molecular interactions determining broad-spectrum partial late blight
resistance in potato
D. HALTERMAN (1), Y. Chen (2)
(1) USDA/ARS, Madison, WI, U.S.A.; (2) Madison, WI, U.S.A.
Phytopathology 100:S46
The potato late blight resistance (R) gene RB (Rpi-blb1) belongs to the
valuable class of plant R genes that confer resistance to a broad spectrum of
pathogen isolates. RB protein recognizes the presence of members of the
Phytophthora infestans effector family IPI-O to elicit resistance. We have
studied IpiO diversity from 40 different P. infestans isolates collected from
Guatemala, Thailand, and the United States. We have found that all of the
isolates contain IPI-O variants that can be recognized by RB. However, some
of these isolates contain an extraordinarily large number of variants.
A few isolates also contain an IPI-O variant (IPI-O4) that is not recognized by
RB. Isolates containing IPI-O4 are able to overcome resistance in RBcontaining potato leaves to cause significantly more disease than isolates
that do not contain IPI-O4, even when other IPI-O proteins are present. We
show that the presence of IPI-O4 blocks the ability of RB to recognize the
presence of other IPI-O variants through direct interaction with the resistance
protein, thereby preventing programmed cell death related to the resistance
response.
Identification of novel regulatory genes of Burkholderia glumae for
virulence factors
J. HAM (1), I. Barphagha (1), H. Karki (1), B. Shrestha (1), R. Melanson (1),
R. Chen (1)
(1) Louisiana State University AgCenter, Baton Rouge, LA, U.S.A.
Phytopathology 100:S47
Burkholderia glumae is the major causative agent of bacterial panicle blight of
rice. Symptoms of this disease include panicle blight, sheath rot and seedling
blight. The phytotoxin, toxoflavin, and lipase are the two most important
virulence factors of this pathogen. B. glumae also produces extracellular
polysaccharides (EPS), which play an important role in bacterial pathogenesis
in many plant or animal pathogenic bacteria. The quorum-sensing mechanism
involving acyl-homoserine lactones is known to be a major regulatory system,
which governs the production of virulence factors and the motility of B.
glumae. In an attempt to identify additional regulatory genes of this pathogen
for virulence, the B. glumae 336-gr1 genome was randomly mutagenized with
a mini-Tn5 derivative, mini-Tn5gus, and the mutants showing altered
phenotypes in the production of known virulence factors were screened. From
more than 20,000 random mutants, 56 mutants have shown reduced or
increased production of toxoflavin, lipase or EPS. As the complete DNA
sequence of the B. glumae BGR1 genome is publicly available, the genes
mutated with mini-Tn5gus could be easily identified by sequencing the
flanking regions of the transposon. From this study, several novel regulatory
elements for the B. glumae virulence factors have been identified. Currently,
we are investigating the functions of these newly discovered regulators on the
expression of various virulence genes and the bacterial pathogenesis of B.
glumae.
Over-expression of a maize WRKY transcription factor and its effect on
the responses of Arabidopsis to biotic and abiotic stresses
P. HAN (1), Y. Xie (2), Z. Chen (2)
(1) Louisiana State University, Baton Rouge, LA, U.S.A.; (2) Baton Rouge,
LA, U.S.A.
Phytopathology 100:S47
WRKY proteins are comprised of a large family of transcription factors
identified specifically from the plant kingdom. WRKY transcription factors
contain one or two conserved WRKY domains. Evidence is showing that the
transcription of WRKY gene plays important roles in many biological
processes, such as senescence, abiotic stress and pathogen- triggered signal
transduction cascades in numerous plant species. In this study, we selected
one maize WRKY transcription factor (PTZm 631) that showed a significant
upregulation in response to Aspergillus flavus infection than other maize
WRKY transcription factors in a preliminary field inoculation study. Two
over-expression vectors of this WRKY transcription factor under the control
of d35S and ZmPR10 promoters (pCambia 1302-d35S-WRKY and pCambia
1302-PZmPR10-WRKY) were constructed. After transformation into
Arabidopsis, homozygous transgenic plants were selected and the WRKY
expression was determined using real time PCR. We are currently testing the
responses of these transgenic Arabidopsis plants over-expressing WRKY to
abiotic stresses including SA, H2O2, NaCl, ABA, KT, heat, and biotic stress
such as Pseudomonas syringae.
Study on the genetic diversity within Phytophthora capsici with nuclear,
mitochondria and SNPs markers in New Mexico
S. Hanson (1), M. PEIMAN WILLIAMS (1)
(1) New Mexico State University, Las Cruces, New Mexico, U.S.A.
Phytopathology 100:S47
Phytophthora species are devastating plant pathogens in both agricultural and
natural environments. Phytophthora capsici is a severe pathogen on chile
peppers grown in the desert southwest where it can cause up to 100% losses in
severely affected fields. Pathogenicity and phenotypic studies have indicated a
high level of diversity among P. capsici strains isolated from chiles in the
desert Southwest. Prior work with several commonly used phylogenetic
molecular markers such as ITS regions, B-tubulin, LUS, and Cox-II showed
that these markers were uninformative as they were unable to distinguish
strains of P. capsici based on location, time, or phenotypes such as
pathogenicity. The development of informative molecular markers for
distinguishing different strains of P. capsici would be useful for resistance
breeding and epidemiological studies and is the long term goal of this
project. Herein we report the evaluation of several single-nucleotide
polymorphism (SNP) markers for their utility as molecular markers that are
predictive of P. capsici phenotypic diversity. Ongoing studies suggest that
several of these markers may be useful as markers to identify different strains
of P. capsici.
Sugar beet seedling damping-off in Michigan
L. E. HANSON (1), J. McGrath (1)
(1) USDA ARS, East Lansing, MI, U.S.A.
Phytopathology 100:S47
Seedling damping-off is a constraint to sugar beet production, particularly in
the humid eastern U.S. growing region. On average, only 60% of the seed
planted in Michigan produces a final plant stand. A large part of this loss has
been attributed Aphanomyces cochlioides, but little screening has been done.
To improve understanding of damping-off, seedlings with symptoms were
collected from commercial beet fields and research plots in 2008 and 2009.
Hypocotyls and roots were surface disinfested, cut into sections, and plated on
potato dextrose agar with antibiotics to restrict bacterial contamination or in
sterile distilled water with sterile millet seeds to bait for oomycetes. Hyphal
tip transfer or single spore isolation were used to obtain pure cultures.
Morphological and molecular methods were used to identify and characterize
isolates. Rhizoctonia solani was the most commonly isolated pathogen in
2008, followed by Fusarium oxysporum, A. choclioides, Phoma betae and
Pythium spp. in the order listed. In 2009, A. cochlioides was the most
commonly isolated pathogen, followed in decreasing frequency by Fusarium
spp., Pythium spp., Rhizoctonia solani and Phoma betae. Isolates of all the
above genera were pathogenic on beet seedlings in the greenhouse. The R.
solani isolates collected included AG 4, AG 2-2 IV and AG 2-2 IIIB. Thus, in
addition to Aphanomyces, a complex of seedling diseases contribute to stand
problems in Michigan.
Effect of temperature on survival of chlamydospores and oospores of
Phytophthora species in irrigation water
W. HAO (1), C. Hong (1)
(1) VPI & State University, Virginia Beach, VA, U.S.A.
Phytopathology 100:S47
Plant pathogens, especially Phytophthora species in re-circulating irrigation
water, present significant health risks to floral crops. One of current
technologies for water decontamination is heat treatment, which is effective
and has minimal human health and environmental hazards. The primary
objective of this study was to examine whether water temperature required to
inactivate major pathogens in re-circulated irrigation water can be lowered
from 95°C as recommended in the current protocols. Specifically, we
investigated the effect of water temperature on survival of chlamydospores
and oospores using two of the most destructive species, P. nicotianae and P.
pini. Oospores of P. pini did not survive after 12 h of heat treatment at about
42°C, and the majority of chlamydospores of P. nicotianae did not survive 24
h at the same temperature. These results suggest that water temperature for
heat treatment may be lowered substantially from 95°C without sacrificing
efficacy. This research is being expanded to include other stages of the life
cycle of Phytophthora species and other major groups of pathogens as well as
trials in greenhouse settings.
Characterization of Salmonella enterica genes, STM0978 and STM0693,
with a role in plant colonization
L. HAO (1), J. Barak (1)
(1) University of Wisconsin-Madison, Madison, WI, U.S.A.
Phytopathology 100:S47
Salmonellosis outbreaks caused by consumption of produce are increasing.
The causal agent of the disease, Salmonella enterica, is not a plant pathogen,
but it can utilize specific molecular mechanisms to attach and colonize plants
rather than passive contamination which can be rinsed away. In our study, two
S. enterica serovar Typhimurium function unknown genes were studied,
STM0978 and STM0693. In individual bacterial strain inoculation
experiments, ΔSTM0978 was defective in alfalfa sprout colonization at 48 h,
with 2 logs reduction in population compared to the wild type. ΔSTM0693 had
no significant difference from the wild-type. However, in a co-inoculation
experiment with ΔSTM0693 and the wild type, ΔSTM0693 had a significant
lower population than the wild-type with 1 log reduction. Such defective
phenotypes were not simply due to a growth defect, since growth of both
mutants had no obvious difference from the wild type in 48 h alfalfa exudates.
Further examination of ΔSTM0978 found it defective in growth in M9
minimal media, swimming, and swarming compared to the wild-type. The
function of both STM0978 and STM0693 is still underway. We are interested
in targeting the essential compounds that are needed by the pathogen to
survive in plants. Characterization of such genes will provide critical
Vol. 100, No. 6 (Supplement), 2010
S47
understanding of the pathogen survival mechanism outside its warm-blood
host and help to develop useful strategies to prevent plant contamination in the
future.
Oxidized forms of silver as safe, effective seed treatments
M. W. HARDING (1), D. A. Sowa (2), R. J. Howard (3), M. E. Olson (2)
(1) Innovotech Inc., Brooks, AB, CANADA; (2) Innovotech Inc., Edmonton,
AB, CANADA; (3) Alberta Agriculture and Rural Development, Brooks, AB,
CANADA
Phytopathology 100:S48
Metallic fungicides have a long history of agricultural use, beginning with
copper in the late 1800’s in France for control of grape downy mildew. Silver,
on the other hand, was not registered as a pesticide until 1954 and, at the time
of this report, no silver-based fungicides were currently registered for
agricultural use in North America. Innovotech Inc. has demonstrated that
highly oxidized, silver-based compounds are excellent seed treatment
compounds for eradicating seed-borne bacteria and fungi. For example,
control of seed-borne bacterial blights on dry beans caused by Pseudomonas
syringae pv. syringae, P. syringae pv. phaseolicola, Xanthomonas axonopodis
pv. phaseoli and bacterial wilt caused by Curtobacterium flaccumfaciens pv.
flaccumfaciens can be readily controlled or eradicated when seeds are coated
with a 0.5% aqueous solution of oxysilver nitrate. Furthermore, silver-based
seed treatments are effective on other crops, including soybean, tomato,
ginseng and potato. These silver-based seed treatments are manufactured as
wettable powders, which are effective at very low concentrations, i.e. 10 to
100 times lower than copper. Silver powders are compatible when tank mixed
with many existing seed treatment fungicides and are very convenient to mix
with flowable formulations such as Apron Maxx® RTA.
Development of a field inoculation method for Pythium blight of snap
beans useful for field efficacy trials
L. HARRISON (1), S. L. Rideout (1)
(1) Virginia Tech, Painter, VA, U.S.A.
Phytopathology 100:S48
Pythium blight has become a severe disease threatening snap beans production
in important growing areas, such as the Eastern Shore of Virginia (ESV).
However, no effective in-season foliar fungicides currently have a Section 3
Federal label for control of Pythium blight. Labeling of fungicides for control
is hindered by the difficulty associated with conducting trials with the
pathogen(s), which occurs sporadically and clustered in the field. Different
inoculum substrates and concentrations were evaluated in order to develop an
inoculation technique that produces more uniform disease in field trials. Over
3 summers, substrates inoculated with an ESV Pythium aphanidermatum
isolate were evaluated in the field, including sterilized soil/oatmeal (2% by
weight), vermiculite/V8 juice (5:3 weight to volume), and long grain
rice/water (5:3.6 weight to volume). Each inoculum substrate was applied at
rates of 0, 2,500, 5,000, and 10,000 cfu at plant flower bloom. Disease
incidence was recorded as percentage of diseased 0.3 m segments (foot rows).
The V8/vermiculite inoculum substrate (5,000 cfu) consistently caused ~50%
disease in each field trial. This treatment was used to provide disease pressure
in a Pythium blight fungicide efficacy trial in 2009. Results indicated that
azoxystrobin, cyazofamid, pyraclostrobin, and materials in the phosphonate
class were most effective at reducing disease incidence and improving
marketable yields when compared to the non-treated control. Field efficacy
trials will be repeated in 2010.
Managing Rhizoctonia root and crown rot in Nebraska utilizing
azoxystrobin applications based on soil temperature measurements
R. M. HARVESON (1)
(1) University of Nebraska, Scottsbluff, NE, U.S.A.
Phytopathology 100:S48
Rhizoctonia root and crown rot, caused by Rhizoctonia solani, is the most
widespread, consistently damaging sugar beet disease in Nebraska, and causes
both a seedling disease and two different phases of root rot later in season.
These two phases include a crown rot, and a tip rot of the tap root beneath the
soil surface. Because of the diversity of pathogen forms observed, making
fungicide recommendations based on plant growth stage or chronological time
of the season is difficult and impractical. Therefore, a study was begun in
2009 with the purpose of evaluating optimal timing for making fungicide
applications based on measurement of soil temperature. Spray treatments were
applied based when 10 cm soil temperatures averaged 15°, 18°, 21°, and 24°C
for 3 sequential days, with two additional treatments consisting of an
untreated check and spraying at symptom expression. Data collected included
multiple disease counts, disease severity ratings assigned at harvest, and
sucrose and root yield determinations. Sugar yields and numbers of diseased
plants were significantly improved with the use of azoxystrobin when soil
temperatures reached 15°, 18° and 21°C, compared to controls and spraying
after symptom development. Therefore the general concept appears to work
S48
PHYTOPATHOLOGY
adequately for Nebraska conditions; however improvements may still be
realized with some further modifications.
A survey to determine predominant diseases found in Nebraska
sunflower production
R. M. HARVESON (1)
(1) University of Nebraska, Scottsbluff, NE, U.S.A.
Phytopathology 100:S48
Sunflower is a well-adapted crop for the Central High Plains and can be
successfully cultivated in both dry-land and irrigated areas. It additionally fits
well in many production systems as an alternative crop in dry-land wheat
rotations. Sunflowers are also being increasingly used to lengthen the
traditional irrigated rotations of dry beans, corn and sugar beets. As would be
expected, the increase in acreage also increases the potential for disease
problems to occur. Thus it would be important to develop a knowledge base
for the prominent diseases found in the state. Thus a comprehensive disease
survey was conducted in Nebraska production fields during the 2009 season.
The survey consisted of 30 different fields, including 20 that were irrigated
and 10 that were non-irrigated. Twenty-five of the fields were surveyed at
least twice to correspond with different plant growth stages. Rust was the
most commonly found, widespread disease throughout Nebraska production
fields in 2009. Other familiar, expected diseases were identified including
white mold, and Rhizopus head rot, but not at high levels. Several new and/or
unknown diseases were additionally found including Verticillium wilt, apical
chlorosis, downy mildew, several distinct leaf spots, and numerous root and
stem/stalk rots.
Thousand cankers disease of walnut: Status in California
J. K. HASEY (1), R. M. Bostock (2), T. J. Michailides (3)
(1) University of California, Yuba City, CA, U.S.A.; (2) University of
California, Davis, CA, U.S.A.; (3) University of California, Parlier, CA,
U.S.A.
Phytopathology 100:S48
Thousand cankers disease of eastern black walnut (Juglans nigra) and related
Juglans spp. has emerged as a disease of significant concern in the western
U.S. The disease is caused by a newly described fungal pathogen, with a
proposed name of Geosmithia morbida, and is spread from attacks by the
walnut twig beetle (WTB, Pityophthorus juglandis) with subsequent canker
formation in the phloem. These cankers eventually coalesce to girdle the
stems and branches. Trees usually die within three years of initial symptom
development that include upper crown yellowing and dark bark staining. The
thousand cankers disease was first confirmed in CA in June, 2008 in Yolo Co.
Since then, the beetle-fungus complex has been confirmed in many counties
distributed across the state on four black walnut species, English walnut,
and/or seedling Paradox hybrid walnut rootstock. English walnut planted for
commercial nut production does not appear to be a preferred host for the
beetle. Botryosphaeria spp. that can cause cankers and limb dieback on
English walnut have also been isolated from some branches infected with
thousand cankers. WTB is a native bark beetle first collected in 1959 in Los
Angeles Co., but its association with Geosmithia spp. in CA has only recently
been documented. What is unclear is why thousand cankers disease has
emerged on such a wide scale in California and the western U.S.
A multiplexed, probe-based quantitative PCR assay for DNA of
Phytophthora sojae
J. S. HAUDENSHIELD (1), G. L. Hartman (1)
(1) USDA ARS, Urbana, IL, U.S.A.
Phytopathology 100:S48
Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and postemergence damping-off, and sometimes foliar blight in soybean (Glycine
max). Crop loss may approach 100% with susceptible cultivars. We report
here the development of a unique quantitative PCR assay specific to DNA of
P. sojae, and a matching exogenous control, suitable for multiplexing with
other similar pathogen assays. The primers (previously reported) and probe
for this fluorogenic, 5’-exonuclease assay target the DNA sequences of a
gypsy-like transposable retroelement present in P. sojae. The assay exhibited
a limit of detection of under 34 fg total P. sojae DNA (0.5 genome) in serial
dilutions, and suggested that up to 10 copies of the target retroelement were
present per genome. Losses during DNA extraction effected a practical
detection limit of four zoospores per sample. The assay positively detected
DNA from 13 different P. sojae isolates pathogenic on soybean, and excluded
from detection 17 other species of Phytophthora, as well as 13 fungal species
pathogenic on soybean. The exogenous internal control target validated
negative calls, and the assay was successfully multiplexed with two additional
assays to simultaneously detect DNA from the fungus Fusarium virguliforme
and the nematode Heterodera glycines. P. sojae DNA is readily extracted
from infested soil, seed and plant debris, and may now be quantified by realtime PCR for diagnostic, forensic or research purposes.
Investigating altered triazole sensitivity in Rhynchosporium secalis
N. Hawkins (1), H. J. Cools (1), M. W. Shaw (2), H. Sierotzki (3), B. A.
FRAAIJE (1)
(1) Rothamsted Research, Harpenden, UNITED KINGDOM; (2) University
of Reading, Reading, UNITED KINGDOM; (3) Syngenta Crop Protection,
Stein, SWITZERLAND
Phytopathology 100:S49
Rhynchosporium secalis is a fungal pathogen causing barley leaf blotch or
scald. It is highly economically damaging, causing yield losses of up to 40%
when untreated. Control strategies generally rely on a combination of more
resistant barley varieties and fungicide use, and the triazoles are a major
fungicide group for the control of this disease. However, a reduction in
sensitivity to some triazole fungicides had been reported in the field. This
study aimed to characterise the in vitro triazole sensitivity of R. secalis
isolates, and to identify the molecular mechanisms associated with sensitivity
differences. A fungicide sensitivity bioassay was developed for R. secalis.
Sensitivity tests revealed ten- to 100-fold reductions in in vitro sensitivity to
some triazoles over the last 10–15 years. Rhynchosporium secalis has two
copies of the gene, CYP51, encoding the triazole target site. Investigations
into the roles of these genes in triazole sensitivity will be presented.
Identification and application of the rice broad-spectrum blast resistance
gene Pigm
Z. HE (1), Y. Deng (2)
(1) Shanghai Institutes for Biological Sciences, CAS, Shanghai, PRC
PEOPLES REP OF CHINA; (2) Shanghai, PRC PEOPLES REP OF CHINA
Phytopathology 100:S49
Rice blast is one of the most destructive diseases of rice. Identification and
utilization of broad-spectrum resistance genes has been the most effective and
economical approach to control the disease. A native Chinese variety, GM4,
was identified with broad-spectrum and durable resistance. Map-based
cloning identified that the locus Pigm with 13 NBS-LRR members confers
broad-spectrum blast resistance and had undergone duplication during the
evolution of the resistance gene cluster. In the Pigm locus, Pigm1 and Pigm2
control whole stage resistance to rice blast including and leaf and neck blast
on the seedling and mature rice stage, and Pigm3 confers the resistance to
neck blast that leads to large loss of grain yield. Genetic and transcriptional
analysis suggested that broad-spectrum resistance might be attributed to the
different expression pattern of three R genes pyramided in the Pigm locus.
Meanwhile we have succeeded in developing elite hybrid rice lines with
broad-spectrum blast resistance using molecular markers-assisted selection for
Pigm, indicating good potential of the gene in rice breeding.
Evaluation of leaf removal timing and method and gibberellic acid for
grape bunch rot management
B. HED (1), H. K. Ngugi (2), J. W. Travis (2)
(1) Lake Erie Regional Grape Res & Ext Ctr, North East, PA, U.S.A.; (2)
Penn State Fruit Research and Extension Center, Biglerville, PA, U.S.A.
Phytopathology 100:S49
Bunch rot caused by Botrytis cinerea is an important disease of wine grapes
worldwide. Bunch rot development is strongly influenced by the compactness
of clusters, which can be modified to reduce the disease. For 3 seasons, we
evaluated cluster zone leaf removal timing (trace bloom, 2–3 weeks post
bloom, or veraison) and method (by hand or Gallagher leaf blower) and bloom
gibberellin sprays (5 or 10 ppm), for effects on cluster compactness and
Botrytis bunch rot development on Vitis vinifera ‘Chardonnay’ grapevines.
All treatments including the check received two Botrytis specific fungicides;
cyprodinil at pre-close and fenhexamid at veraison. Botrytis bunch rot
decreased the earlier the leaf removal was performed. Compared to the check,
leaf removal at trace bloom significantly reduced (P < 0.05) bunch rot
incidence in every season (by an average of 80 percent), and reduced cluster
compactness and bunch rot severity in 2 of 3 seasons. Leaf removal at trace
bloom was as effective at reducing bunch rot as 2 additional fungicides
(cyprodinil at bloom and fenhexamid at pre-harvest), suggesting potential for
reducing fungicide use. There was no significant effect of leaf removal
method on bunch rot. Gibberellin reduced bunch rot by an average of 33 to 35
percent over the check, but the reductions were significant only at 5 ppm in 1
of 3 seasons. Gibberellin at 10 ppm significantly reduced cluster compactness
in 2 of 3 seasons, when compared to the check.
Management of bunch rot of Vignoles and Chardonnay grapes with
gibberellic acid
B. HED (1), H. K. Ngugi (2), J. W. Travis (2)
(1) Lake Erie Regional Grape Res & Ext Ctr, North East, PA, U.S.A.; (2)
Penn State Fruit Research and Extension Center, Biglerville, PA, U.S.A.
Phytopathology 100:S49
Harvest bunch rot of wine grapes caused by Botrytis cinerea is a perennial
problem in the eastern U.S. that can severely impact wine quality. Previously,
we documented a strong relationship between cluster compactness and the
incidence and severity of bunch rot on Vignoles grapes. The goal of this study
was to investigate the effectiveness of gibberellic acid (GA) applications to
loosen clusters as a means for controlling bunch rot on Vignoles and
Chardonnay grapes. On Chardonnay, GA applications at 10 or 25 ppm
significantly (0.0422 ≤ P ≤ 0.0062) reduced cluster compactness in a 3-year
study with bloom applications being more effective than pre-bloom
applications. On Vignoles, all bloom applications and the 25 ppm pre-bloom
treatments reduced compactness in every year. Depending on the year, GA
applications had mixed effects on the incidence and severity of bunch rot on
Chardonnay. On Vignoles, combining GA treatments with two fungicide
applications resulted in better control of harvest rots than the use of two
additional fungicides in 2006 and 2008. Cluster compactness accounted for
52.7% of the variation in the incidence of bunch rot on Chardonnay, between
77.2 and 89.4% on Vignoles, and was negatively correlated (0.731 ≤ r ≤
0.857; P < 0.0001) with percent spray coverage on berries. In spite of
repeated treatment of the same vines for four consecutive years, no negative
effects on yield from GA treatments were noted on Vignoles in three of the
four years.
Ethylene biosynthesis and its effect on rice resistance to fungal infection
E. E. HELLIWELL (1), Q. Wang (2), Y. Yang (2)
(1) Department of Plant Pathology and Huck Institutes of Life Sciences,
Pennsylvania State University, State College, PA, U.S.A.; (2) Department of
Plant Pathology and Huck Institutes of Life Sciences, Pennsylvania State
University, University Park, PA, U.S.A.
Phytopathology 100:S49
Recent evidences suggest that ethylene (ET) may play a positive role in host
resistance to Magnaporthe oryzae, the causal agent of rice blast disease. In the
ET biosynthetic pathway, the rate-limiting step is the conversion of Sadenosylmethionine (AdoMet) to 1-aminocyclopropane-1-carboxylic acid
(ACC) by ACC synthase (ACS). Among the six ACS genes in rice,
OsACS1 and OsACS2 are induced by M. oryzae within 24 hours of
inoculation. In this study, we have taken the transgenic approach to further
determine the role of ET biosynthesis in rice resistance to fungal infection. Using the RNA interference (RNAi) method, transgenic rice lines
were generated with suppressed expression of single (OsACS2) and double
(OsACS1+2) ACS genes. These RNAi lines have much lower levels of
ethylene production as compared to nontransformed control lines. In
addition, transgenic rice lines with inducible overexpression of OsACS2 and
increased levels of ET production were generated by placing the transgene
under the control of a strong pathogen-inducible promoter. Currently, both
ET-deficient and ET-overproducing lines are being evaluated for
altered defense gene expression and host resistance to rice blast and
sheath blight (Rhizoctonia solani). These transgenic lines will be valuable
tools in elucidating the importance of ethylene biosynthesis in rice disease
resistance.
Effect of an at tassel fungicide application on Aspergillus and Fusarium
spp. infestations in harvested field corn in Mississippi
A. Henn (1), T. W. ALLEN (2), D. M. Ingram (3)
(1) Mississippi State University, Starkville, MS, U.S.A.; (2) Mississippi State
University, Stoneville, MS, U.S.A.; (3) Mississippi State University, Raymon,
MS, U.S.A.
Phytopathology 100:S49
Currently, fungicide use in field corn (Zea mays) to prevent diseases caused
by Aspergillus flavus and Fusarium spp. relies on seed treatments. The recent
shift in Mississippi production hectares from cotton to corn has been
associated with increased marketing of strobilurin fungicides for numerous
non-disease related issues and aflatoxin reduction. This marketing strategy
occurred in response to a reported “plant health” benefit from a carefully
timed, tassel (VT) fungicide application. However, unless an application
reduced the levels of toxin producing fungi then the resultant toxin would
likely still occur in the corn plant. As part of a larger project conducted from
2007 to 2009 to determine yield response to a VT strobilurin application, the
presence of Aspergillus, Fusarium and other fungi were quantified from
harvested corn. Large plot trials were conducted on producers’ fields in the
Mississippi Delta and small plot trials on experiment stations. Fungicide
applications were made at VT using azoxystrobin + propiconazole (as Quilt),
propiconazole (as Propimax), and pyraclostrobin (as Headline) along with an
untreated control by airplane (n = 15) or by ground (n = 6). Trial locations
contained at least four replicates. Propiconazole and pyraclostrobin
significantly reduced Aspergillus and Fusarium colony numbers compared to
the untreated. However, Aspergillus colony numbers were significantly
increased with an azoxystrobin + propiconazole treatment.
Vol. 100, No. 6 (Supplement), 2010
S49
Epidemiology of potato zebra chip in the Texas Panhandle
D. C. HENNE (1), F. Workneh (1), C. Rush (1)
(1) Texas AgriLife Research, Bushland, TX, U.S.A.
Phytopathology 100:S50
“Zebra Chip” (ZC), an emerging disease of potato in the U.S., is caused by the
phloem-limited fastidious bacterial endosymbiont Candidatus Liberibacter
solanacearum (CLs), which is vectored by the potato psyllid, Bactericera
cockerelli. ZC leads to early plant death and causes internal tuber necrosis that
renders them unmarketable. Although first observed in south Texas as early as
2000, no information was available concerning spatial and temporal incidence
of ZC in potato fields. Therefore, to assess the incidence of ZC, studies were
performed from 2007–2009 in potato fields at two commercial production
areas in the Texas Panhandle. It was determined that, under field conditions,
ZC-affected plants typically do not express symptoms of the disease until after
flowering (although the vector is present much earlier), whereupon symptoms
increase monotonically over several weeks to a plateau. Spatial incidence of
ZC-affected plants generally follows a negative binomial distribution, with
groups of diseased plants occurring close together. In most cases, incidence of
ZC across potato fields yielded no discernable pattern. However, a regularly
repeating pattern of disease incidence was present across at least one field.
Incidence of ZC in potato fields did not correlate with psyllid abundance, or
percentage of potato psyllids testing positive for CLs.
The impact of a strobilurin fungicide and other pesticides on soybean
yield and yield components
R. HENRY (1), K. Wise (1)
(1) Purdue University, West Lafayette, IN, U.S.A.
Phytopathology 100:S50
The use of strobilurin fungicides has increased in recent years in the United
States. These fungicides reportedly improve plant health as well as control
foliar diseases, and fungicide applications have been touted as a means to
increase yield in many field crops. The objective of this study is to determine
the impact of a strobilurin fungicide on soybean disease control, yield, and
yield components, alone and in combination with other pesticides, in Indiana.
Field research trials were established in Wanatah, West Lafayette, and
Butlerville, Indiana in 2009. Foliar treatments consisted of glyphosate,
manganese, pyraclostrobin, and a lambda-cyhalothrin insecticide, applied
alone or in various combinations to two glyphosate-resistant soybean
varieties. Control treatments were included. Disease incidence and insect
populations were low throughout the season at all three locations. Fungicide
treatment did not significantly (P < 0.05) increase yield of the newest class of
glyphosate-resistant soybean at any location. Fungicide did have a significant
effect on yield of the conventional glyphosate-resistant soybean at the West
Lafayette and Butlerville locations. Inclusion of any other pesticide or
micronutrient with a fungicide did not significantly improve the yield of either
variety. The results of this study demonstrate the variability in the yield
response due to a strobilurin fungicide application.
Evaluation of winter wheat cultivars for resistance to Fusarium head
blight and deoxynivalenol
J. HERNANDEZ NOPSA (1), S. N. Wegulo (1)
(1) University of Nebraska, Lincoln, NE, U.S.A.
Phytopathology 100:S50
Fusarium head blight (FHB) caused by Fusarium graminearum is a
destructive disease of wheat. F. graminearum also produces the mycotoxin
deoxynivalenol (DON) which contaminates grain. One strategy for managing
FHB is to plant resistant cultivars. Field trials were conducted in Nebraska,
U.S.A. in 2008 at Mead and in 2009 at Paxton to evaluate winter wheat
cultivars for resistance to FHB and DON. Cultivars were arranged in a
randomized complete block design with 3 or 4 replications. FHB index
(percent) and DON (ppm) were measured. DON was measured in grain from
symptomatic heads (DONtag) and in grain bulked from each plot (DONplot).
Cultivars differed in FHB index at Paxton (20 cultivars, P < 0.0001) and
Mead (12 cultivars P < 0.0001). Cultivars differed in DONtag at Mead (P =
0.0015), but not at Paxton (P = 0.1249). DONplot differed (P < 0.0001)
among cultivars at Mead, but not at Paxton (P = 0.1330). DONtag averaged
across cultivars was higher (P < 0.0001) than DONplot at both locations. At
Paxton, FHB index ranged from 4 (Goodstreak) to 60 (Overley). DONtag
ranged from 4.7 (Art) to 12.6 (Hatcher). DONplot ranged from 0.2 (Overland)
to 4.3 (Postrock). At Mead, FHB index ranged from 13 (Harry) to 64
(Overley). DONtag ranged from 5 (2137) to 19 (Overley). DONplot ranged
from 4 (Hondo) to 10 (Harry).
Analysis of viral DNA accumulation in pepper plants with two different
strains and chimeras of Pepper golden mosaic virus
C. HERNANDEZ-ZEPEDA (1), A. M. Idris (1), J. K. Brown (1)
(1) University of Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S50
S50
PHYTOPATHOLOGY
Two strains of the bipartite begomovirus Pepper golden mosaic virus
(PepGMV) cause different symptom phenotypes in pepper and tomato plants.
The mosaic strain, PepGMV-Mo, causes severe, systemic yellow mosaic
symptoms in pepper and mild symptoms in tomato, whereas, the distortion
strain, PepGMV-Di, causes leaf distortion symptoms on inoculated leaves
subsequent recovery in developing leaves, whereas it causes severe symptoms
in tomato. We have mapped the ‘remission’ phenotype of the Di strain to a
defective upstream non-coding (putative promoter) region of the PepGMV-Di
cell-to-cell movement protein (BL1). Chimeric viruses were previously
obtained by exchanging the BL1 promoter between the Mo and Di strains. We
inoculated pepper (Capsicum annum var. Anaheim) plants with the wild type
PepGMV-Mo and PepGMV-Di and with ‘promoter chimeric’ viruses. Plants
were grown under environmentally controlled conditions for 21 days. Total
DNA was isolated from pooled leaves (1 to 6) above the point of inoculation.
Radioactive labeled viral DNA was used as probe to assess viral DNA
accumulation. Results indicated there was no significant detectable decrease
in viral DNA levels in the symptomatic, compared to recovered, leaves. This
supports the hypothesis that the recovery DI phenotype is not due to reduced
viral replication in systemically infected leaves, but rather to cell-to-cell and
(likely) systemic movement owing to aberrant BL1 expression by a possibly
defective promoter.
MeloCon WG® and SoilGard 12G® used in a program as a methyl
bromide alternative to control nematodes and soil borne diseases in
vegetable production
H. HIGHLAND (1)
(1) Certis USA, Nokomis, FL, U.S.A.
Phytopathology 100:S50
In reaction to the Montreal Accord of 2007 on restricting ozone depleting
gases, effective and safe alternative treatments are being investigated, labeled
and used in commercial production. The loss of fumigants is especially
deleterious to the production of fresh market vegetables. In the southeastern
U.S., soil borne diseases and nematodes can be of particular concern. A
program of MeloCon® WG and SoilGard® 12 G, marketed by Certis USA,
have been shown to be very effective when used alone or in combination to
control nematodes and soil pathogens in field trials in the U.S. MeloCon® WG
is a naturally occurring and beneficial soil fungus (Paecilomyces lilacinus
strain 251) that controls a wide range of plant parasitic nematodes. MeloCon®
WG has been shown in replicated field trials to control both southern root
knot nematodes (Meloidogyne incognita) and stubby root nematodes
(Trichodorus spp. and Paratrichodorus spp.), as well as many others.
SoilGard® 12G is also a naturally occurring and beneficial soil fungus
(Gliocladium (Trichoderma) virens strain GL-21) that controls a wide range of
soil borne pathogens, including southern blight (Sclerotium rolfsii), Fusarium
crown rot, and pepper blight (Phytophthora capsici). Replicated field
trials using various fresh market vegetables with these products in conjunction
with soil applied herbicides resulted in improved plant growth, increased
survival, and increased yields, similar to methyl bromide and other chemical
standards.
Characterization of the cytochrome b gene from three stone fruit
infecting Monilinia species
J. HILY (1), S. Singer (1), K. Cox (1)
(1) Department of Plant Pathology and Plant-Microbe Biology, Cornell
University, Geneva, NY, U.S.A.
Phytopathology 100:S50
Single amino acid substitutions in the cytochrome b (cyt b) protein are known
to be responsible for resistance to Qo inhibitor (QoI) fungicides in many plant
pathogenic fungi. In many instances this results from specific point mutation
in the cyt b gene, leading to the replacement of glycine with alanine at
position 143 within the protein (G143A). To date, little is known about the
structure cyt b gene and its relation to QoI sensitivity in Monilinia species.
The goal of this study was to clone and characterize the cyt b gene of
three agriculturally important Monilinia species that cause brown rot of stone
fruit: Monilinia fructicola, M. fructigena and M. laxa. Full-length genes
were cloned from genomic DNA, while mRNA sequences were cloned
from cDNA, and were compared to previously determined cyt b sequences
from other fungal pathogens. The three Monilinia species were nearly
identical at the protein level, but differed in the number and size of introns.
M. laxa appears to have three amino acid differences in the Qo regions
compared to M. fructicola, which may explain the differences in QoI
sensitivity between the two species in regional populations. Finally, we
sought to improve the available molecular detection methods for Monilinia
spp. We developed a single set of cyt b-specific PCR primers that
amplify differentially sized fragments allowing discrimination of the three
Monilinia species commonly responsible for brown rot in North America and
Europe.
Efficacy of Streptomyces lydicus and cover crops for management of
Fusarium wilt of watermelon
J. HIMMELSTEIN (1), K. Everts (2), J. Maul (3)
(1) University of Maryland, College Park, MD, U.S.A.; (2) University of
Maryland and University of Delaware, College Park, MD, U.S.A.; (3)
Sustainable Agriculture Systems Lab. USDA-ARS, Beltsville, MD, U.S.A.
Phytopathology 100:S51
Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (FON) is a reemerging threat to watermelon production in the eastern U.S. due in part to
production changes such as the increase in triploid watermelon acreage. New
management practices are necessary because triploid cultivars have little
resistance to Fusarium wilt. Use of cover crops and biocontrol soil
inoculations are potential ways farmers can improve the sustainability of their
production systems. Efficacy of the biofungicide Actinovate (Natural
Industries, Inc.), which has an active ingredient of Streptomyces lydicus, used
alone and in combination with a tilled cover crop was evaluated in two
locations in Maryland. The cover crops Vicia villosa (hairy vetch), Trifolium
incarnatum (crimson clover), and Secale cereale (rye) were grown, rolled, and
incorporated as a green manure into the soil prior to planting watermelon.
FON was inoculated at the base of each seedling three days after transplanting
at the Salisbury location and five days after transplanting in Beltsville.
Actinovate was first applied to watermelon seedlings two weeks before
transplanting and again six days after the FON inoculation. No visible wilt
symptoms were observed at either location. However Actinovate significantly
increased marketable fruit yield in plots inoculated with FON compared to
inoculated plots with no Actinovate, or plots with no inoculation. Evaluation
of Actinovate in greenhouse trials is underway.
Derivation and validation of a model to predict selection for fungicide
resistance
P. H. HOBBELEN (1), N. D. Paveley (2), B. A. Fraaije (1), J. A. Lucas (1), F.
Van den Bosch (1)
(1) Rothamsted Research, Harpenden, UNITED KINGDOM; (2) ADAS, High
Mowthorpe, Duggleby, Malton, UNITED KINGDOM
Phytopathology 100:S51
The evaluation of fungicide resistance management strategies using
mathematical models can help select options that are worth testing in the field.
However, the usefulness of model predictions depends on their predictive
power. To the best of our knowledge, none of the published fungicide
resistance models have been validated. In this study, we aimed to derive and
validate a mathematical model that predicts the selection for fungicide
resistance in foliar pathogens of cereal crops. The model was validated against
independent data from four field experiments quantifying selection for a
mutation conferring resistance to a quinone outside inhibitor (QoI) fungicide
in powdery mildew (Blumeria graminis f. sp. hordei) on spring barley
(Hordeum vulgare). Fungicide treatments with azoxystrobin differed in the
total applied dose and spray number. For each treatment, we calculated the
observed selection ratio as the ratio of the frequency of the resistant strain at
the end of the season and its frequency at the start of the season. On a scatter
plot of log observed selection ratios on log predicted selection ratios, for all
four experiments, the 45° line through the origin explained 89–90% of the
variance in the observed selection ratios. We believe this is the first fungicide
resistance model for plant pathogens to be rigorously validated. The model
can now be used with some degree of confidence to identify potential antiresistance treatment strategies.
The usefulness of mixtures of a single-site and a multi-site fungicide as
resistance management strategy
P. H. HOBBELEN (1), N. D. Paveley (2), F. Van den Bosch (1)
(1) Rothamsted Research, Harpenden, UNITED KINGDOM; (2) ADAS, High
Mowtorpe, Duggleby, Malton, UNITED KINGDOM
Phytopathology 100:S51
Single-site fungicides that attack one site in the genome of a fungal pathogen
give sufficient disease control but are at-risk of resistance development, while
the reverse is often true for multi-site fungicides. Aim of this study was to use
mathematical modelling to predict whether mixtures of a single- and a multisite fungicide delay the development of resistance against the single-site
fungicide while maintaining a reasonable minimum level of disease control.
We focused on a host-pathogen system consisting of winter wheat and
Septoria tritici with pyraclostrobin as single-site and chlorothalonil as multisite fungicide. The usefulness of the mixing of a single- and a multi-site
fungicide as resistance management strategy was defined as the number of
seasons that the disease-induced reduction of the green leaf area duration was
less than 5%. We determined this measure for scenarios in which the dose rate
1) was constant for both chlorothalonil and pyraclostrobin, 2) was constant for
chlorothalonil, but could increase for pyraclostrobin and 3) could increase for
both fungicides but their ratio in the mixture was fixed. Doses could increase
in between growing seasons as to maintain disease control. The effective life
of pyraclostrobin was predicted to increase with increasing dose of
chlorothalonil in the mixture from 3–4 years when applied alone to 9–12 years
in mixtures with chlorothalonil close to or at the recommended dose.
Effect of chemical and biological control treatments against brown rot
blossom blight in an organic sour cherry orchard
I. J. HOLB (1), M. Fazekas (2), F. Abonyi (2), P. Lakatos (2)
(1) University of Debrecen, Centre for Agricultural Sciences and Engineering,
Debrecen, HUNGARY and Plant Protection Institute, Hungarian Academy of
Sciences, Budapest, HUNGARY; (2) University of Debrecen, Centre for
Agricultural Sciences and Engineering, Debrecen, HUNGARY
Phytopathology 100:S51
In a three-year study, the effect of lime sulfur, copper hydroxide, potassium
carbonate and Aureobasidium pullulans treatments was evaluated against
brown rot blossom blight in a Hungarian organic sour cherry orchard.
Treatments were applied at recommended dosages at three times (closed
blossom, full bloom, petal fall) during bloom on cultivar Érdi Bőtermő. In all
years, copper hydroxide and lime sulfur alone were most effective for blossom
blight control. Both treatments were not as effective as the conventional
standard (penconazole) and caused significantly more damage on spur-leaf
clusters and blossom during wet weather conditions. The effect of treatments
of Aureobasidium pullulans and potassium carbonate alone were significantly
better against blossom blight than untreated control. These treatments caused
no damage on spur-leaf clusters and blossom but their efficacies against the
disease were significantly lower than treatments of copper hydroxide and lime
sulfur. A reduced dosage of copper hydroxide in combination with
Aureobasidium pullulans increased efficacy against blossom blight but also
increased phytotoxicity compared to treatment of Aureobasidium pullulans
alone. Either efficacy or phytotoxicity of this treatment combination was not
significantly higher compared to treatments of copper hydroxide and lime
sulfur alone.
To what extent can winter pruning intensity reduce apple powdery
mildew in integrated and organic apple orchards?
I. J. HOLB (1), M. Fazekas (2), B. Balla (2), F. Abonyi (2)
(1) University of Debrecen, Centre for Agricultural Sciences and Engineering,
Debrecen, HUNGARY and Plant Protection Institute, Hungarian Academy of
Sciences, Budapest, HUNGARY; (2) University of Debrecen, Centre for
Agricultural Sciences and Engineering, Debrecen, HUNGARY
Phytopathology 100:S51
The effectiveness of winter pruning intensity against apple powdery mildew
was investigated on a susceptible (cv. Jonathan), a moderately susceptible (cv.
Idared) and a lowly susceptible cultivar (cv. Mutsu) in organic and integrated
apple orchards. The area under the disease progress curve for shoot and fruit
incidences was calculated to evaluate three winter pruning treatments
(unpruned, weakly pruned and strongly pruned) from 2007 until 2009. The
lowly susceptible cultivar (cv. Mutsu) showed no significant effect of pruning
treatments on apple powdery mildew in either orchard. In the organic orchard,
both strong and weak pruning significantly decreased mildew incidence on
shoot and fruit on the susceptible cultivar (cv. Jonathan) compared to
unpruned ones. The effects of pruning treatments on mildew incidence were
significant only on shoot of the moderately susceptible cultivar (cv. Idared), in
all years in the organic orchard. In the integrated orchard, strong pruning
treatment showed significant effect only on shoot of the susceptible and of the
moderately susceptible cultivars (cvs. Jonathan and Idared) compared to
unpruned ones. Our findings indicated that winter pruning intensity is an
essential element of powdery mildew control on susceptible and moderately
cultivars in organic apple growing.
Two new homothallic species of Phytophthora from irrigation reservoirs
and natural waterways in Virginia
C. HONG (1), P. Richardson (1), S. Ghimire (1), W. Hao (1), P. Kong (1), G.
Moorman (2), J. Lea-Cox (3), D. Ross (3)
(1) VPI & State University, Virginia Beach, VA, U.S.A.; (2) Penn State
University, University Park, PA, U.S.A.; (3) University of Maryland, College
Park, MD, U.S.A.
Phytopathology 100:S51
Two distinct genotypes (L2 and A-2) were recovered from irrigation
reservoirs and a stream in Virginia, U.S.A. Following molecular, morphological and physiological examinations, the ‘L2’ genotype was named
Phytophthora aquimorbida and the ‘A-2’ designated Phytophthora taxon
‘aquatilis’. Both species are homothallic. P. aquimorbida is characterized by
its noncaducous and nonpapillate sporangia, lateral and intercalary chlamydospores, catenulate and radiating hyphal swellings, and plerotic oospores
formed in globose oogonia mostly in the absence of an antheridium. P. taxon
‘aquatilis’ produces plerotic oospores in globose oogonia mostly with a
diclinous, paragynous antheridium, and semi-papillate, caducous sporangia
with variable pedicel lengths but it does not produce chlamydospores nor
Vol. 100, No. 6 (Supplement), 2010
S51
hyphal swellings. Sequence analyses revealed that the closest relatives are P.
hydropathica, P. irrigata and P. parsiana for P. aquimorbida and P.
multivesiculata for P. taxon ‘aquatilis’. The optimum temperature for culture
growth is 30 and 20°C for P. aquimorbida and P. taxon ‘aquatilis’,
respectively. Both P. aquimorbida and P. taxon ‘aquatilis’ were pathogenic to
rhododendron plants and caused root discoloration, pale leaves, wilting, tip
necrosis and dieback. Their plant biosecurity risk is also discussed.
Biological control with Xylella fastidiosa strain EB92-1 for the prevention
of Pierce’s disease development in mature, producing grapevines
D. HOPKINS (1)
(1) University of Florida, Apopka, FL, U.S.A.
Phytopathology 100:S52
Pierce’s disease (PD) of grapevine, caused by Xylella fastidiosa, limits the
grape industry in much of the southern U.S. Injection of a benign strain
(EB92-1) of X. fastidiosa into transplants has controlled PD in new plantings
of Vitis vinifera cultivars. While strain EB92-1 has been shown to be effective
in preventing PD in new grape plantings, there are mature vineyards that are
rapidly being destroyed by PD. A treatment is needed to protect mature vines
already in fruit production against PD. In vineyards in Georgia with moderate
PD pressure (<30% new infections/yr) developing in the untreated vines,
injection of EB92-1 by pin-pricking new branches resulted in significantly
lower PD incidence in the cv. Mourvedre. Four years after treatment, PD
incidence was 23% in the untreated vines and 8% in the EB92-1 treated vines.
Injection of EB92-1 by pin-pricking in vineyards with chronic PD and
abundant vectors did not reduce PD incidence (>30% per year) and does not
appear to work in this high pressure situation. However, drill and syringe
injection of the main trunk in these vineyards with chronic PD did effectively
reduce PD incidence in the cvs. Mourvedre and Merlot. In North Carolina in
2009, drill and syringe injection of the main trunk was a more effective means
of treating mature hybrid grapevines than pin-pricking. Drill and syringe
injection of strain EB92-1 appears to have the potential to control PD in
mature, producing vineyards.
BDM1 regulates virulence in Fusarium graminearum
P. HOREVAJ (1), J. Xu (2), B. H. Bluhm (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) Purdue Universtiy,
West Lafayette, IN, U.S.A.
Phytopathology 100:S52
Fusarium graminearum causes head blight of wheat, as well as ear, kernel,
and stalk rots of corn. During pathogenesis, it can produce numerous
mycotoxins, including zearalenone and trichothecenes such as
deoxynivalenol. Although the genes encoding the biosynthetic enzymes
required for mycotoxin biosynthesis have been identified in the fungus, the
molecular mechanisms underlying pathogenesis and mycotoxigenesis are not
fully understood. In this study, an ortholog of BDM1, a virulence factor in the
chestnut blight pathogen Cryphonectria parasitica, was disrupted in F.
graminearum via split-marker homologous recombination. The phenotype of
the disruption mutant was pleiotropic, including morphological abnormalities
(e.g., the formation of knotted hyphae) and a substantial reduction in virulence
on wheat heads. The functional characterization of BDM1 in F. graminearum
expands the current working model of how pathogenesis is regulated and
suggests that orthologs of BDM1 serve as virulence factors in taxonomically
diverse fungal pathogens.
A virulence factor of phytoplasma inducing witches’ broom and
dwarfism symptoms
A. Hoshi (1), S. Kakizawa (1), Y. Ishii (1), N. Kojima (1), K. Sugawara (1),
Y. Okano (1), K. Maejima (1), K. Oshima (1), S. NAMBA (1)
(1) Laboratory of Plant Pathology, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Tokyo, JAPAN
Phytopathology 100:S52
Phytoplasmas are bacterial plant pathogens that can cause devastating yield
losses in diverse crops worldwide. They reside inside of host cells, and are
transmitted by insect vectors. Phytoplasma-infected plants show symptoms of
witches’ broom and dwarfism called Tengu-su disease in Japan. The
molecular mechanisms of symptoms such as Tengu-su disease have been
unclear. We have determined the complete genomic sequence of Candidatus
Phytoplasma asteris, and revealed that the phytoplasma genome encodes very
few metabolic functions, implying that phytoplasma is highly dependent on
metabolic compounds from its hosts. We also revealed that Amp, a surface
membrane protein of phytoplasma, formed a complex with insect
microfilament proteins, and the formation of Amp-microfilament complex
was observed only with the phytoplasma-transmissible insects, suggesting that
this complex have a major role in determining the transmissibility of
phytoplasmas. Here, we show that a secreted protein of phytoplasma, named
TENGU, induces witches’ broom and dwarfism symptoms. Nicotiana
benthamiana and Arabidopsis thaliana plants expressing TENGU showed
S52
PHYTOPATHOLOGY
symptoms of witches’ broom and dwarfism, which are typical of phytoplasma
infection. Microarray analyses showed that auxin-responsive genes were
significantly down-regulated in the tengu-transgenic plants. These results
suggest that TENGU inhibits auxin-related pathways, thereby affecting plant
development.
Genetic structure of Phytophthora infestans population in eastern North
America, 2002–2009
C. HU (1), F. G. Perez (2), R. Donahoo (3), A. McLeod (4), K. L. Myers (4),
K. L. Ivors (5), P. D. Roberts (3), W. E. Fry (4), K. L. Deah (2), J. B. Ristaino (6)
(1) North Carolina State University, Raleigh, NC, U.S.A.; (2) USDAARS/PSI-Genetic Improvement of Fruits and Vegetables Laboratory,
Beltsville, MD, U.S.A.; (3) IFAS-SWFREC, University of Florida, Immokalee, FL, U.S.A.; (4) Dept. of Plant Pathology, Cornell University, Ithaca, NY,
U.S.A.; (5) MHCREC, NC State University, Mills River, NC, U.S.A.; (6)
Dept. of Plant Pathology, NC State University, Raleigh, NC, U.S.A.
Phytopathology 100:S52
Late blight caused by Phytophthora infestans, reemerged in the U.S. in 2009,
and was the worst in modern history due to a “perfect storm” of widespread
inoculum distribution and conducive weather. Two new genotypes named US20 and US-21 were found on tomato in FL and NC between 2002 and 2007.
More than 80 isolates of P. infestans were collected from 11 states and
Canada. The US-8 genotype was found in potato crops in 5 states. In addition,
three new genotypes, US-22, US-23 and US-24, were recovered from both
potato and tomato. US-22 (A2; Gpi: 100/122) was widespread on tomato
transplants sold in home garden centers that later spread to nearby commercial
tomato or potato fields. US-23 (A1; Gpi: 100/100) was found in four states on
both tomato and potato. US-24 (A1; Gpi: 100/100/111) was found only on
potato in ND. P. infestans populations between tomato and potato were
genetically differentiated. Migration analysis suggested that gene flow
occurred from tomato to potato in the eastern U.S. populations. Isolates from a
home garden in TN, a single site in NY in 2007, and FL in 2008 were also
US-22. Coalescent analysis documented that the 2009 populations were
derived from the 2007 US-22 population. The data indicate that the US-22
existed before the epidemics of 2009. Genotype diversity was greatest in PA
and both mating types occurred there. The severe late blight epidemics of
2009 underscore the need for an improved web-based tracking and monitoring
system for the pathogen in the U.S.
Development and use of an efficient and temperature-insensitive virusinduced gene silencing system in Nicotiana tabacum
C. HUANG (1), X. Zhou (1)
(1) Institute of Biotechnology, Zhejiang University, Hangzhou, PRC
PEOPLES REP OF CHINA
Phytopathology 100:S52
Virus-induced gene silencing (VIGS) is a recently developed technique for
characterizing the function of plant genes by gene transcript suppression and
is increasingly used to generate transient loss-of-function assays. Recently we
described that the geminivirus satellite vectors (2mDNA1 and DNAm ) can
trigger efficient gene silencing in many permissive host plants excluding
Nicotiana tabacum when co-inoculated with the helper virus Tomato yellow
leaf curl China virus. Here we report that the 2mDNA1 vector can induce
efficient gene silencing in N. tabacum with Tobacco curly shoot virus. We
have successfully silenced the β-glucuronidase (GUS) gene in the GUS
transgenic N. tabacum plants and sulphur desaturase (Su) gene in the five
different N. tabacum cultivars. These pronounced and severe knockout
phenotypes are persistent and ubiquitous. Once initiated in seedlings, the
silencing phenotype lasted for the entire life span of the plants and silencing
could be induced in a variety of tissues and organs including leaf, shoot, stem,
root and flower, and could be achieved at any growth stage. This system
works well between 18–32°C. We also silenced NtEDS1 gene and
demonstrated that NtEDS1 is essential for N gene mediated resistance against
Tobacco mosaic virus (TMV) in N. tabacum. The above results indicate that
this system has great potential as a versatile VIGS system for routine
functional analysis of genes in N. tabacum.
Suppression of Fusarium crown and root rot in tomato by silicon
C. Huang (1), P. ROBERTS (2), L. Datnoff (3)
(1) University of Florida, GCREC, Wimauma, FL, U.S.A.; (2) University of
Florida, SWFREC, Immokalee, FL, U.S.A.; (3) Department of Plant
Pathology & Crop Physiology, LSU, Baton Rouge, LA, U.S.A.
Phytopathology 100:S52
Fusarium oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium
crown and root rot (FCRR), is an important soilborne pathogen of tomato in
south Florida. Although fumigation and host resistance may reduce the impact
of this disease, other alternative management strategies are needed. Since
silicon (Si) has been shown to reduce a number of fungal plant diseases, the
purpose of this study was to determine if this element could suppress the
development of FCRR. Si significantly reduced the severity of FCRR on the
stem of tomato 4-weeks after inoculation. Further analysis of disease progress
suggested that the decrease in FCRR disease severity by Si amendment
probably resulted from delaying the initial infection in roots and the
movement of the pathogen from roots to stems. Si contents of roots and shoots
were significantly higher in tomato plants with Si in comparison to those
treatments without Si. Moreover, the increase in the Si content of roots was
significantly correlated with the reduction of disease severity of root, crown,
and stem, indicating a silicon-induced resistance and/or reduction of fungal
colonization. Si treatments probably limited the basipetal spread of FORL
from infected roots to stems. Although laboratory experiments including this
study have shown that Si can alleviate biotic and abiotic stresses in tomato,
further research in applying Si fertilizers for field-grown tomatoes needs to be
conducted to further elucidate the effects of Si.
Microarray analysis identified Puccinia striiformis f. sp. tritici genes
involved in infection and sporulation
X. Huang (1), X. CHEN (2), T. Coram (2), M. Wang (3), Z. Kang (1)
(1) Northwest A&F University, Yangling, Shaanxi, PRC PEOPLES REP OF
CHINA; (2) USDA ARS, Pullman, WA, U.S.A.; (3) Washington State
University, Pullman, WA, U.S.A.
Phytopathology 100:S53
Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, one of the most
important diseases of wheat worldwide. To identify Pst genes involved in
infection and sporulation, a custom oligonucleotide Genechip was made using
sequences of 442 genes selected from Pst cDNA libraries. Microarray analysis
was conducted by hybridizing the Genechip with cDNA from urediniospores
(Ure), germinated Ure, and Pst-infected and mock-inoculated leaves sampled
at 12 h, 24 h, 48 h, 7 d, and 14 d after inoculation. The time course study
identified 55 genes that were differentially induced during the infection
process. Nine of the genes were induced in both Ure and the sporulation stage
of infection. Genes in this group mostly have functions in carbohydrate and
lipid metabolism. Six genes, including a mitochondrial ATP synthase, a
copper-induced metallothionein, a differentiation-related protein Infp, and a
hypothetical cell wall mannoprotein, were induced during the early infection
process and therefore, likely involved in pathogenicity. Four genes, including
an exo-1,3-Beta-glucanase, a chitin deacetylase, and a meiotic recombinationrelated protein, were induced in both early infection and sporulation stages.
Thirty five genes were induced in the sporulation stage, but not in Ure,
indicating that they are involved in reproduction. One gene from the haustorial
library with unknown function was only induced in infected leaves.
Identification and characterization of the causal agent of a new viral
disease on sweet pepper in Taiwan
C. HUANG (1), Y. Zheng (1), Y. Cheng (2), C. Chen (1), F. Jan (1)
(1) National Chung Hsing University, Taichung, TAIWAN; (2) Division of
Plant Pathology, Agricultural Research Institute, Taichung, TAIWAN
Phytopathology 100:S53
A virus culture TwPep1 was isolated from leaves of a sweet pepper plant
(Capsicum annuum cv. Andalus) showing viral disease-like symptoms of
chlorosis, chlorotic spots in the fields in central Taiwan in July, 2009. A
partial L gene of tospovirus was amplified from the total RNA isolated from
TwPep1-infected plant by RT-PCR with a degenerate primer pair for
tospoviruses. The 819-nt L RNA conserved region of TwPep1 shared 94.4–
97.7% nucleotide identity with those of Tomato spotted wilt virus (TSWV)
available in GenBank. A 29 KDa protein of TwPep1 reacted positively with
antiserum against the N protein of TSWV using a western blot. The
nucleocapsid (NP) gene of the TwPep1 was amplified by RT-PCR using
primers for the N gene of TSWV. The 777-bp N gene of TwPep1 shared 98.3–
99.1% nucleotide and 98.5–99.6% amino acid identity with that of 21 TSWV
isolates with full-length sequences of S RNA available in GenBank. The
TwPep1 isolate was back-inoculated onto sweet pepper plants for
pathogenicity test. The inoculated plants showed symptoms of chlorosis and
chlorotic spots which were similar to that observed in the field. Electron
microscopic examination showed about 80–100 nm in diameter in ultrathin
sections of TwPep1-infected sweet pepper. Taken together, it is concluded
that the causal agent of the new sweet pepper disease in Taiwan is indeed an
isolate of TSWV. This is also the first demonstration of isolation and
characterization of TSWV in Taiwan.
Molecular characterization of the Enterobacter cloacae-onion (Allium
cepa) interaction and the search for pathogenicity determinants
J. L. Humann (1), S. Dossey (1), J. Peña (1), N. Peterson (1), A. A. Bates (1),
R. Liesche (1), T. Peever (1), B. K. SCHROEDER (1)
(1) Washington State University, Pullman, WA, U.S.A.
Phytopathology 100:S53
Enterobacter cloacae causes Enterobacter decay of onion bulbs in storage and
is an emerging bacterial pathogen of onion (Allium cepa). E. cloacae is
ubiquitous in nature and is an opportunistic pathogen of humans. A multilocus
phylogeny demonstrated that strains of E. cloacae obtained from onion bulbs
occupy a well-supported clade distinct from isolates of medical origin. Little
is known about the E. cloacae-onion interaction and our investigations have
demonstrated that E. cloacae can move through onion leaf tissue at a rate of
approx. 2 cm per week. In addition, preliminary evidence suggests that E.
cloacae biofilm mutants move faster in planta than wild-type strains. SEM
studies have indicated that a matrix forms in the phloem of E. cloacae
inoculated onion potentially reducing the size of the plant. Analysis of E.
cloacae culture filtrates determined that a necrosis-inducing product is
secreted into the medium that is both heat-instable and sensitive to proteinase
K. In an effort to identify genes involved in the production of this necrosisinducing product and pathogenicity, an onion slice assay was developed. This
assay enabled the high-throughput screening of mini-Tn5 mutants of E.
cloacae and the identification of putative pathogenicity-minus mutants. These
mutants are currently being characterized and genetic analysis of these E.
cloacae mutants will be discussed.
Etiology of tomato yellow leaf curl disease complex in the Sultanate of
Oman involves two helper begomoviruses, a betasatellite, and a DNA-2
satellite
A. M. Idris (1), J. K. BROWN (1)
(1) University of Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S53
Rolling circle amplification was used to amplify, clone, and sequence the
genomes of suspect begomoviruses and satellites from field grown tomato
plants collected in 2005 in Oman. Tomato yellow leaf curl virus from Oman
(TYLCV-OM), and an associated beta satellite have been reported previously
by our group to be associated with symptomatic tomato plants. From the same
samples, we have recently cloned a second, recombinant begomovirus that is
sufficiently divergent from other begomoviruses to warrant its’ classification
as the distinct species, Tomato leaf curl Oman virus (ToLCOMV). Also
cloned from the same sample was an alphasatellite-like molecule, referred to
herein as TYLCDNA2 that is surprisingly closely related to another
alphasatellite (AYVDNA2) from Ageratum in Singapore. The cloned
ToLCOMV induced mild leaf curl symptoms in Nicotiana benthamiana and
tomato, while TYLCV-OM infected plants developed more severe symptoms
than those infected by TYLCV-Om. The addition of the betasatellite
intensified the symptoms of both of the helper viruses. However, coinoculation of plants with either of the helper viruses, plus the beta satellite
and the TYLCDNA2 satellite, resulted in amelioration of symptom severity in
both test plants. This is the first report of amelioration of disease symptoms in
a natural and experimental host by a DNA-2 satellite in the presence of a
helper virus and associated beta satellite.
Virulence and molecular characterization of Verticillium species from spinach
A. IGLESIAS-GARCIA (1), C. Feng (1), L. du Toit (2), K. Subbarao (3), J.
Correll (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) Washington State
University Mount Vernon NWREC, Mount Vernon, WA, U.S.A.; (3)
University of California, Davis, Salinas, CA, U.S.A.
Phytopathology 100:S53
Verticillium wilt, caused by Verticillium dahliae, is a widespread,
economically important disease affecting >200 plant species. V. dahliae is a
pathogen of spinach seed crops, in which symptoms develop only after the
initiation of bolting (onset of reproductive growth). The objective of this study
was to characterize molecular diversity, parasitic ability (root infection,
vascular colonization), and host specificity among a diverse collection of V.
dahliae, V. tricorpus, Gibellulopsis nigrescens (= V. nigrescens), V. alboatrum, and Lecanicillium fungicola (= V. fungicola) isolates. Isolates of
Verticillium species from spinach and several other hosts were evaluated for
variability using the ITS region, mtDNA RFLPs, and putative species-specific
markers. Pathogenicity tests in the greenhouse on spinach, tomato, and cotton
plants with root-dip or root-drench inoculations revealed a wide range of host
specificity and virulence among isolates. Isolates of V. dahliae that originated
from spinach were pathogenic on spinach, whereas some isolates that
originated from other hosts were not pathogenic on spinach. Isolates of V.
tricorpus and G. nigrescens recovered from spinach seed, and isolates of V.
albo-atrum and L. fungicola were not pathogenic on spinach. Together, these
results indicate that Verticillium and related species associated with spinach
display substantial variability in parasitic ability, virulence, and pathogenicity
to spinach.
Species limits and evolution in Verticillium, a group of vascular wiltpathogens of global importance
P. INDERBITZIN (1), R. M. Bostock (1), R. M. Davis (1), K. V. Subbarao (1)
(1) University of California, Davis, CA, U.S.A.
Phytopathology 100:S53
Vol. 100, No. 6 (Supplement), 2010
S53
Verticillium is a diverse group of ascomycete pathogens of mushrooms,
insects and plants. Here we focus on Verticillium sensu strictu (s.s.), a
monophyletic group of plant pathogens closely related to Glomerella
(anamorph Colletotrichum). Like many strains of Fusarium oxysporum,
Verticillium s.s. causes vascular wilts that result in severe crop losses. In
California, Verticillium dahliae affects many different hosts including lettuce,
tomato and strawberry. Of lesser importance are V. albo-atrum, V. tricorpus,
V. longisporum as well as V. nubilum which is only infrequently isolated from
diseased plant materials. Verticillium spp. form thick-walled, highly
melanized resting structures that can survive in the soil for years. Species
identification is largely based on the kind of resting structures produced, i.e.,
microsclerotia in V. dahliae, resting mycelium in V. albo-atrum,
chlamydospores in V. nubilum, and all three kinds of resting structures in V.
tricorpus. In this study, we used multilocus phylogenetic and morphological
analyses of type material and a global sample of Verticillium with emphasis
on California, to investigate species limits and evolution in Verticillium.
Molecular data indicate that resting structure morphology might be a poor
indicator of species limits, as V. albo-atrum-like morphology is present in at
least two different, unrelated phylogenetic groups.
Flutriafol for control of cotton root rot, caused by Phymatotrichopsis
omnivora
T. ISAKEIT (1), R. R. Minzenmayer (2), A. Abrameit (3)
(1) Texas A&M University, College Station, TX, U.S.A.; (2) Texas AgriLIFE
Extension Service, Ballinger, TX, U.S.A.; (3) Texas AgriLIFE Extension
Service, Thrall, TX, U.S.A.
Phytopathology 100:S54
Cotton root rot (CRR), caused by the fungus Phymatotrichopsis omnivora, is a
serious disease in many of the cotton production areas of Texas and other
southwestern states. The objective of this study was to evaluate flutriafol at
several rates for control of CRR in field experiments. The fungicide was
applied prior to flowering, via drip irrigation or as a spray directed towards the
lower stem. When applied via drip irrigation in a San Angelo trial, 0.125 lb
a.i./A significantly (P < 0.05) reduced CRR incidence to 18%, in comparison
to 52% incidence with the control. A yield (lint + seed) of 7405 lb/A was
significantly (P < 0.05) greater with this rate, in comparison to the control
yield, which was 5016 lb/A. Disease incidence was lower (33%, NS) with two
applications each of 0.0625 lb a.i./A, separated by three weeks, but the yield
of 6519 lb/A was significantly (P < 0.05) greater than the control. Rates of
0.0625, 0.125 and 0.25 lb a.i./A applied as a spray in 40 gpa to the stem
significantly (P < 0.05) reduced CRR to 20%, 22%, and 12% incidence,
respectively, in comparison with the control, 55%, in a trial in
Williamson county. The disease incidences were also lower with the same
rates of stem-directed sprays applied in San Angelo, 37%, 43%, and 32%,
respectively, but these differences were not significantly (P < 0.05) less
than the control, which was 57%. Flutriafol shows promise for economical
control of CRR and further field experiments are underway to optimize
effectiveness.
Involvement of chloroplast localized reactive oxygen species in promoting
host and nonhost bacterial pathogens induced cell death
Y. ISHIGA (1), T. Ishiga (1), T. Wangdi (1), C. Ryu (1), K. S. Mysore (1), S.
R. Uppalapati (1)
(1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.
Phytopathology 100:S54
Coronatine (COR), a virulence factor produced by Pseudomonas syringae pv.
tomato (Pst DC3000), is known to induce chlorosis during disease
development. Recently we demonstrated that COR targets chloroplast to
modulate reactive oxygen species (ROS) homeostasis in promoting diseaseassociated necrotic cell death. To further understand the role of COR in
symptom development, we utilized Nicotiana benthamiana and virus-induced
gene silencing (VIGS) as a forward genetics approach and identified several
genes with altered responses to COR leading to necrotic cell death. One of the
identified genes is chloroplast Peroxiredoxin (Prx). Prx-silenced N.
benthamiana and tomato plants showed necrosis-like phenotype in response to
COR. To investigate the involvement of chloroplast-localized Prx during the
Pst DC3000-host interaction, we used Arabidopsis prx mutants. In
pathogenicity assays, mutations in any of the five chloroplast prx genes did
not resulted in altered disease symptom development. However, mutations in
NADPH-dependent thioredoxin reductase (NTRC), an electron donor for Prxs
in the NADPH-dependent thioredoxin (Trx) system resulted in accelerated Pst
DC3000 disease-associated necrotic cell death in Arabidopsis. Furthermore,
ntrc mutant showed accelerated cell death in response to nonhost pathogens,
including Pseudomonas syringae pv. tabaci, pv. glycinea, and pv. tomato T1.
These results suggest chloroplast ROS might play a key regulatory role in
promoting cell death during both host and nonhost interactions.
S54
PHYTOPATHOLOGY
A Medicago truncatula resistance to rust (rer) mutant displays enhanced
resistance to Phakopsora pachyrhizi but not to necrotrophic fungal
pathogens
Y. ISHIGA (1), S. Mittal (1), S. R. Uppalapati (1), V. Doraiswamy (1), H.
Schultheiss (2), K. S. Mysore (1)
(1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.; (2) BASF
Plant Sci GmbH, Limburgerhof, GERMANY
Phytopathology 100:S54
Asian soybean rust caused by the fungus Phakopsora pachyrhizi is one of the
most devastating foliar diseases affecting soybeans grown worldwide.
Understanding the plant defense mechanisms and signaling pathways to P.
pachyrhizi would assist in development of resistant plants. The most common
form of plant defense mechanism against potential pathogens in nature is
nonhost resistance. Medicago truncatula is a model plant species for legumes,
and shows nonhost resistance response to P. pachyrhizi. To identify M.
truncatula mutants with altered resistance to P. pachyrhizi, we have
established a forward-genetic screen of M. truncatula Tnt1 insertion lines.
Screening of more than 1000 Tnt1 lines identified several interesting mutant
phenotypes. Tnt1 insertion mutant of one of the mutant, rer (Resistance to
rust), showed resistance to P. pachyrhizi by supporting less spore adhesion
and germ-tube elongation. Further, rer mutant showed resistance to
hemibiotrophic pathogen, Colletotrichum trifolii, but not to necrotrophic
pathogens, Phoma medicaginis and Sclerotinia sclerotiorum. Interestingly, rer
mutant has five leaves and shows less leaf surface hydrophobicity, suggesting
that chemical or physical surface signals of rer mutant may play an important
role in altered P. pachyrhizi spore adhesion and infection structure formation
leading to enhanced resistance.
Pantocin A antibiotic produced by Pantoea vagans C9-1: Chemical and
genetic characterization
C. A. ISHIMARU (1), T. Lansdell (2), J. Gross (3), J. Clardy (3), T. H. Smits
(4), B. Duffy (4)
(1) University of Minnesota, St. Paul, MN, U.S.A.; (2) Colorado State
University, Fort Collins, CO, U.S.A.; (3) Harvard Medical School, Boston,
MA, U.S.A.; (4) Agroscope Changins-Wädenswil ACW, Wädenswil,
SWITZERLAND
Phytopathology 100:S54
Pantoea vagans C9-1 is one of the most effective and reliable biocontrol
agents against fire blight, and has been commercialized as Blight Ban C9-1.
Production of multiple antibiotics contributes to antagonism. Here we describe
the genetics, chemical isolation, and structure of pantocin A (formerly
reported as herbicolin O), the histidine sensitive antibiotic produced by P.
vagans C9-1. Mutational analysis indicated that biosynthesis of pantocin A
required paaAB and a sequence encoding the peptide precursor of pantocin A.
Complete genome sequencing of P. vagans C9-1 identified the paaABC gene
cluster encoding biosynthesis and autoresistance, located on a 28 kb
chromosomal genomic island. PCR analysis indicated absence of paaABC in
other P. vagans and presence in some P. agglomerans strains. The cluster was
cloned in E. coli and purified antibiotic was isolated using improved methods
for small peptides. The 1H NMR spectra of the C9-1 antibiotic closely
resembled those of pantocin A produced by P. agglomerans. Detailed analysis
of the proton spin systems via a dqf-COSY NMR and HMQC NMR
experiments showed that the chemical shift values and coupling constants of
the protons in C9-1 herbicolin O correspond exactly to those of pantocin A.
Based on these genetic and chemical analyses, herbicolin O and pantocin A
are the same molecule.
Phenology of natural inoculation of apples by sooty blotch and flyspeck
fungi in Iowa apple orchards
S. ISMAIL (1), J. C. Batzer (1), D. A. Mayfield (1), M. L. Gleason (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S54
The sooty blotch and flyspeck (SBFS) complex is comprised of epiphytic
fungi that cause dark blemishes on apples. In 2009, we investigated the timing
of natural inoculation of apple fruit by SBFS species in six Iowa orchards.
Five trees in each orchard received no fungicide sprays after first cover.
Within 1 week after first cover, ‘Golden Delicious’ apples in each orchard
were covered with Japanese fruit bags to exclude SBFS inoculum.
Subsequently, five apples per tree were exposed for 2-week periods and then
re-bagged for the remainder of the growing season, for a total of seven
exposure periods. Controls included apples that were bagged for the entire
season or exposed all season. At harvest, fruit bags were removed and apples
were sorted by exposure period and stored at 4°C for 6 weeks. Colonies of
SBFS fungi on each fruit were counted, then excised with the subtending
peels and pressed between paper towels. During each of the first three
exposure periods, about 70% of the apples developed colonies, with an
average of 3.0 SBFS colonies per apple. During the seventh exposure period,
21% of apples were infested, with a mean of 1.3 colonies per apple.
Approximately 4% of apples that were bagged all season had 1 or 2 SBFS
colonies located close to the stem end. Results of molecular-based
identification of SBFS species will yield species-specific patterns for timing
of apple inoculation.
Detecting resistance to QoI fungicides in Alternaria solani isolates
collected from tomatoes in North Carolina
K. IVORS (1), L. Lacey (1), D. Milks (1), G. Olaya (2)
(1) North Carolina State University, Mills River, NC, U.S.A.; (2) Syngenta
Crop Protection, Vero Beach, FL, U.S.A.
Phytopathology 100:S55
During the 2007 and 2008 growing seasons, some North Carolina (NC)
growers observed unsatisfactory levels of early blight control after standard
applications of QoI fungicides. Alternaria solani isolates were collected from
four tomato fields in NC during the 2008 growing season. DNA extracts from
75 isolates were subjected to cytochrome b sequencing to determine if they
possessed any of the mutations correlated with reduced sensitivity to QoI
fungicides. Twenty-seven isolates with the F129L mutation (substitution of
the amino acid phenylalanine for leucine at position 129 of the cytochrome b
gene) were detected in 3 of the 4 fields. This F129L mutation can be produced
by the 3 different single nucleotide polymorphisms (SNPs) TTA, CTC and
TTG. Among these 27 isolates, the most common SNP was CTC followed by
TTA; TTG was not detected. All 20 isolates collected from one of these
commercial fields with a long history of consecutive tomato cropping
possessed the F129L mutation with either the CTC or TTA SNP. We are
currently conducting in vivo sensitivity tests on all isolates using the QoI
fungicide azoxystrobin on Mountain Fresh tomato plants to combine results
from both molecular and plant assays.
Diversity of Phytophthora capsici from vegetable crops in Georgia
K. JACKSON (1), J. Yin (1), A. Csinos (1), H. Scherm (2), P. Ji (1)
(1) University of Georgia, Tifton, GA, U.S.A.; (2) University of Georgia,
Athens, GA, U.S.A.
Phytopathology 100:S55
Phytophthora blight caused by Phytophthora capsici is a major concern in
vegetable production in Georgia and other southeastern states. Studies were
conducted to determine the diversity of P. capsici from vegetable crops in
Georgia. Sporangia of the isolates ranged from 38.5 to 55.8 µm in length with
length to width ratios ranging from 1.4 to 2.0. The diameters of oospores
ranged from 24.8 to 30.4 µm, with no considerable differences among isolates
from different hosts. All the isolates tested could grow at 35°C, but growth
rates of the isolates differed at all the temperatures evaluated. Studies with
susceptible and tolerant bell pepper cultivars under greenhouse conditions
indicated that there were significant differences among the isolates in
aggressiveness. The majority of the isolates were sensitive to 100 ppm of
mefenoxam but insensitive to 100 ppm of cyazofamid, while all the isolates
were sensitive to fluopicolide or mandipropamid. EC50 values in suppressing
mycelial growth, zoospore germination, and sporangium production averaged
0.2, 2.7 and 1.7 ppm for fluopicolide and 0.02, 6.6 and 0.02 ppm for
mandipropamid. Analysis of the variability of P. capsici isolates in Georgia
using different molecular markers indicated that the isolates were genetically
distinct. These results suggest that P. capsici populations infecting vegetable
crops in Georgia are genetically diverse, which should be considered in
developing resistant cultivars or other disease management programs.
In planta expression profiling reveals Ralstonia solanacearum physiology
and the importance of sucrose metabolism during bacterial wilt of tomato
J. M. JACOBS (1), F. Meng (1), L. Babujee (1), C. Allen (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S55
At early stages of disease development, cells of the bacterial wilt pathogen
Ralstonia solanacearum (Rs) reach densities of 1e8-1e9 CFU/g stem and
remain xylem-limited. Little is known about what compounds Rs metabolizes
to colonize the low-nutrient xylem environment. A comparative microarray
analysis of two ecologically distinct Rs strains, tropical GMI1000 (phylotype
I) and temperate UW551 (phylotype II), gave a global overview of the cellular
processes used during tomato pathogenesis. Most known virulence factors
(e.g. cell wall degrading enzymes, exopolysaccharide, protein secretion) were
highly expressed in planta. Several central primary metabolic pathways (such
as sucrose, nitrate and myo-inositol metabolism) were induced in tomato
stems. Notably, a sucrose-specific phosphoenolpyruvate phosphotransferase
(PTS) metabolic cluster (scrRABYK) was highly expressed in both strains,
suggesting Rs encounters sucrose in the xylem environment. Rs is the only
known -proteobacterium with a sucrose-specific PTS cluster. Mutants
lacking ScrA (sucrose-6-phosphotransferase) could not use sucrose as sole
carbon source. Following naturalistic soil-soak inoculation of tomato plants,
scrA mutants were delayed in both virulence and host colonization when
compared to wild-type. This functional genomic analysis suggests Rs depends
on host sucrose to thrive in the host environment.
Comparative in planta microarray analysis modifies the regulatory model
for the type three secretion system in Ralstonia solanacearum
J. M. JACOBS (1), F. Meng (1), L. Babujee (1), A. Milling (1), C. Allen (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S55
The regulatory mechanisms that govern virulence factor gene expression in
the bacterial wilt pathogen Ralstonia solanacearum (Rs) are complex and
have been primarily studied in vitro. We used transcriptome analysis to
compare gene expression in tomato stem tissue and rich medium of two
distinct Rs strains: GMI1000 (phylotype I) and UW551 (phylotype II). About
25% of the approximately 3500 shared orthologous genes were differentially
expressed (P < .05) during early symptom development of tomato (~5e8
CFU/g stem) when compared to rich medium (6e8 CFU/g). Our in planta
microarray data agreed with previous findings that the virulence factor
exopolysaccharide is abundantly expressed during early symptom
development at high cell densitites (>5e8 CFU/g stem). However, genes
encoding the type three secretion system (T3SS) and effectors were also
strongly expressed at high cell densities in planta, contrary to the model based
on in vitro data. In culture the global virulence regulator PhcA represses the
expression of T3SS regulator hrpB at high cell densities; the current model
posits that T3SS is important early in disease but not later. In contrast, our in
planta study showed that both EPS and T3SS genes were highly expressed
even at later stages of disease development and thus at high cell densities.
This suggests that the biological role of T3SS extends beyond early disease,
and that in vitro regulatory studies can yield misleading results.
Influence of crop rotation on persistence of the atoxigenic strain
Aspergillus flavus AF36 in Arizona
R. JAIME-GARCIA (1), P. J. Cotty (2)
(1) University of Arizona, Tucson, AZ, U.S.A.; (2) USDA ARS/University of
Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S55
Aflatoxins are toxic and carcinogenic secondary metabolites produced by
fungi of the genus Aspergillus and frequently contaminate cottonseed in
Arizona. Several methods to contain aflatoxins have been devised. The use of
atoxigenic strains of A. flavus, including strain AF36, to displace aflatoxin
producers is a commercially proven technology. Previous work indicates that
atoxigenic strain applications can benefit subsequent crops in treated fields
and that applications have beneficial influences for several years. However,
factors that influence atoxigenic strain persistence are not well documented.
The current study sought to determine influences of cropping practices and
rotation on persistence of AF36 in desert production areas of Arizona.
Quantities of A. flavus in fields treated with the AF36 biocontrol were highest
immediately after harvest, declining significantly once winter crops were
planted. However, the percent of the A. flavus community composed of AF36
was not significantly affected. Structures of A. flavus communities were
significantly affected by spring and summer crops. Fields cropped to wheat
had significantly lower percentage of aflatoxin producer strains and highest of
AF36 than fields planted to other crops. Results indicate that agronomic
practices influence both the quantity and quality of A. flavus resident in fields
and that practices might be optimized to maximize long-term displacement of
aflatoxin producers by atoxigenic biocontrols.
Comparative genomic analysis of Xanthomonas axonopodis pv. citri str.
Aw 12879 and Xanthomonas axonopodis pv. citri str. 5208 (Miami)
N. JALAN (1), N. Wang (1)
(1) Microbiology and Cell Science Dept, CREC, University of Florida, Lake
Alfred, FL, U.S.A.
Phytopathology 100:S55
Xanthomonas axonopodis pv. citri (XAC) is the causal agent of citrus canker,
which has a significant impact on citrus production. There are distinct types of
canker that can be caused by various pathovars and variants of XAC. Because
symptoms are generally similar, separation of these forms is based on host
range and other phenotypic or genotypic characteristics of the strains. The
Asiatic type of canker (Canker A), caused by Asian strain XAC, is by far the
most widespread and severe form of the disease. Genetic variations of Astrain in Florida, resulted in local variants with either similar host range- XAC
str. 5208 (Miami) or limited host range- XAC str. Aw 12879. Complete
genome of XAC strain Aw 12879 was sequenced using 454-Titanium FLX
sequencing (25x coverage) and Illumina paired-end 75bp Solexa run (288x
coverage). The contigs were annotated and compared with previously
published Xanthomonas axonopodis pv. citri str. 306 genome. Several
singleton and shared features of the genome were identified, which hold
potential for exploitation to get insights into virulence and host specificity of
the strains. Solexa run was used to obtain the sequence of XAC strain 5208
Vol. 100, No. 6 (Supplement), 2010
S55
Miami (367x coverage). Whole genome comparison with XAC str. 306
revealed that the strains share 99.99% sequence identity and only differ in 211
polymorphic sites. The polymorphisms offer a view into the adaptive
evolution of the genus in different environments.
Characteristics of Monilinia fructicola isolates from decayed stone fruits
in eastern West Virginia
W. J. JANISIEWICZ (1), A. R. Biggs (2), W. M. Jurick II (3), I. Vico (3), W.
S. Conway (3)
(1) USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV,
U.S.A.; (2) West Virginia University, KTFRC, Kearneysville, WV, U.S.A.;
(3) Food Quality Laboratory, USDA-ARS, BARC-East, Beltsville, MD,
U.S.A.
Phytopathology 100:S56
Thirty eight isolates of Monilinia fructicola were obtained from decayed stone
fruits (peach, plum, and nectarine) collected from trees growing in eleven
eastern West Virginia orchards. The isolates were characterized
phenotypically for growth characteristics, including: growth rate under
different temperatures, sporulation, and resistance to fenbuconazole, the most
commonly used preharvest fungicide in the region. There were five distinct
culture phenotypes, ranging from albino to dark, melanized cultures. On PDA
media, the growth rate per day of the isolates differed greatly at all
temperature tested and ranged from 0.4 to 3.2 mm at 4°C, from 2.9 to 7.6 mm
at 10°C, and from 6.5-15.5 at 24°C. Sporulation on peach agar at 24°C varied
from profuse to no sporulation on three-day-old cultures, with some cultures
sporulating only sparsely even on 10-day-old cultures. In spiral dilution tests,
the ED50 for fenbuconazol ranged from 0.01 µg/µL to 0.137 µg/µL, indicating
the development of resistance to the fungicide in some orchards. The identity
of the isolates was confirmed genetically using sequences from the ITS region
of the nuclear ribosomal RNA gene repeat. In addition, a sequence of a
repetitive element, ‘Mona’, associated with resistance of M. fructicola to DMI
fungicides, was detected in the isolates.
Development of infectious full-length cDNA clone of Grapevine leafrollassociated virus 3
S. JARUGULA (1), S. Gowda (2), W. O. Dawson (2), R. A. Naidu (1)
(1) Department of Plant Pathology, Washington State University, IAREC,
Prosser, WA, U.S.A.; (2) Citrus Research and Education Center, University of
Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S56
Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus and
family Closteroviridae) is associated with grapevine leafroll disease (GLRD).
Different segments of the 18,498 nucleotide genomic RNA of GLRaV-3 were
amplified using double-stranded RNA isolated from virus-infected grapevines
showing GLRD symptoms. A multistep cloning strategy was used to assemble
full-length genomic cDNA and its integrity was verified by sequencing. The
full-length cDNA was cloned into an agro-binary vector, pCambia1380,
between an enhanced 35S promoter and NOS poly-A terminator. Ribozyme
sequence was added at the 3’ termini of the cDNA clone to enhance
infectivity. Infectivity of several cDNA constructs in Nicotiana benthamiana
leaves was tested by agro-infiltration assays in the presence of RNA silencing
suppressors. Total RNA analyzed by Northern blot hybridization using genespecific non-radioactive riboprobes showed that six of the ten putative 3’coterminal subgenomic (sg) RNAs were abundantly present only in leaves coinfiltrated with GLRaV-3 and silencing suppressor constructs. The sgRNA
profile was similar to that obtained from total RNA extracted from grapevines
naturally infected with GLRaV-3. Filamentous virion particles ranging from
1000–2000 nm long were observed in agro-infiltrated leaves, further
demonstrating infectivity of agro-infiltrated cDNA clone. Availability of
infectious cDNA clone enables to study molecular biology of GLRaV-3 and
etiology of GLRD.
Xanthomonas type III secretion system based analysis of candidate
effectors from Blumeria graminis f. sp. hordei
A. S. JAYASENA (1), Y. Meng (1), L. Bindschedler (2), P. Spanu (3), R.
Wise (4), A. Bogdanove (1)
(1) Iowa State University, Ames, IA, U.S.A.; (2) University of Reading,
Reading, UNITED KINGDOM; (3) Imperial College London, London,
UNITED KINGDOM; (4) USDA-ARS / Iowa State University, Ames, IA,
U.S.A.
Phytopathology 100:S56
Powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) is a major
disease of barley. Better understanding the Bgh-barley interaction will
facilitate new control strategies. However, the obligate biotrophic lifestyle of
Bgh hinders genetic studies. We have used the Xanthomonas type III secretion
(T3S) delivery system to examine the effects of Bgh effector candidates
(BECs) in barley, maize and rice. Vector pYM5 was constructed to fuse BECs
to the T3S signal avrBS2. Confirming the effectiveness of this approach,
S56
PHYTOPATHOLOGY
CI16149 (Mla10) but not Manchuria (mla10) plants showed hypersensitive
reaction (HR) to Xanthomonas campestris pv. armoraciae 756C carrying
pYM5: AVRa10. Therefore a set of 7 haustorial proteins from Bgh DH14 was
screened on a panel of near-isogenic barley lines carrying different R genes,
and on maize, B73 and MO17, parents of the maize IBM mapping population.
In preliminary experiments, we identified one putative HR elicitor in
HOR11358 (Mla9), two BECs that elicited an HR-like phenotype in MO17
and one that produced an HR-like necrosis in B73. Our data suggest that the
bacterial T3S based assay is an effective screening method for identifying and
characterizing fungal effectors that elicit necrosis. Screening of BECs in a
compatible interaction between Xanthomonas oryzae and rice is in progress to
identify effectors that affect plant disease susceptibility.
Elimination of Black raspberry necrosis virus (BRNV) from Rubus
occidentalis by in vitro thermotherapy
A. JEON (1), E. Cheong (1), R. Mock (1)
(1) USDA, ARS, NGRL, Beltsville, MD, U.S.A.
Phytopathology 100:S56
Plants of the genus Rubus entering the U.S. are considered ‘prohibited’ plant
germplasm and must be tested for exotic pathogens in the USDA quarantine
program. Pathogens detected must be eradicated from host plants prior to
germplasm distribution. Black raspberry necrosis virus (BRNV) infection in
black raspberry (Rubus occidentalis L) results in yield and quality losses.
Axillary buds of black raspberry cv. Munger infected with BRNV were grown
on an in vitro culture medium at 23°C (room temperature - RT) or in a heat
treatment (HT) regime of 4-hr periods of alternating 29°C/38°C with a 14 hr
photoperiod. In vitro explants, removed from heat after 5 weeks and biweekly thereafter up to 13 weeks, were tested for BRNV using RT-PCR. All
explants with heat treatments of 5–13 weeks tested free of BRNV. The
explants were transplanted to soil, and leaf tissue was tested at monthly
intervals. After two months in soil culture all explants have tested negative for
BRNV. Testing of these and additional BRNV-HT plants will continue, to
verify pathogen elimination. Introduction of the anti-viral chemical ribavirin
(15mg/L) to the medium proved fatal to all explants (RT and HT). Rubus sp.
plants with other viruses are being tested to determine optimum pathogen
elimination protocols. This protocol will aid in the processing of valuable
genetic resources through quarantine, and in broadening the genepool from
which breeders can develop new and improved varieties.
Role of photoreceptors in R protein-mediated resistance to Turnip crinkle
virus
R. JEONG (1), A. Chandra-Shekara (2), A. Kachroo (1), P. Kachroo (1)
(1) University of Kentucky, Lexington, KY, U.S.A.; (2) Dow AgroSciences,
Indianapolis, IN, U.S.A.
Phytopathology 100:S56
Light harvested by plants is essential for the survival of most life forms. This
light-perception ability requires the activities of proteins termed
photoreceptors. In addition to various growth and developmental processes,
light also plays a role in plant defense against pathogens and is required for
activation of several defense genes and regulation of the cell death response.
However, the molecular or biochemical basis of light modulated regulation of
defense signaling is largely unclear. Previously we have shown that
incompatible interaction between Arabidopsis-Turnip Crinkle Virus (TCV)
pathosystem is dependent on light. Resistance to TCV is also dependent on
the Resistance (R) protein, HRT. To determine the molecular and biochemical
basis of light-dependent defense pathway, we studied the role of various
photoreceptors in HRT-mediated resistance to TCV, HRT protein levels and
its localization. Interestingly, mutation in certain photoreceptors led to
degradation of HRT via a proteosome-dependent pathway and resulted in
susceptibility to TCV. Exogenous application of salicylic acid induced
transcription of HRT, which restored HRT levels in some, but not all, mutant
backgrounds. These results show that different photoreceptors function
distinctly in maintaining post-transcriptional stability of HRT. The current
focus is to determine biochemical basis of photoreceptor-mediated stability of
R proteins.
Suppression of sheath blight of rice by cow urine
R. JEYARAMAN (1)
(1) Annamalai University, Annamalainagar, Tamil Nadu, INDIA
Phytopathology 100:S56
Most rice growing countries are infested with Rhizoctonia solani, the causal
agent of sheath blight. Although fungicides control the disease, they are not
always economically or environmentally sustainable. Present activities to find
alternatives that are safe to humans and environment. Utilization of cattle
urine may provide an alternative for disease management. Studies were
conducted to determine the stages of cow urine to control sheath blight of rice.
Cow urine, bull urine, and bullock urine completely inhibit the growth of R.
solani under in vitro. The toxicity of urine was not affected during storage.
Urine sprayed at 200 liter ha–1 was found significantly reduced the disease
severity and increases the grain yield under greenhouse and field conditions.
Urine treated plants showed more number of fungal and bacterial populations.
Species of Bacillus, Pseudomonas, and Streptomyces were identified in urine
treated plants. Urine treated plants showed enhanced synthesis of phenol and
activities of phenylalanine ammonia lyase, peroxidase, -1,3-glucanase, and
chitinase relative to control. Our in vitro tests demonstrated that cow urine
contains ammonia and nitrous acid are highly toxic to R. solani and, therefore,
at least partially responsible for pathogen inhibition in urine treated rice
plants. Thus, cow urine has the potential to suppress R. solani through
chemical and microbial agents.
remains unclear. This study tested the effects of several commonly used
surfactants on the germination of aerial conidia of M. verrucaria on PDA
plates. Surfactants used in this study were Kinetic HV, Sorbitan Monolaurate,
Tween 40, Silwet L-77 and Latron AG 98. Conidia were also suspended in DI
water and then spread on PDA plates as control. Incubation was conducted at
25°C. Germination percents were taken at 6 hours and 9 hours, respectively.
Results indicated that Silwet L-77 and Tween 40 significantly (P < 0.05)
promoted initial germination in the first 6 hours when compared to the
control. At 9 hours of germination process, there were no significant
differences among all treatments. Quicker germination of conidia after
application may contribute to the enhanced bioherbicidal efficacy.
A new fungicide for control of Phytophthora capsici on vegetable crops
P. JI (1), J. Yin (1), M. Purvis (1), A. S. Csinos (1), L. J. Newsom (2)
(1) Department of Plant Pathology, University of Georgia, Tifton, GA,
U.S.A.; (2) BASF Corporation, Tifton, GA, U.S.A.
Phytopathology 100:S57
First characterization of a new Exserohilum foliar disease on warm
season turfgrasses
Y. JO (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S57
Phytophthora blight, incited by Phytophthora capsici, causes severe yield and
quality losses in production of peppers, cucurbits and other vegetable crops.
Application of effective chemical fungicides continues to be a significant
component in integrated management of this disease. A new fungicide,
Zampro®, was evaluated in laboratory, greenhouse and field studies for
control of P. capsici. In lab studies, EC50 values of this product in suppressing
mycelial growth, zoospore germination and sporangium production averaged
0.69, 0.13 and 0.23 ppm, respectively. Foliar applications of Zampro at 5 to
25 ppm under greenhouse conditions significantly reduced Phytophthora
blight severity on squash. Field studies were conducted in Tifton, GA, to
evaluate Zampro for control of Phytophthora blight on squash and bell pepper.
In 2008 studies, soil treatment with mefenoxam in conjunction with foliar
sprays of Zampro proved most effective and provided greater disease
suppression compared with mefenoxam applied alone. In 2009 field trials,
Zampro and fluopicolide provided the greatest disease reductions among the
treatments tested. Squash and pepper yield was significantly increased by
Zampro treatments, compared with the non-treated control, in both years. The
results suggest that Zampro has the promise to be used as an effective
component in integrated programs for managing Phytophthora blight on
vegetables. Zampro is expected to be registered by the US EPA in 2012.
An Exserohilum sp. has been isolated from foliar diseases observed on
bermudagrass (Cynodon sp.) and zoysiagrass (Zoysia sp.) fairways and putting
greens at golf courses in Houston, Texas since 2007. Disease symptoms on
individual leaves exhibited prominent elliptical black lesions along the
margin. Symptoms on the closely mowed turfgrass appeared as dark brownish
to black spots of about 5 cm in diameter. As the disease progressed, individual
spots coalesced into larger, irregular patches. Koch’s postulates were verified
for pathogenicity of the fungus on warm season turfgrasses through
experiments in the growth chamber, and confirmation of the etiological agent
causing the foliar disease was achieved. Mycelium was dark gray to black on
potato dextrose agar medium, and produced no sexual fruiting structure or
conidia unlike other Exserohilum spp. The fungus grew best on corn meal
agar medium, and showed an optimal growth at 25°C and pH 7. Phylogeny
trees were constructed based on internal transcribed spacer (ITS), Brn1
(reductase gene involved in melanin biosynthesis) and glyceraldehyde-3phosphate dehydrogenase (gpd) gene sequences using PHYLIP. The
sequences of the fungus isolated in the study showed no complete matching
but high similarity with other known Exserohilum spp., indicating the fungus
associated with a novel foliar disease on warm season turfgrasses is a new
species of Exserohilum.
Morphological and physiological alteration of maize root architectures on
drought stress
T. JIANG (1), B. Scully (1), R. Kemerait (2), R. D. Lee (3), B. Guo (1)
(1) USDA-ARS, Crop Protection and Management Research Unit, Tifton,
GA, U.S.A.; (2) Department of Plant Pathology, the University of Georgia,
Tifton, GA, U.S.A.; (3) Department of Crop and Soil Sciences, the University
of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S57
Effect of diverse cropping systems on arbuscular mycorrhizal fungal
community diversity and structure
E. G. JOHNSON (1), J. H. Graham (1), D. O. Chellemi (2)
(1) University of Florida, Lake Alfred, FL, U.S.A.; (2) USDA-ARS-USHRL,
Ft. Pierce, FL, U.S.A.
Phytopathology 100:S57
Drought tolerance is a complex agronomic trait and root characteristics
logically play an important role in determining the response of plants to
drought stress. Studies were conducted to investigate genotypic variations in
morphological and physiological responses of roots to drought stress in corn.
Two inbred lines, Lo964 and Lo1016, were planted in the field, greenhouse,
and in the laboratory growth chamber for examination of the morphological
and physiological alteration of root traits under drought stress versus no stress
(well-water) conditions. The results revealed that Lo964 had a strong lateral
root system, a high root/shoot ratio, and a high production of ABA in
comparison with Lo1016 under drought stressed condition. The root systems
of Lo1016 were much shallower and smaller than those of Lo964 under water
and drought conditions. After 7 d of drought treatment, fresh root weights
were significantly lower than that of well-watered plants for both Lo964 and
Lo1016. ABA synthesis increased in both Lo964 and Lo1016 under drought
stresses. The ABA contents increased by 9.6 and 3.1 times in the leaves of
Lo964 and Lo1016, and 1.8 and 1.2 times in the root of Lo964 and Lo1016,
respectively. Myo-inositol 1-phosphate synthase (MIPS) gene expression was
7 times higher in leaves of Lo964 than that of Lo1016 under well-water
condition, which was decreased significantly to the level in Lo1016 under
drought-stressed conditions.
Effects of surfactants on conidial germination of Myrothecium verrucaria
X. JIN (1)
(1) USDA ARS, Stoneville, MS, U.S.A.
Phytopathology 100:S57
Myrothecium verrucaria has been employed as a unique biological control
agent because it is highly effective against several annual and perennial
weeds, including red vine, trumpet creeper, redroot pigweed, kudzu,
hempsesbania and sicklepod. Although aerial conidia of M. verrucaria are
hydrophilic, surfactants are still needed to improve the biological control
efficacy on weeds, such as kudzu. The mechanism of surfactants, mainly
wetting agents, such as Silwet L-77 that enhance bioherbicidal efficacy
In agricultural systems, AMF are greatly affected by soil disruption, lack of
host tissue, inhibition due to fertilization, and fumigation. To determine the
effect of diverse crop production practices on AMF community structure and
diversity, five tomato crop production systems consisting of bahiagrass
pasture, conventional, continuous vegetation removal (disk fallow), organic,
and undisturbed (weed fallow) were initiated. The plots were adjusted to the
new management regime for three or four years. Tomato production occurred
for the next two years and soil DNA samples were taken prior to cultivation
and after harvest. AMF phylotypes based on sequence combined with
nonparametric multivariate statistical analysis were used to compare
community structure and diversity. Bahiagrass, weed fallow, and organic
management support diverse, but different AMF communities with multiple
phylotypes lacking described sporotypes, while disk fallow and conventional
management systems greatly reduce AMF populations. Tomato cropping
tends to shift AMF communities to a low diversity community with
phylotypes containing species that sporulate prolifically. After one year
organic and conventional AMF communities are indistinguishable. Weed
fallow communities are more resilient in absence of stress while bahiagrass
communities are very stable. Comparison of the AMF communities in these
cropping systems to environmental, disease, and yield data is underway.
Role of Soybean mosaic virus (SMV) genes in seed transmission in
soybean
S. JOSSEY (1)
(1) University of Illinois, Urbana, IL, U.S.A.
Phytopathology 100:S57
SMV infection can cause severe crop losses and seed coat mottling. In North
America, seed transmission serves as the primary source of inoculum for
SMV as there are very few alternative hosts for the pathogen. Secondary
spread of the virus is by aphids. To study the role of SMV coding regions in
seed transmission, recombinant viruses were constructed using infectious
clones of strains SMV 413 and SMV G2, which show high and low seed
transmission, respectively. Using two restriction enzyme sites common in the
two strains, reciprocal recombinants were made between the 5’ and 3’ regions
Vol. 100, No. 6 (Supplement), 2010
S57
of the genomes of SMV 413 and SMV G2. Site-specific mutagenesis was
used to create a recombinant virus in which the helper component proteinase
of SMV 413 was replaced with that of SMV G2. The amino acid sequence of
SMV G2 coat protein (CP) lacks a motif, DAG, which is important for aphid
transmission. The role of this motif in seed transmission was examined by
mutating the DAG motif in SMV 413 to DAD. Soybean seedlings, cv.
‘Itachi’, were inoculated with recombinant viruses, and seed transmission was
assayed using seed grow-out tests. Seed transmission rates of 20 to 40% were
recorded for SMV 413 and 0% for SMV G2. Recombinant viruses in which
the 5’ and 3’ regions had been exchanged between the two strains failed to
show any seed transmission. The single amino acid change in the DAG motif
of the CP of SMV 413 reduced seed transmission by about 57%.
indicated that the expression of the heat shock protein, AtBiP3, is selectively
up-regulated in AtBAG7 null mutants upon heat and cold stress. Our results
reveal an unexpected diversity of the plants BAG gene family and suggest that
AtBAG7 is essential component of the UPR during heat and cold tolerance,
thus confirming the cytoprotective role of plant BAGs.
Purification and biochemical characterization of a polygalacturonase
produced during Penicillium solitum decay of ‘Anjou’ pear fruit
W. JURICK (1), I. Vico (1), B. Whitaker (1), V. Gaskins (1), W. J.
Janisiewicz (2), W. Conway (1)
(1) USDA ARS - Food Quality Laboratory, Beltsville, MD, U.S.A.; (2)
USDA-ARS, AFRS, Kearneysville, WV, U.S.A.
Phytopathology 100:S58
Silicon (Si) is known to have many positive effects in defending plants against
important pathogens. The effects of Si amendment in banana, an Siaccumulating plant, on its susceptibility to the airborne fungus
Mycosphaerella fijiensis and to the soilborne fungus Cylindrocladium
spathiphylli were studied under controlled conditions. The four last fully
unfolded leaves of adult banana (Musa acuminata, cv Grande Naine) plants
grown under a hydroponic culture system in the presence of Si (1,66mM) or
not, were inoculated with M. fijiensis by spraying conidial suspensions or by
brushing mycelial fragments. The foliar symptoms increased more rapidly on
the non Si-supplied plants than on the Si-supplied plants and the severity was
weaker on plants grown in the presence of Si. In another experiment
conducted in the greenhouse, ten week-old plantlets supplied or not supplied
with 2 mM of Si were inoculated by dipping the root system into a conidial
suspension of C. spathiphylli and then transplanted in a Si-deficient ferralsol.
Two weeks after inoculation, the root lesion severity of each plant was
assessed using the image analysis program WinRHIZO. A reduction of about
50% of the root necrosis was observed for the Si-supplied plants compared
with the non Si-supplied plants. The Si amendment also alleviated the growth
reduction caused by the pathogen. These results suggest that Si might have a
substantial role as a sustainable and non-pollutant means of managing
important fungal diseases in banana.
Polygalacturonase (PG) was isolated and purified from Penicillium solitumdecayed ‘Anjou’ pear fruit. Ammonium sulfate precipitation, followed by gel
filtration and cation exchange chromatography, were used to purify the
enzyme. Both chromatographic methods revealed a single peak corresponding
to PG activity. The enzyme had a molecular mass of 43 kDa and a pI of 5.3.
PG activity was not associated with a glycosylated protein and the enzyme
was active from pH 3.5 to 6, with an optimum at 4.5. The greatest PG activity
was observed at 50°C but was also detectable at 0, 5, 10, 20, 37, and 70°C. In
vitro PG activity was inhibited by addition of the divalent cations Ca, Mg,
Mn, and Fe to varying levels. Thin-layer chromatographic analysis of
hydrolysis products showed that the enzyme exhibits endo and exo activity,
and is unable to cleave dimer or trimers of polygalacturonate. The purified PG
macerated fruit in vitro and produced ~1.2 fold more soluble polyuronides on
pear than on apple tissue. This is the first report of the production of a PG by
P. solitum during postharvest decay of pear fruit, which is biochemically
distinct from the previously characterized PG produced during apple fruit
decay by the same fungus.
Sclerotinia sclerotiorum induces redox changes in the host cellular
environment via oxalic acid
M. KABBAGE (1), B. Williams (1), H. Kim (1), R. Britt (1), M. Dickman (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S58
The necrotrophic fungal pathogen Sclerotinia sclerotiorum produces the nonspecific phytotoxin and pathogenicity factor, oxalic acid (OA). Transgenic
plants expressing a redox-regulated GFP reporter, provided real-time evidence
that Sclerotinia initially induces reducing conditions that suppresses the host
oxidative burst and callose deposition, but subsequently promotes plant ROS
generation leading to programmed cell death. Our non-pathogenic OA- mutant
strain is unable to alter host redox status, however chemical induction of
reducing conditions in host cells with DTT, remarkably restores its ability to
cause disease. OA thus appears to have dual opposing functions, by creating
reducing conditions, OA inhibits the plant oxidative burst defense response
and cell death much like in biotrophic interactions, and then subsequently promotes cell death and disease. The reduction of the host cellular environment
may be a key strategy for establishment of necrotrophic fungal infection.
AtBAG7, an Arabidopsis Bcl2-associated athanogene resides in the
endoplasmic reticulum and is involved in the unfolded protein response
M. KABBAGE (1), B. Williams (1), R. Britt (1), M. Dickman (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S58
The Bcl-2-associated athanogene (BAG) family is an evolutionarily
conserved, multifunctional group of co-chaperones that perform diverse
cellular functions ranging from proliferation to growth arrest and cell death in
yeast, mammals and recently plants. The Arabidopsis genome contains seven
homologs of the BAG family, including four with domain organization similar
to animal BAGs. In the present study we show that AtBAG7 is a uniquely
localized Endoplasmic Reticulum BAG that is necessary for the proper
maintenance of the Unfolded Protein Response (UPR). AtBAG7 was shown
to directly interact in vivo with the molecular chaperone, AtBiP2, by
Bimolecular Fluorescence Complementation (BiFC) assays, and confirmed by
yeast two hybrid assay. Treatment with an inducer of UPR, Tunicamycin
(Tm), resulted in accelerated cell death of AtBAG7 null mutants.
Furthermore, AtBAG7 knockouts were sensitive to known ER-stress stimuli,
heat and cold. Heat sensitivity was successfully reverted in these knockouts to
the wild-type phenotype with the addition of the chemical chaperone,
Tauroursodexycholic acid (TUDCA). Real-time PCR of ER-stress proteins
S58
PHYTOPATHOLOGY
Effects of silicon amendment on diseases caused by Mycosphaerella
fijiensis and Cylindrocladium spathiphylli in banana
L. KABLAN (1), M. Vermeire (1), J. Risede (2), M. Dorel (2), B. Delvaux
(1), A. Legreve (1)
(1) Université catholique de Louvain, Louvain-la-Neuve, BELGIUM; (2)
CIRAD-FLHOR, Capesterre-Belle-Eau, GUADELOUPE
Phytopathology 100:S58
Oleate-regulated signaling and plant defense
P. Kachroo (1), A. Kachroo (1), M. K. MANDAL (1)
(1) University of Kentucky, Dept. of Plant Pathology, Lexington, KY, U.S.A.
Phytopathology 100:S58
Oleate-regulated signaling and plant defenseMihir K Mandal, Aardra
Kachroo, Pradeep Kachroo Department of Plant Pathology, University of
Kentucky, Lexington, KY 40546Oleic acid (18:1) is one of the major
monounsaturated fatty acid (FA) which plays an important regulatory role in
animal cells. In plants, changes in the levels of 18:1 results in the alteration of
salicylic acid (SA)- and jasmonic acid (JA)-mediated defense responses. This
is evident in the Arabidopsis ssi2/fab2 mutant, which encodes a defective
stearoyl-acyl carrier protein-desaturase (S-ACP-DES) and consequently
accumulates high levels of stearic acid (18:0) and low levels of 18:1.
Consequently replenishing 18:1 levels results in restoration of wild-type-like
signaling in the ssi2 mutant. We have identified several genes, which either
participate in the prokaryotic fatty acid (FA) or generalized defense pathways
and loss-of-function of which restores various phenotypes in ssi2 plants. A
reduction in 18:1 levels induces defense response by upregulating the
expression of several R genes in an SA-independent manner (1). More
recently, we have shown that 18:1 regulated induction of R proteins is
dependent on EDS1 or SA and that EDS1 and SA act in a redundant manner
to regulate R protein derived signaling regardless of the structure of R protein.
Further characterization has led to the isolation of several 18:1 binding
proteins, whose enzymatic activities are regulated by 18:1 levels. Detailed
analysis of these proteins will be presented.
Functional analysis of soybean genes for a role in soybean cyst nematode
resistance
P. KAITHERI KANDOTH (1), N. Ithal (1), J. Recknor (2), D. Nettleton (2),
T. Maier (3), T. Baum (3), M. G. Mitchum (1)
(1) Division of Plant Sciences, Bond Life Science Center, University of
Missouri, Columbia, MO, U.S.A.; (2) Department of Statistics, Iowa State
University, Ames, IA, U.S.A.; (3) Department of Plant Patholgy, Iowa State
University, Ames, IA, U.S.A.
Phytopathology 100:S58
Soybean cyst nematode (SCN ; Heterodera glycines) is one of the most
important pathogens of soybean. The resistance against this pathogen is
controlled by two major QTLs, Rhg1 and Rhg4, in soybean. In order to better
understand the resistance mechanism against this pathogen, we performed
comparative transcript profiling of laser-microdissected SCN feeding sites
(syncytia) isolated from resistant and susceptible soybean NILs differing at
the Rhg1 locus. This resulted in a highly specific library of differentially
expressed genes in syncytia undergoing a resistant reaction and identified a
number of genes potentially involved in the resistance phenotype against this
pathogen. Also, with the recent release of the complete soybean genome
sequence, it is now possible to identify soybean homologs of known plant
defense gene and test them for a role in the resistant reaction against this
pathogen. A third pool of genes includes those mapped to resistance QTL loci.
We are undertaking a large scale functional analysis of these genes in hairy
roots by RNAi in composite plants of soybean. Moreover, the differentially
expressed gene set is a suitable library for identifying nematode-inducible
promoters which will be useful for highly targeted syncytia-specific gene
silencing and overexpression studies.
Reaction to Plasmodiophora brassicae, pathotype 6 in lines of Brassica
species
K. Kalpana (1), M. MCDONALD (1), B. D. Gossen (2)
(1) University of Guelph, Guelph, ON, CANADA; (2) Agriculture and AgriFood Canada, Saskatoon, SK, CANADA
Phytopathology 100:S59
Clubroot of Brassica spp., caused by Plasmodiophora brassicae Woronin, is
an important disease of vegetable Brassica crops worldwide, and has spread
rapidly on canola (B. napus) in western Canada since first reported in 2003.
Trials to evaluate clubroot incidence and severity on lines of selected Brassica
spp. were established on naturally infested organic soil (pH 6.7) in 2008 and
2009 near Bradford, Ontario, Canada. Pathoptype 6 is predominant at this site.
The study focused on the Rapid Cycling Brassica Collection (RCBC),
including lines of B. carinata, B. juncea, B. napus, B. nigra, B. oleracea, B.
rapa, and Raphanus sativum), which have potential as model systems for
controlled environment studies and as differential hosts in pathotype
assessment, but also included lines of canola and Asian vegetables (B. rapa)
such as pak choy (var. communis), Chinese flowering cabbage (var. utilis) and
napa cabbage (ssp. Pekinensis). Clubroot incidence and severity were higher
in 2008 than in 2009, but the pattern of response was similar each year.
Several of the RCBC lines were susceptible to pathotype 6, as were all of the
Asian vegetables. RCBC lines of B. carinata and B. juncea, as well as pak
choy and flowering cabbage, are good candidates for use as model crops. The
susceptible canola lines will be useful for studies with pathotype 6, such as
studies of fungicides and symptom development. Canola lines 5202 LL and
04- 2 and napa cabbage Deneko had no symptoms of clubroot.
Characterization of the antibacterial peptide herbicolin I biosynthetic
operon in the fire blight biocontrol agent Pantoea vagans C9-1
T. Kamber (1), T. Lansdell (2), T. H. Smits (1), V. O. Stockwell (3), C.
ISHIMARU (4), B. Duffy (1)
(1) Agroscope Changins-Wädenswil ACW, Wädenswil, SWITZERLAND; (2)
Michigan State University, East Lansing, MN, U.S.A.; (3) Oregon State
University, Corvallis, OR, U.S.A.; (4) University of Minnesota, St. Paul, MN,
U.S.A.
Phytopathology 100:S59
Pantoea vagans strain C9-1 (ex. P. agglomerans, E. herbicola) is one of the
most effective and reliable biological control agents against fire blight, caused
by Erwinia amylovora. It is registered for commercial use in the U.S.A. and
Canada as BlightBan C9-1 (Nufarms Americas). Understanding C9-1
mechanisms of action is critical for improving biocontrol performance and
regulatory evaluation in Europe. Competition and production of the antibiotic
pantocin A are known mechanisms of pathogen suppression in the infection
court (flower stigmas). We have characterized the peptide antibiotic herbicolin
I (syn. dapdiamide E), which may also contribute to C9-1 biocontrol activity.
A plasposon library was screened using novel pathogen biosensors. Insertion
sequences for 16 herbicolin I-negative mutants were used to locate the
herbicolin I operon in our complete genome of C9-1. The operon consists of
10 ORFs on a 166 kb plasmid. Comparative genomics identified a
homologous gene cluster in Serratia proteamaculans 568, and absence in our
sequence of the commercial strain P. agglomerans E325 (Northwest
Agricultural Products). Prevalence of herbicolin I and diversity of biosynthetic
genes among Pantoea biocontrol, environmental and clinical isolates, and
pathogen resistance will be presented.
Microbial Rosetta Stone Central Agricultural Database: A database for
high consequence plant pathogens
S. KAMENIDOU (1), A. Simonson (2), R. Jain (2), K. Hari (2), J. Robertson
(3), J. Fletcher (1)
(1) Oklahoma State University, National Institute of Microbial Forensics &
Food and Agricultural Biosecurity, Stillwater, OK, U.S.A.; (2) cBIO Inc., Freemont, CA, U.S.A.; (3) Federal Bureau of Investigation, Quantico, VA, U.S.A.
Phytopathology 100:S59
Plant pathogens, if used as agents of biowarfare, bioterrorism or biocrime,
could have a serious impact on the U.S. food supply, environment and society.
The Microbial Rosetta Stone (MRS) Central, a database developed for use by
federal investigators, contains key information on high priority pathogens of
humans and animals. The goal of this work was to select and provide curated
information for 100 high consequence plant pathogens for the establishment
of a MRS Central Agricultural Database. Plant pathogens were chosen based
on their potential for damage to U.S agricultural and natural ecosystems, their
inclusion on several existing plant pathogen threat lists and their
recommendation by experts in the American Phytopathological Society’s
Microbial Forensics Interest Group (representing academia, government and
industry). Information on these pathogens was collected using a multiple
database search and keyword curation was performed for categories of
taxonomy, synonyms, symptoms, detection and diagnosis, laboratory and field
protocols, sample collection, geographic distribution, plant hosts, insect
vectors and epidemiology. The resulting MRS Central Agricultural Database
links curated data for high consequence plant pathogens and important plant
diseases to relevant scientific literature and internet resources, thereby
providing law enforcement, forensic and intelligence communities with an
additional tool to counteract agricultural emergencies.
Development of Cucumber mosaic virus coat protein- and replicasemediated resistant Gladiolus plants
K. Kamo (1), R. JORDAN (2), M. Guaragna (2), H. Hsu (1), P. Ueng (2)
(1) Floral and Nursery Plants Research Unit, USDA-ARS, U.S. National
Arboretum, Beltsville, MD, U.S.A.; (2) Molecular Plant Pathology Lab, PSI,
USDA-ARS, Beltsville, MD, U.S.A.
Phytopathology 100:S59
Cucumber mosaic virus (CMV) is one of the most important plant viruses
because it infects about 1000 plant species, including food crops and
ornamentals. Infection of various flower bulb crops with CMV results in
dramatic streaking of the flowers making the flowers unmarketable, and
infected plants have decreased vigor resulting in a poor bulb yield. There are
two subgroups of CMV (I and II) that are distinguished by serotype, biology,
and molecular analysis. Transgenic Gladiolus plants that contain either CMV
subgroup I coat protein (CMV CP I), subgroup II coat protein (CMV CP II),
subgroup I replicase (CMV Rep), a combination of the CMV CP I and CMV
CP II, or a combination of the CMV CP II and CMV Rep genes were
developed. These plants were multiplied in vitro and challenged with purified
CMV I and II Gladiolus isolates using a hand-held gene gun. Three out of 19
independently transformed plants expressing the CMV Rep gene under
control of the duplicated CaMV 35S promoter were found to be resistant to
CMV I. Three out of 21 independently transformed plants with the CMV CP
II gene under control of the Arabidopsis UBQ3 promoter were resistant to
CMV II. Eighteen independently transformed plants with either the CMV CP I
or a combination of CMV CP I and CP II genes were found to be susceptible
to both CMV I and II. This work will facilitate the evaluation of virus
resistance in transgenic Gladiolus plants to yield improved floral quality and
productivity.
Response of hard red spring wheat germplasm to the bacterial leaf streak
pathogen (Xanthomonas campestris pv. translucens)
Y. R. KANDEL (1), L. E. Osborne (1), K. D. Glover (1), C. Tande (1)
(1) South Dakota State University, Brookings, SD, U.S.A.
Phytopathology 100:S59
Bacterial leaf streak, caused by Xanthomonas campestris pv. translucens has
emerged as an important disease of wheat in the Northern Great Plains. The
potential for loss is not well known in the U.S. however the disease has been
indicated to cause potential yield losses up to 20% in Mexico. As with many
foliar bacterial diseases, in-season control is difficult and development of
resistant genotypes is an urgent need. Limited information is available on
sources of resistance to this disease. The objective of the study was to evaluate
spring wheat germplasm against bacterial leaf streak. Field experiments were
conducted near Aurora and Watertown, SD in 2009. Forty five HRSW
genotypes with diverse genetic backgrounds were inoculated at tillering stage
with a virulent isolate (XtSD-017). Disease severity was assessed at 7-day
intervals from heading through dough stage. Differences in disease severity
were observed, although no genotypes were immune. Of the 45 genotypes,
most were susceptible however line SD4205 showed a high level of resistance
whereas SD4148 and SD4176 were moderately resistant. Resistant genotypes
had slower disease progression with shorter streak length. Resistant genotypes
identified in this study can potentially be used in breeding programs to
incorporate resistance genes into adopted hard red spring wheat genotypes.
Additionally, these findings may have implications for the further study of
bacterial leaf streak resistance genes.
A Ustilago maydis homolog of Aspergillus veA is required for hyphal
proliferation in maize
B. KARAKKAT (1), S. Covert (2)
(1) University of Georgia, Athens, GA, U.S.A.; (2) University of Georgia,
Warnell School of Forestry and Natural Resources, Athens, GA, U.S.A.
Phytopathology 100:S59
Vol. 100, No. 6 (Supplement), 2010
S59
Members of the fungal-specific velvetA (veA) gene family affect spore
production in saprobic Ascomycetes. However, the functions of similar
proteins in Basidiomycetes have not been established. We predicted that veA
gene homologs in the basidiomycete plant pathogen Ustilago maydis might
regulate spore formation, spore viability, and disease progression. To pursue
these hypotheses, three U. maydis genes Um00893, Um04203 and Um01146,
were identified by BLAST searches as veA family members. Using a gene
replacement strategy, deletion mutants were made in all three genes. None of
the mutants showed any phenotypic alteration during yeast-like, in vitro
growth. However, the Um00893 mutants failed to induce gall or teliospore
formation in maize. Chlorazol staining of leaves infected with Um00893
mutants revealed that the mutant hyphae did not proliferate normally during
the early stages of infection. The Um01146 mutants were able to induce galls,
but had a slight decrease in virulence while the Um04203 mutants were not
affected in disease progression. These data suggest that at least one veA family
member is essential for U. maydis virulence.
Effect of Trichoderma harzianum on soil-borne pathogens affecting
French beans in Kenya
G. M. KARIUKI (1), Z. M. Kinyua (2)
(1) Kenyatta University, Nairobi, KENYA; (2) Kenya Agricultural Research
Institute, Nairobi, KENYA
Phytopathology 100:S60
The effect of Trichoderma harzianum T-22 on French beans has been studied
in Kenya. A total of four trials were carried out during a period of one year in
Naivasha, Kenya (under greenhouse conditions) and Mwea, Kenya (under
field conditions) to evaluate the efficacy of T. harzianum in the management
of soil-borne diseases of French beans caused by Fusarium, Pythium and
Rhizoctonia spp. Preliminary work had shown that both sites had high
incidence of these pathogens. A total of six treatments were applied in each of
these sites and the trial was repeated once in each site. The treatments were:
30 g of T. harzianum per 1000 planting bags, 15g T. harzianum per 1000
planting bags, 5g of T. harzianum per 1kg of seed (seed treatment), 15 g of
rootgard per 1000 planting bags, 23 ml of carbendazim per 2000 planting bags
and the untreated control. The second application for each of the treatments
except for the carbendazim was done again after 35 days. There were
significant differences in terms of root/shoot growth and root colonization by
T. harzianum in all the treated plots. The study confirms that T. harzianum
enhances root growth of plants, and therefore renders the soil-borne pathogens
ineffective. Root samples that were taken for plant pathogen DNA analysis
further confirmed that T. harzianum is antagonistic to the soil-borne
pathogens.
Host-pathogen interactions in the Mustard-White rust pathosystem:
Protein expression profiling
P. KAUR (1), K. Sivasithamparam (1), M. Barbetti (1)
(1) The University of Western Australia, Perth, AUSTRALIA
Phytopathology 100:S60
Albugo candida (white rust) is a serious disease of cruciferous vegetable and
oilseed crops. Leaf and inflorescence infection causes yield losses up to 60%
or more in India, and losses of up to 20% in Australia. Presently, canolaquality B. juncea is being developed to extend Brassica oilseed production to
the lower rainfall areas of the southern Australian grainbelt. Unfortunately, the
varieties of B. juncea available in Australia appear highly susceptible to white
rust. To understand the biochemistry of the host-pathogen interaction a
proteomic study was undertaken to analyse changes in protein expression
levels at different time points following inoculation, during the interaction of
A. candida with resistant and susceptible cultivars, respectively. Some key
regulators contributing to host resistance towards A. candida were determined
that can be used to design genetic markers to screen for resistance available in
B. juncea germplasm. Transcriptional expression changes shown by some
enzymes reflect on how the pathogen manipulates the host plant to access its
resources/nutrient reserves. These results provide a new insight into the
mechanisms of resistance in this pathosystem.
A new product for Peronospora and Pseudoperonospora control in
ornamentals
R. J. KEESE (1), K. E. Kalmowitz (2), C. Palmer (3)
(1) BASF Corp., Res Triangle Park, NC, U.S.A.; (2) BASF Corp., Raleigh,
NC, U.S.A.; (3) IR-4 Project, Rutgers, The State University of NJ, Princeton,
NJ, U.S.A.
Phytopathology 100:S60
Orvego™ fungicide, with the active ingredients ametoctradin and
dimethomorph, will be a new experimental entry into the ornamentals market.
Orvego is a combination of two modes of action – a mitochondrial respiration
inhibitor (FRAC Group 45) and a FRAC Group 40 cell wall synthesis
inhibitor. This combination has also been shown in field trials to be an
effective resistance management tool. The range of rates tested for
S60
PHYTOPATHOLOGY
ornamentals, including phytotoxicity testing, were between 11 and 56 fl
oz/100 gal. Efficacy has been demonstrated in research trials across the
country with universities and through the IR-4 program, against a number of
downy mildews and excellent control is achieved with these rates. Crop safety
at a 4X safety factor has been established in many ornamentals and a plant list
will be reviewed. Efficacy studies on Coleus, deadnettle, rose and others will
be discussed. Registration is expected in 2012.
Controlling powdery mildew in the greenhouse on hybrid Cucurbita
seedlings used as rootstocks for grafting watermelon
A. P. KEINATH (1), G. V. Baccari (1), V. B. DuBose (1)
(1) Clemson University, Charleston, SC, U.S.A.
Phytopathology 100:S60
‘Strong Tosa’ interspecific hybrid squash (Cucurbita moschata x C. maxima)
is used as a rootstock for grafting seedless watermelon scions. ‘Strong Tosa’
seedlings may become infected by powdery mildew Podosphaera xanthii
before grafting or during the healing phase after grafting. The objective was to
test 5 biofungicides, 6 conventional synthetic, and 3 organic-approved
fungicides to prevent powdery mildew on ‘Strong Tosa.’ Fungicides at labeled
rates per 935 l/ha water were applied to seedlings. In the first two
experiments, seedlings were sprayed three times at 5-day intervals and
exposed to powdery mildew inoculum continuously after the first application.
In the second two experiments, seedlings were exposed to inoculum for 7
days, sprayed once, and held in a humidity chamber for 7 days to simulate
healing conditions. Powdery mildew incidence and severity were greater with
than without humidity. In all experiments, conventional fungicides were more
effective than organic-approved fungicides, which were more effective than
biofungicides. Hydrogen dioxide, skim milk, and thiophanate-methyl were
ineffective compared to water. Penthiopyrad (LEM-17), cyprodinil plus
fludioxonil, tebuconazole, and myclobutanil were very effective and did not
differ from each other. Parafinic oil (JMS Stylet Oil), sulfur, and potassium
bicarbonate were as effective as conventional fungicides in three of four
experiments (P = 0.01). Fungicides should be applied before exposure to
powdery mildew.
Effect essential oils on inhibition of Phytophthora capsici
E. KELLEY (1), J. Hao (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S60
Phytophthora capsici is an economically important pathogen that infects
many plants, including cucurbit and solanaceous species. Laboratory studies
were conducted to determine the effect of essential oils (bay, cinnamon leaf,
clove bud, rosemary, and red thyme) on P. capsici (1) mycelial growth, (2)
zoospore production, motility and mortality, and (3) oospore production. (1) A
mycelial plug was transferred to a Petri plate containing V8 agar medium,
amended with an essential oil (V8-O) and colony diameter was recorded after
3 days. (2) P. capsici was grown on V8-O plates for 5 days in the dark,
followed by 3 days in constant light. Released zoospores were counted using a
hemacytometer. Zoospore suspensions were treated with essential oils and
aliquots were pipetted onto V8 plates, with colonies counted 2 days later.
Treated zoospores were also viewed under a compound microscope for
movement at 3, 10, 30, and 60 minutes. (3) Two strains of P. capsici with
opposite mating types were co-cultured on V8-O plates, and oospore
production was observed after 1 week. All tests were done utilizing 5 oil
concentrations, in the range of 0.005 to 0.8 ul/ml. Effective concentration for
50% growth inhibition of the pathogen was calculated. At a concentration of
0.15 ul/ml, red thyme was the only oil to fully inhibit all mycelia growth, but
bay clove bud and cinnamon leaf inhibited growth at 0.3 ul/ml. All oils
suppressed the movement of zoospores within three minutes of treatment at
0.15 ul/ml.
Structural studies of filamentous plant viruses by X-ray fiber diffraction
and cryo-electron microscopy
A. KENDALL (1), J. Groom (1), W. Bian (1), M. McDonald (1), D. Williams
(1), P. Stewart (1), S. Ghabrial (2), G. Stubbs (1)
(1) Vanderbilt University, Nashville, TN, U.S.A.; (2) University of Kentucky,
Lexington, KY, U.S.A.
Phytopathology 100:S60
The filamentous plant viruses are important agricultural pathogens that cause
significant economic losses, particularly in developing countries. Although
there have been extensive genetic and plant pathological studies of these
viruses, little detailed structural information has been available until recently.
Using a combination of X-ray fiber diffraction and cryo-electron microscopy,
we have produced the first medium resolution models of several filamentous
plant viruses including the potexvirus potato virus X and the potyvirus
soybean mosaic virus. We have also extended our studies to other filamentous
plant viruses including the hordeiviruses and the closteroviruses. Improving
the resolution of these models will allow us to phase our fiber diffraction data
and enable the production of high resolution models. These models will give
us insight into viral-host interactions and virus assembly and disassembly, and
will facilitate the use of these viruses in biotechnological applications.
Supported by NSF grant MCB-0743931.
Screening for differential resistance responses to Phakopsora pachyrhizi
between Rpp3, Rpp?(Hyuuga), and 12 additional soybean accessions
M. D. KENDRICK (1), K. F. Pedley (1), R. D. Frederick (1), D. L. Hyten (2),
P. B. Cregan (2), D. K. Harris (3), B. Ha (3), H. R. Boerma (3)
(1) USDA-ARS, Fort Detrick, MD, U.S.A.; (2) USDA-ARS, Beltsville, MD,
U.S.A.; (3) University of Georgia, Athens, GA, U.S.A.
Phytopathology 100:S61
necrosis in tobacco. Yukon Gold, a North American potato cultivar was
shown to produce HR against infection with PVYNTN isolates, as well as with
L26 and, thus, to carry the Nz resistance gene. Based on genetic responses in
potato cultivars carrying Nz, Ny and Nc genes, and molecular make-up of their
genomes we propose to group PVYZ and PVYNTN isolates into a single
PVYZ/NTN strain group.
CorA, a magnesium/nickel/cobalt transporter is required for full
virulence in the soft rot pathogen, Pectobacterium carotovorum
C. KERSEY (1), C. K. Dumenyo (1)
(1) Tennessee State University, Nashville, TN, U.S.A.
Phytopathology 100:S61
Asian soybean rust (ASR) is an economically significant disease caused by
the fungus Phakopsora pachyrhizi. Five soybean genes that confer resistance
to specific isolates of P. pachyrhizi (Rpp1 – Rpp5) were previously identified.
More recently, the soybean cultivar Hyuuga (PI506764) was found to be
resistant to field isolates of the pathogen. The genes Rpp?(Hyuuga) and Rpp3
map to the same region of chromosome 6, between markers Satt460 and
Satt307, and Satt460 and Sat_263 respectively. Twelve additional soybean
accessions with resistance to P. pachyrhizi and mapping within 5 cM of the
Rpp3/Rpp?(Hyuuga) locus have been identified by a bulk-segregant approach.
It is unknown whether the resistance genes in PI506764(Hyuuga),
PI462312(Rpp3), and these 12 lines are identical, allelic, or independent
genes. Previously it was reported that PI462312 (Rpp3) and PI506764
(Hyuuga) responded similarly when inoculated with 10 P. pachyrhizi isolates.
However, when challenged with a Brazilian isolate, Rpp3 plants were
susceptible while Rpp?(Hyuuga) were resistant, leading to the possibility that
‘Hyuuga’ may carry a unique allele at the Rpp3 locus or another resistance
gene. To further characterize these 14 lines, we inoculated each with
additional isolates. The differential responses that we observed suggest that
resistance is conditioned either by two closely linked loci or two different
alleles at the same locus.
Pectobacterium carotovorum (Erwinia carotovora subsp. carotovora) is a soft
rot plant pathogen that produces tissue maceration in host by secreting
exoenzymes that degrade polysaccharide-based plant cell wall components.
Although many genes are known to be involved in the regulation of virulence
and exoenzyme production, the function of approximately 20% of P.
carotovorum’s predicted proteome still remains unknown. We have isolated a
mini-Tn5 lacZ1 transposon mutant of P. carotovorum Ecc 71 (CKD_A12)
that is altered in the production of exoenzymes and virulence. Compared to
the parent, the mutant produced less extracellular protease (Prt), cellulose
(Cel), pectate lyase (Pel) and polygalacturonase (Peh) and macerated less
plant tissues. The mutated gene in CKD_A12 was identified as corA, a
magnesium/nickel/cobalt transporter. The mutant phenotype was confirmed in
parental strain P. carotovorum Ecc 71 by marker exchange inactivation of
corA. The marker exchanged mutant was tested for exoenzyme levels, and as
in mutant CKD_A12, Pel, Prt, Peh, and Cel levels were all reduced in the
corA mutants. Complementation of both the original and the marker exchange
corA mutant strains with a functional corA+ gene from P. carotovorum Ecc 71
restored exoenzymes levels to parental levels and pathogenicity in celery and
carrot. These results indicate that, like in Salmonella enterica, CorA is
involved in the virulence of P. carotovorum.
USDA-APHIS plant pest permitting policy pertaining to containment
facilities for plant pathogens
M. J. KENNEY (1)
(1) USDA APHIS, Riverdale, MD, U.S.A.
Phytopathology 100:S61
Gene expression profiling of two Citrus cultivars in response to
huanglongbing (HLB) using the Agilent Citrus custom microarray Chip
A. A. KHALAF (1), F. G. Gmitter (2), R. H. Brlansky (2), J. Fan (2), G. A.
Moore (1)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) Citrus Research and
Education Center, University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S61
The research community has interest in new and emerging plant diseases,
agrobioterrorism, and exotic biocontrol organisms; research in these areas
frequently must be conducted in containment facilities in order to safeguard
American agriculture and the environment. The Animal and Plant Health
Inspection Service (APHIS) recently implemented several new policies and
standard conditions pertaining to the application, inspection, approval and
maintenance of containment facilities. The level of biocontainment security
required is based on risk of escape and possible establishment of plant pests.
Containment facilities can consist of laboratories, growth chambers, and
greenhouses, singly or combined. Containment facilities can be expensive to
design, build, and maintain. APHIS provides assistance to permittees during
this process; it also evaluates all containment facilities before permits are
issued for pathogen research. Periodic re-evaluations are also required. Only a
small percentage of the more than 2,000 APHIS-approved containment
facilities are adequate to do work with high risk pathogens such as Puccinia
graminis race UG99, Phytophthora ramorum, and plum pox virus. In addition,
to ensuring that appropriate structural safeguards are established and
maintained, there may be additional permits conditions that restrict the
movement and use of plant pathogens.
Identification of the molecular make-up of the Potato virus Y strain PVYZ
C. KERLAN (1), O. Nikolaeva (1), X. Hu (1), T. Meacham (1), S. Gray (2),
A. Karasev (1)
(1) University of Idaho, Moscow, ID, U.S.A.; (2) USDA-ARS, Cornell
University, Ithaca, NY, U.S.A.
Phytopathology 100:S61
Potato virus Y (PVY) strains were defined by interactions with different
resistance genes in potato cultivars. Five distinct strain groups were
distinguished that caused local and/or systemic hypersensitive response in the
genetic background with a corresponding N gene, these were PVYO, PVYN,
PVYC, PVYZ and PVYE. Here, we report that a recently found recombinant
isolate PVY-L26 induces a hypersensitive response in a potato cultivar
carrying the Nz gene, and is not recognized by two other resistance genes, Ny
and Nc. These genetic responses in potato, combined with the inability of
PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate
from the PVYZ strain group and provides the first information on genome
structure and sequence of this PVYZ strain. The genome of PVY-L26 isolate
displays typical features of PVYEU-NTN isolates, i.e. European NTN type with
three recombinant junctions. Two PVYNTN isolates elicited similar HR
reactions in cv Maris Bard carrying Nz gene, but, unlike L26, induced vein
‘Candidatus Liberibacter’, a phloem-limited bacterium, is associated with
citrus greening disease also known as huanglongbing (HLB). HLB infects all
citrus types and causes rapid decline of trees. There is no good source of
genetic resistance to HLB in the genus ‘Citrus’. A microarray experiment was
designed to compare gene expression response of two citrus cultivars to
Liberibacter infection, sweet orange (C. sinensis) that is extremely susceptible
and rough lemon (C. jambhiri), a more tolerant variety according to field
observations. Plants from both cultivars were graft-inoculated with HLB. A
similar set of plants of the same size and age from each cultivar was mock
graft-inoculated. RNA was extracted from leaves collected at four time-points
post inoculation (0 time, 9, 17, 27 wpi) from both inoculated sweet orange and
mock inoculated plants. The arrays were repeated three times with different
independent biological replicates from infected and mock-inoculated plants of
both cultivars. The resultant complimentary RNA (cRNA) was hybridized to
our custom Citrus Agilent GeneChip Array (4x44K format). Gene expression
analysis results will be presented.
Synergistic agents to reduce fungicide resistance and health risks
N. I. KHAN (1), J. A. Trogolo (1)
(1) Agion-Technology Inc., Wakefield, MA, U.S.A.
Phytopathology 100:S61
Agion Technologies Inc. has developed highly effective microbiocides with
levels of mixed metals up to 1,000 times lower than the early fungicides.
Agion’s formulation C200, may function as a stand alone biocide and may be
most valuable as a powerful synergist with conventional fungicide thereby
reducing the use levels and their environmental impact, as well as may result
in efficacy against the resistant strains. Laboratory test results indicate
that C200 alone at 100pm, 50 ppm or 25 ppm or combining with
Mancozeb (EBDC) or Daconil (Cholorothalonil) at 50%, 25% or 12.5% can
be as effective as Mancozeb or Daconil at 100% recommended dose level
against both Alternaria solani and Botrytis cineria (P > 0.05). Combining
C200 at 50 ppm and 25 ppm with Daconil or Mancozeb at 50% and 25%
of there recommended doses showed additive or synergistic effect that
was not significantly different from the 100% recommended dose of
Mancozeb. In repeated detached tomato leaf bioassay experiments, C200
alone at 100ppm, or combined with Mancozeb 50% of the recommended
dose showed significant reduction in mean disease severity over control (P >
0.05). There was no significant difference among, C200 at 100 ppm,
Vol. 100, No. 6 (Supplement), 2010
S61
50 ppm, and 25 ppm, Mancozeb at 100%, 50% and 25% of the recommended dose, and the combination treatments where C200 100ppm was
combined with Mancozeb at 50% or 25% of the recommended dose (P >
0.05). Greenhouse and field experiments will be performed in the next
phase.
Prevalence of benzimidazole resistance in Botrytis cinerea isolates from
Rose greenhouses in Center of Iran
P. KHAZAELI (1), H. Zamanizadeh (1), B. Morid (2), S. Hajmansoor (1)
(1) Department of Plant Pathology, Science and Research Branch, Islamic
Azad University, Tehran, IRAN; (2) Department of Plant Protection, Takestan
Branch, Islamic Azad University, Takestan, IRAN
Phytopathology 100:S62
Botrytis cinerea Pers. is a phytopathogenic fungus which causes grey mould
on over 230 hosts. Development of fungicide resistance is now one of the
major problems in plant disease control. The widespread use of fungicides,
such as benzimidazoles, in the prevention or elimination of fungal attack has
resulted in the appearance of resistant strains. This study was conducted to
evaluate the sensitivity and resistance response to benzimidazole of 51 isolates
of B. cinerea were obtained from Roses are cultivated under greenhouses in
Tehran and Markazi provinces of Iran. The isolates were identified at the
species level by their morphological characteristics and molecular method
with used specific primer. Threshold concentrations for evaluating the
resistance were 1 ppm for sensitive, 50 ppm for resistance and 500 ppm for
high resistance. Results showed that 42% of isolates grew on PDA containing
500 ppm benomyl and showed the high resistance phenotypes, whereas all
other isolates were inhibited by this concentration and grew on PDA
containing 1 ppm benomyl and showed that sensitive phenotypes. According
to the results, the resistance of Botrytis cinerea isolates to benzimidazole is
increasing in rose producing greenhouses of Iran and this widespread
occurrence of resistance to benzimidazole fungicides suggests that their
efficacy against grey mould might be impaired and that more aggressive
resistance management is needed.
A semi-automated system for quantitative analysis of Meliodogyne
reproduction
J. KILCREASE (1), F. Solano (1), A. Rascon (1), S. Hanson (1)
(1) New Mexico State University, Las Cruces, NM, U.S.A.
Phytopathology 100:S62
types of X. fastidiosa cells which are required to the virulence and
transmission.
Molecular characterization of genes differentially expressed during
conidiation by Magnaporthe oryzae
K. KIM (1), Y. Lee (1)
(1) Department of Agricultural Biotechnology, Center for Fungal Genetic
Resources, and Center for Fungal Pathogenesis, Seoul National University,
Seoul, SOUTH KOREA
Phytopathology 100:S62
Like most fungal pathogens, conidia (asexual spores) of Magnaporthe oryzae
play a key role in disease cycle. Briefly, this includes conidial attachment to
the surface of host plants, conidial germination, appressorial development,
colonization of host cells, and asexual reproduction (conidiation) for
conidium-mediated dissemination and new infections. This cycle occurs
several times during rice-growing season, causing severe loss of rice
production. Therefore, understanding the molecular mechanisms involved in
conidiation would be a priority to develop novel strategies for disease
management. However, relatively little is known about molecular mechanisms
that regulate conidiation in M. oryzae. Recently, our lab revealed that a
homeodomain transcription factor, MoHOX2 is a key regulator, specifically
involved in conidiation. To better understand the molecular mechanism of
conidiation-mediated by MoHOX2 in M. oryzae, we performed a microarray
analysis using RNAs from conidiating and nonconidiating mycelia of the
wild-type, and the conidiation-defective mutant, ΔMohox2. This analysis
resulted in the identification of genes differentially expressed during
conidation and in the conidiation-defective mutant. Many were revealed to
encode potential regulators such as transcipriton factors, protein kinase,
phosphatases, and etc. Ongoing works on identified genes from a microarray
analysis will be presented.
Fsr1-interacting proteins in Fusarium verticillioides are required for stalk
rot virulence on maize
J. KIM (1), C. Wang (1), B. D. Shaw (1), W. Shim (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S62
Quantification of eggs produced by Meloidogyne sp. as an indicator of
infection severity is a long accepted and practiced technique in nematology
and plant pathology. Since resistance to Meloidogyne is often partial rather
than complete, accurate quantitative measurements are essential for evaluation
of resistance in plants. Current methods used to count eggs produced are
tedious, time consuming, and prone to error. Herein we describe the
assessment of several methods for quantifying Meloidogyne reproduction in
plants and the development of a semi-automated system for quantification of
nematode eggs. We also show the use of this system in the evaluation of
infection severity in a nematode resistance development project. The system
uses computer assisted image analysis to count eggs captured in micrographs
of egg samples. Evaluation of several parameters demonstrates that this
system is substantially faster and more accurate than manual counting
methods. The system is also free of human bias that can skew results of
manual counting schemes. The data presented here includes methods for
extraction of eggs from infected plants and a description for how similar
quantification systems can be set-up on a minimal budget.
Maize stalk rot is a complicated disease primarily caused by fungal pathogens.
Using Fusarium verticillioides, one of the key stalk rot pathogens, we
discovered that a protein, designated Fsr1, plays an important role in stalk rot
virulence. The predicted Fsr1 protein contains multiple protein-binding
domains, and the coiled-coil (CC) domain in the N-terminus was determined
essential for virulence. The CC domain is known to mediate protein-protein
interactions, and our premise is that this interaction triggers downstream gene
signaling associated with stalk rot virulence. The aim of this study was to
identify putative Fsr1-binding proteins and determine their role in stalk
rot virulence. In particular, we used the N-terminal region of Fsr1 as bait
and performed yeast two-hybrid experiments. We identified two putative
Fsr1-binding proteins, Wor1 and Pex14, in F. verticillioides, and
these interactions were further verified through co-immunoprecipitation. For
further characterization, we generated wor1 and pex14 knockout mutants,
Δwor1 and Δpex14, respectively. Interestingly, while both mutants showed
reduced stalk rot virulence, they were not as severe as the symptom caused by
fsr1 deletion mutant. Our data suggest that Wor1 and Pex14 play a role in
stalk rot virulence by interacting with Fsr1. Further in vivo confirmation of
Fsr1-Wor1/Pex14 interactions with the use of fluorescent proteins are in
progress.
Role of structural polysaccharides in the virulence and transmission of
Xylella fastidiosa
N. KILLINY (1), R. Almeida (1)
(1) University of California-Berkeley, Berkeley, CA, U.S.A.
Phytopathology 100:S62
Functional characterization of FST1 from Fusarium verticillioides
H. KIM (1), C. P. Woloshuk (1)
(1) Purdue University, Department of Botany and Plant Pathology, West
Lafayette, IN, U.S.A.
Phytopathology 100:S62
Xylella fastidiosa, is the causal agent of Peirce’s disease in grape and
other emergent diseases. It is transmitted by the xylem sap feeding
sharpshooters where it colonizes the foregut. Previous work showed that, the
host structural carbohydrates, pectin and glucan provoke significant
changes in gene regulation resulting in inducing the transmission by vector.
Here, we report the effect of the vector polysaccharide, chitin on X.
fastidiosa growth. Similarly to pectin, chitin affects the gene regulation. It upregulates the adhesion proteins and down-regulates the movement genes.
Cells grown in the presence of chitin showed are more attached to glass
rather than planktonic. Additionally, X. fastidiosa cells were able to form a
biofilm on the citinaceous surface of insect wings and use chitin as a
carbon source. We propose that chitin in insect foregut is an alternative
environmental sensor to pectin. Our data show that, the environmental
sensors (polysaccharides) and the diffusible signaling factor (DSF) contributes jointly in the gene regulation to produce attached and movable
Fusarium verticillioides causes an ear rot disease in maize and produces the
mycotoxin fumonisin B1 (FB1). FST1, a putative hexose transporter gene in
F. verticillioides, is highly expressed in endosperm compared with germ.
When inoculated onto ears, a disruption-mutant (Δfst1) grew at half the rate as
the wild type, disease symptoms were delayed, and no FB1 was detectable. A
functional characterization of the FST1 promoter revealed that regulation of
FST1 expression is similar to fumonisin (FUM) genes; the expression was the
highest during growth on endosperm and repressed by high levels of
ammonium. Fluorescence microscopy with a strain expressing a fluorescenttagged FST1 suggested that FST1 is localized to the plasma membrane.
Expression of FST1 failed to complement growth of a yeast strain that lacks
hexose transporter genes. From the results, we hypothesize that FST1
functions as an environmental sensor that is important for initiating mycotoxin
production and for colonization of maize kernels.
S62
PHYTOPATHOLOGY
Stability and fitness of pyraclostrobin- and boscalid-resistant phenotypes
in field isolates of Botrytis cinerea from apple
Y. KIM (1), C. Xiao (1)
(1) Washington State University, TFREC, Wenatchee, WA, U.S.A.
Phytopathology 100:S63
Phenotype stability and fitness of pyraclostrobin (PYR)- and boscalid (BOS)resistant isolates of Botrytis cinerea from apple were evaluated. Stability of
resistance to PYR and BOS was determined after consecutive transfers on
potato dextrose agar (PDA) or being cycled on apple fruit. Various fitness
components including mycelial growth, osmotic sensitivity, conidial
germination, pathogenicity and virulence on apple fruit, sporulation in vitro
and in vivo and competitive ability on apple fruit were evaluated. After 20 and
10 transfers on PDA and 5 and 3 cycles on apple fruit at 20 and 0°C,
respectively, resistance to PYR and BOS retained at the similar levels as the
initial generation. There were no significant differences in mycelial growth,
osmotic sensitivity, conidial germination and sporulation between resistant
and sensitive isolates except that isolates resistant only to BOS produced
fewer conidia in vitro. Resistant isolates were as pathogenic and virulent on
apple fruit as sensitive isolates. After four disease cycles on apple fruit
inoculated with a mixture of a sensitive isolate and one of the three resistant
phenotypes, the frequency of PYR-resistant individuals was significantly
decreased compared to the initial generation and no BOS-resistant individuals
were detected. The results indicate that resistance to PYR and BOS in B.
cinerea was stable and that PYR- and BOS-resistant isolates did not compete
well with sensitive isolates in vivo.
Sensitivity to pyraclostrobin and boscalid in Penicillium expansum
populations from apple in Washington State
Y. KIM (1), Y. Yin (1), C. Xiao (1)
(1) Washington State University, TFREC, Wenatchee, WA, U.S.A.
Phytopathology 100:S63
Blue mold caused by Penicillium expansum is a major postharvest disease of
apples. Pristine (pyraclostrobin + boscalid) as a preharvest treatment is
effective in controlling blue mold. To establish baseline sensitivity to
pyraclostrobin and boscalid, 50 isolates of P. expansum collected prior to the
registration of Pristine were tested for sensitivity to the fungicides using
conidial germination assays. EC50 values of pyraclostrobin ranged from
0.004 to 0.009 µg/ml, with a mean of 0.006 µg/ml. Boscalid only delayed
conidial germination compared to the control while most conidia were able to
germinate after 30 h of incubation at all concentrations (up to 100 µg/ml)
tested. The minimum inhibitory concentration of pyraclostrobin was 1 µg/ml.
To monitor resistance to pyraclostrobin, 145 isolates from decayed
apples originating from orchards where Pristine had been used for 4
consecutive years were collected from a commercial packinghouse and
screened for resistance to pyraclostrobin. EC50 values of pyraclostrobin for a
subset of 30 isolates from the exposed population were determined. All
exposed isolates remained sensitive to pyraclostrobin. There was no
significant difference in mean EC50 values of pyraclostrobin between the
baseline and exposed populations. The results indicated that boscalid was not
effective against P. expansum and that there was no significant shift in
sensitivity to pyraclostrobin in the exposed populations after 4-year use of the
fungicide.
Cytological and genetic responses of near isogenic lines carrying rice blast
resistance genes
H. KITO (1), K. S. Zenbayashi (1), T. Nakajima (1)
(1) National Agricultural Research Organization, Daisen, Akita, JAPAN
Phytopathology 100:S63
The partial resistance gene Pi34 is proposed to confer durable resistance to
blast in rice. To analyze the partial resistance cytologically, we observed
penetration of Magnaporthe oryzae to rice leaf blades with whole cell clearing
method (Koga and Kobayashi 1980). Pi34-NIL and Chubu32, which is a
donor cultivar of Pi34, did not inhibit penetration of M. oryzae. We also
investigated penetration and infection in rice by intact leaf sheath inoculation
method (Koga et al. 2004). The epidermal cells of Pi34-NIL did not prevent
M. oryzae from penetrating but infecting to adjacent rice cells. Cell death and
accumulation of active peroxide in epidermal cells of Pi34-NIL under
appressoria showed similarity to that of Pib-NIL. Consequently, Pi34
presented common cytological phenotypes with Pib, which classified into the
true resistance gene, except for penetration. To investigate mechanisms of the
partial resistance genetically, transcriptome analysis was conducted with
SuperSAGE method (Matsumura et al. 2003). Two tags, OMG-01 and OMG02, presented distinct expression depending on the presence of Pi34. These
cDNAs and genomic fragments corresponding to OMG-01 and OMG-02 were
amplified and sequenced. OMG-01 and OMG-02 had two alleles in the
cultivars carrying and not-carrying Pi34, respectively. All of them were
expressed. Expression pattern of OMG-01 was constitutive, whereas that of
OMG-02 was infection inducible. We speculated that OMG-02 was the
primary candidate of Pi34.
Ametoctradin: A new Oomycete specific fungicide
K. KLAPPACH (1), K. Walker (2)
(1) BASF, Limburgerhof, GERMANY; (2) BASF, Res Triangle Park, NC,
U.S.A.
Phytopathology 100:S63
Ametoctradin is a new Oomycete specific fungicide under development by
BASF Corporation. Ametoctradin belongs to a new class of chemistry the
triazolo-pyrimidylamines in FRAC group 45. It is a strong inhibitor of
mitochondrial respiration in complex III and has shown to be very active
against zoospores and zoosporangia. It is an excellent preventative material
with a high affinity for the waxy layers of the leaf surface. In research trials, it
has show excellent residual activity and rainfastness properties. Ametoctradin
controls major plant pathogens from the Oomycete class of fungi, specifically
downy mildews and Phytophthora spp. on vine, vegetable crops and
ornamentals. Ametoctradin is very active against Plasmopara viticola in grape,
Phytophthora infestans and a broad variety of downy mildews. The compound
has a favorable toxicological and ecotoxicological profile. The active
ingredient trade name for ametoctradin is Initium® Fungicide. EPA
registration is expected in 2012.
Predicting field activity of experimental fungicides on Septoria leaf blotch
of wheat using multiple regression modeling
C. J. KLITTICH (1), Y. Adelfinskaya (1), W. K. Brewster (1), C. Yao (1)
(1) Dow AgroSciences LLC, Indianapolis, IN, U.S.A.
Phytopathology 100:S63
Fungicide invention generally involves greenhouse testing to predict the
efficacy of compounds in the field, but translation of activity from greenhouse
to field can be skewed by many factors (rainfastness, UV stability,
redistribution, etc.) in ways that are difficult to predict. We used multiple
regression modeling to determine which biological and physiochemical
measurements would best predict control of Septoria leaf blotch of wheat in
the field. Nine compounds from a novel chemical class were tested side-byside in field microplots in 2007. Field activity was well modeled (R2 adj. =
0.87) using only two factors; greenhouse protectant IC50 (Prob > |t| = 0.0005)
and UV stability (T½ range 1-150 hr, Prob > |t| = 0.09). Other factors such as
melting point (range 96-167 C) and log Kow (range 3.5-4.2) were not
significant. To test the importance of UV stability to field activity, we selected
compounds with a wider range of UV stability (T½ range 26-1300 hr) for field
testing in 2008. Compounds with high UV stability were less active than the
2007 model predicted, and regression modeling of the 2008 data showed that,
while greenhouse IC50 continued to be a strong predictor of field activity
(Prob > |t| = 0.008), UV stability did not contribute significantly to the model
(Prob > |t| = 0.25). We concluded that greenhouse activity best predicts the
field control of Septoria leaf blotch by this chemistry, and that UV stability is
not a consistent predictor.
Evidence that fern distortion syndrome is caused by fluorescent
pseudomonads
J. W. KLOEPPER (1), F. Saborío (2), J. A. McInroy (1)
(1) Auburn University, Auburn, AL, U.S.A.; (2) Centro de Investigaciones
Agronómicas, Universidad de Costa Rica, San José, COSTA RICA
Phytopathology 100:S63
Fern distortion syndrome (FDS), a newly described disease of Leatherleaf fern
(Rumohra adiantiformis), causes significant damage in ferneries of Costa
Rica. The main aboveground symptoms of FDS are twisting and distortions of
fronds, making fronds unmarketable. A recent report found an association of
symptoms with increased endophytic rhizome populations of fluorescent
pseudomonads (FPs), and constituted the first steps of Koch’s postulates
(demonstrating a constant association of the suspected causal agent with the
disease and isolation of the agent). Here we report that strains of FPs
isolated from rhizomes, roots, and petioles of ferns with FDS symptoms
cause the typical symptoms of FDS in two greenhouse experiments conducted
over a three-year period. In the first test, rhizomes of asymptomatic ferns
were collected in the field and treated at the time of transplanting in the
greenhouse with log 6 and log 8 cfu/ml with 6 groups of FPs isolated from
symptomatic fern plants. In the second test, tissue culture plants were grown
until rhizomes were present, which were treated similarly to the first
experiment, and an additional 2 treatments were also used: log 6 and log 8
cfu/ml of FPs isolated from healthy-appearing ferns. Ferns treated with the
FPs isolated from symptomatic ferns, but not those treated with FPs from
asymptomatic plants, developed distortions typical of FDS, including twisting
of the frond rachis, loss of triangular shape of frond, and reduced size of
fronds.
Vol. 100, No. 6 (Supplement), 2010
S63
Comparative genomics of the plant vascular wilt pathogens, Verticillium
dahliae and Verticllium albo-atrum
S. J. KLOSTERMAN (1), K. V. Subbarao (2), S. Kang (3), P. Veronese (4),
S. E. Gold (5), B. P. Thomma (6), Z. Chen (7), B. Henrissat (8), Y. Lee (9), J.
Park (9), M. D. Garcia-Pedrajas (10), D. Barbara (11), A. Anchieta (1), R. de
Jonge (6), P. Santhanam (6), K. Maruthachalam (2), Z. K. Atallah (2), S.
Amyotte (12), Z. Paz (5), P. Inderbitzen (13), D. Heiman (7), S. Young (7), Q.
Zeng (7), R. Engels (7), M. Koehrsen (7), J. Galagan (7), B. Birren (7), C.
Cuomo (7), K. F. Dobinson (12), L. Ma (7)
(1) USDA ARS, Salinas, CA, U.S.A.; (2) UC Davis, Salinas, CA, U.S.A.; (3)
Pennsylvania State University, University Park, PA, U.S.A.; (4) North
Carolina State University, Raleigh, NC, U.S.A.; (5) University of Georgia,
Athens, GA, U.S.A.; (6) Wageningen University and Research Center,
Wageningen, NETHERLANDS; (7) Broad Institute, Cambridge, MA, U.S.A.;
(8) Architecture et Fonction des Macromolecules Biologiques, CNRS,
Universites Aix-Marseille, Marseille, FRANCE; (9) Department of
Agricultural Biotechnology, Seoul National University, Seoul, KOREA; (10)
Estacion Experimental La Mayora, CSIC, Malaga, SPAIN; (11) University of
Warwick, Warwick, ENGLAND; (12) Agriculture and Agri-Food Canada,
London, ON, CANADA; (13) UC Davis, Davis, CA, U.S.A.
Phytopathology 100:S64
Verticillium dahliae and Verticillium albo-atrum are plant pathogenic fungi
that cause Verticillium wilts worldwide. The 7.5 X sequence of V. dahliae
strain VdLs.17 and the 4 X sequence of V. albo-atrum strain VaMs.102 were
generated and assembled at the Broad Institute using Sanger sequencing. A
comparison of these genomes revealed a high level of synteny between these
two Verticillium species, and led to the identification of a set of potential
effector proteins. In particular, our study revealed higher numbers of
pectinolytic enzymes in the Verticillium species than in other fungi, which
may have direct implications in the ability of these pathogens to colonize a
wide range of plant hosts. Additionally, we identified in the genome assembly
of V. dahliae strain VdLs.17 four lineage-specific (LS) regions which are
absent from VaMs.102. Certain gene families in the transposon-rich LS
regions have undergone expansion, including transcription factors, ferric
reductases, and phospholipases, which collectively may facilitate niche
adaptation. Comparative analyses with another vascular wilt fungus, Fusarium
oxysporum, revealed a conserved set of proteins that may have particular
relevance for these vascular wilt fungi. These findings provide insight into the
molecular determinants that underpin pathogenicity and niche adaptation in
these vascular wilt fungi, and provide a foundation for functional genomics
analyses.
Novel fungicide timings to target important turfgrass diseases in the
upper Midwest
P. KOCH (1), J. Kerns (2)
(1) University of Wisconsin - Madison, Verona, WI, U.S.A.; (2) University of
Wisconsin - Madison, Madison, WI, U.S.A.
Phytopathology 100:S64
The primary diseases of amenity turfgrass throughout the Upper Midwest are
dollar spot, Microdochium patch and Typhula blight. Previous research has
shown that fungicide applications made well before dollar spot symptom
development delays symptom development and reduces overall fungicide
usage when compared to a calendar-based program. Yet early season
applications do not provide season-long control of dollar spot or target most
other turfgrass diseases. This research examines novel fungicide application
timings targeting dollar spot, Microdochium patch and Typhula blight in order
to reduce fungicide expenditures while maximizing disease control.
Applications of boscalid or a tank-mixture of iprodione and chlorothalonil
were made in combinations of four different fungicide timings consisting of
early fall, late fall, early spring, and late spring. Fungicide applications made
at both the early and late spring timings provided the most effective control of
dollar spot, though they did not control Microdochium patch and Typhula
blight. Applications made at all four timings provided the best suppression of
dollar spot, Microdochium patch, and Typhula blight. Although, dollar spot
suppression only lasted until mid-July. Implementing an integrated program in
mid-July would provide season-long dollar spot and snow mold control with
as little as 4 or 5 fungicide applications.
Validation of commercially available ELISA kits for analyzing
chlorothalonil and iprodione residues on creeping bentgrass using gas
chromatography
P. KOCH (1), J. Kerns (2)
(1) University of Wisconsin, Verona, WI, U.S.A.; (2) University of
Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S64
Commercially available enzyme-linked immunosorbent assay (ELISA) kits
(Horiba, Ltd) can determine concentrations of chlorothalonil and iprodione on
S64
PHYTOPATHOLOGY
fruits and grains quickly and accurately. To be used on turfgrasses,
modifications to the ELISA protocol provided by Horiba, Ltd were required to
accommodate smaller quantities of plant material collected from creeping
bentgrass mowed at fairway height. The amount of tissue was reduced from 5
g to 0.2 g and the tissue was pulverized with a solvent for extraction of the
fungicides with/within the plant tissue. Validation of the modified ELISA
method was conducted in the fall of 2009 by comparing fungicide
concentrations collected from ELISA and gas chromatography/flame ionized
detection (GC/FID). This was accomplished by analyzing six samples of
creeping bentgrass for fungicide concentration one hour following the
application of chlorothalonil and iprodione using the altered ELISA method as
well as GC/FID. Chlorothalonil was not accurately detected using the ELISA
method, while measured levels of iprodione were consistently five times
lower than what was detected with GC/FID. The ELISA method was further
modified to be more consistent with the Horiba, Ltd protocol, and subsequent
comparisons between ELISA and GC/FID were completed on April 14 and
May 12. A reliable ELISA method for the detection of fungicides in turfgrass
can be used to provide insight into the role that fungicides play in disease
control.
Search for fungi as potential biological control agents of Salsola tragus
T. M. KOLOMIETS (1), D. K. Berner (2), Z. М. Mukhina (3), А. V.
Alexandrova (4), O. О. Skatenok (1)
(1) All Russian Research Institute of Phytopathology, Moscow region,
Bolshie Vyazemi, RUSSIA; (2) Foreign Disease-Weed Science Research
Unit, USDA, ARS, Frederick, MD, U.S.A.; (3) All Russian Scientific
Research Institute of Rice, Krasnodar, Belozerniy, RUSSIA; (4) M.J.
Lomonosov’s Moscow State University, Moscow, RUSSIA
Phytopathology 100:S64
Weeds of genus Salsola (family Chenopodiaceae) are widespread inhabitants
of many vegetative communities in damp territories and cultivated fields.
Plants of this genus are resistant to drought and are salt tolerant. Eurasia is the
natural habitat for Salsola. Salsola tragus is a widespread invasive weed
problem in the U.S.A. and is found in most states. In Russia Salsola tragus L.
is widespread in the Caucasuses, the southern areas of the European part of
Russia, in Siberia and in Central Asia. This weed infests grain fields, pastures,
rangelands, and roadsides. Because S. tragus is found mostly on low-value
land, control with herbicides is not economical. Thus, there is a necessity to
develop biological means of control. For use in classical biological control. a
complex of fungi were identified on Salsola tragus, These included the
obligate pathogen Uromyces salsolae Reich Colletotrichum gloeosporioides
(Penz.) Penz. And Sacc., Colletotrichum coccodes, Phomopsis oblonga,
Alternaria alternata (Fr.) Keissl., Epicoccum nigrum Link, Pythium sp.,
Sordaria sp, Arthrinium arundinis (Corda) Dyko and B. Sutton, Arthrinium
phaeospermum (Corda) M.B. Ellis., Bipilaris sorokiniana, Phoma herbarum
Westend., Ascochyta caulina (P. Karst.) Aa et Kesteren, Papulaspora
sepedonioides Preuss. Bionectria sp., Nigrospora oryzae (Berk. And Broome)
Petch., Nigrospora sphaerica, Stemphylium herbarum, Fusarium fujikuroi
Nirenberg., and Aspergillus versicolor (Vuill.) Tirab. Of these fungi,
Uromyces salsolae Reich., Colletotrichum gloeosporioides (Penz.) Sacc. and
Phomopsis oblonga (Diaporthe eres Nitschke) have great potential for
classical biological control of Salsola tragus.
Long-term survival of Xanthomonas fragariae in infected strawberry leaf
tissue
M. KONG (1)
(1) Driscoll Strawberry Assoc Inc, Watsonville, CA, U.S.A.
Phytopathology 100:S64
Xanthomonas fragariae is the pathogen that causes Angular Leaf Spot of
Strawberry, a very common and sometimes quarantine-related disease in
many countries. It is a common cause of disruptions in the movement of
strawberry plants across borders. To eliminate or reduce X. fragariae
contamination levels, short-term survival of X. fragariae under different
temperatures has been the subject of a number of studies. At Driscoll’s, we
completed a study of long-term X. fragariae survival in infected leaf material
under relatively favorable storage conditions. During April 1988, we prepared
X. fragariae by surface sterilizing strawberry leaves with Angular Leaf
Spot symptoms. Leaf spots were cut out from symptomatic leaves and air
dried in a laminar flow hood then stored in tape-sealed Petri dishes in a
refrigerator set at 5°C. Actual temperatures may have fluctuated from 0 to
15°C for short periods. We have tested a few of the leaf spots every few years
for viability. This was done by plating the bacterial suspension that oozed
out from the leaf spots and incubating this suspension at room temperature. During March 2009, after almost 21 years of storage we were still
able to recover living X. fragariae. This recovered X. fragariae was still
virulent and Angular Leaf Spot symptoms arose on susceptible plants that
were inoculated.
Genome-wide functional analysis of bZIP transcription factors in the rice
blast fungus
S. KONG (1), S. Park (1), K. Jung (1), Y. Lee (1)
(1) Department of Agricultural Biotechnology, Center for Fungal Genetic
Resources, and Center for Fungal Pathogenesis, Seoul National University,
Seoul, SOUTH KOREA
Phytopathology 100:S65
Basic leucine zipper (bZIP) proteins are major eukaryotic transcription factors
(TFs). Since fungal lifestyles depends on adaptability in environments, it is
pivotal to elucidate the transcriptional programs operating under different
conditions such as physical and chemical stresses, and host-dependent
constraints. In this study, bZIP TFs in Magnaporthe oryzae (MobZIPs) were
systemically characterized using phylogenetic, expression profiling and gene
deletion analyses. bZIP TF sequences from 23 fungal species were identified
and their phylogenetic relationship was analyzed. In total, 9 out of 15 groups
include at least one functional orthologs. Quantitative RT-PCR analysis for
MobZIP genes on 32 different conditions showed dynamic expression
profiles, suggesting their involvement in various stress responses and during
pathogenesis. To link phylogenetic and expression data to phenotypes, gene
deletion was conducted for 2 Magnaporthe-specific (MGG_02865.6,
MGG_07305.6) and 2 having orthologs (MGG_01990.6, MGG_02006.6).
Phenotype changes, however, were not detectable on these mutants for
mycelia growth in several stress conditions and pathogenicity on susceptible
rice plants. These results suggest that fungal bZIP TFs within the same clade
may have different roles depend on the lifestyles, and expression pattern does
not always directly reflect its roles on phenotypes.
Dose-response relationship in UV-C induced disease resistance and
phytoalexin accumulation in stored carrots
N. KOUASSI (1), R. Corcuff (1), R. Tweddell (2), J. Arul (1)
(1) Department of Food Science and Nutrition and Horticultural Research
Center, Universite Laval, Quebec, QC, CANADA; (2) Horticultural Research
Center, Université Laval, Quebec, QC, CANADA
Phytopathology 100:S65
Major limitation in long-term storage of carrots is the diseases caused by
Botrytis cinerea and Sclerotinia sclerotiorum. Intensification of the natural
defenses of the plant can be an attractive approach to protect stored crops
without resorting to chemicals. We have shown previously that UV-C induces
disease resistance in carrots. The objective of this work was to determine the
dose-response relationship in UV-C induced disease resistance and 6methoxymellein (6-MM) accumulation. Carrots (cv. Sun255) were exposed to
UV doses ranging from 0.0 - 10.8 kJ.m–2, and were stored at 4°C under high
relative humidity. After 14 days of storage, treated carrots were sampled to
assay 6-MM in the peel. After 28 days, treated carrots were inoculated with 3day old mycelial plugs of either B. cinerea or S. sclerotiorum and the severity
of infection assessed 21 days after inoculation. The dose-response relationship
for disease resistance to B. cinerea or S. sclerotiorum as well as 6-MM
accumulation in carrots was biphasic. The severity of diseases progressively
decreased compared with control roots up to a dose of about 5.4 kJ.m–2. But
beyond that dose, disease severity increased. Maximum accumulation of 6MM was also observed in carrots treated with the dose of 5.4 kJ.m–2. It was
concluded that beneficial or hormetic UV dose for improving disease
resistance of carrots was 5.4 kJ.m–2 and that the level of 6-MM present in
carrots before inoculation was a significant factor contributing to resistance.
Response of U.S. bottle gourd (Lagenaria siceraria) plant introductions
(PI) to crown rot caused by Phytophthora capsici
C. S. KOUSIK (1), J. A. Thies (2)
(1) USDA ARS, Charleston, SC, U.S.A.; (2) U.S. Vegetable Laboratory,
USDA ARS, Charleston, SC, U.S.A.
Phytopathology 100:S65
Phytophthora capsici can cause severe damage to cucurbit crops grown in
open fields in the southeast regions of U.S.A. In recent years there has been a
growing interest in the U.S.A. in grafting watermelon plants onto various
cucurbit rootstocks including bottle gourds for managing soil borne diseases.
We evaluated over 200 U.S. Plant Introductions (PI) of bottle gourd for
resistance to crown rot caused by P. capsici in the greenhouse by inoculating
four week old seedlings with a zoospore suspension (104/ml/plant). Plants of
watermelon variety ‘Mickey Lee’ were used as the susceptible check. This
trial was conducted twice. Plants were rated on a 1–9 scale of increasing
disease severity where 1 = no symptoms to 9 = plants dead. All the plants of
‘Mickey Lee’ were dead within 2–3 weeks after inoculation. Eleven (5.2%) of
the PIs tested were resistant to P. capsici. Of these 11 four were resistant and
7 were moderately resistant. Variability in the level of resistance of individual
plants within PIs also was observed. Based on the two evaluations, 42 PIs
were evaluated again and rated four times over 50 days after inoculation.
Disease development was significantly (P = 0.05) slower on PI 271352, PI
497351, PI491278 and PI 487482 compared to checks. These PIs can be
considered as potential sources of resistance to P. capsici. Single plant selections
from resistant PIs are being made for use in our rootstock breeding program.
Evaluation of wheat world genetic collections to harmful pathogens
E. D. Kovalenko (1), T. M. Kolomiets (1), M. I. KISELEVA (1), I. F.
Lapochkina (2), I. B. Ablova (3), Z. N. Khudokormova (3), H. Bockelman (4)
(1) All Russian Research Institute of Phytopathology, Moscow region,
Bolshie Vyazemi, RUSSIA; (2) Agriculture Research Institute of Non
Chernozem Zone, RUSSIA; (3) Krasnodar Agriculture Research Institute
named after P.P. Lukyanenko, RUSSIA; (4) USDA-ARS Germplasm
Resources Information Network (GRIN), Aberdeen, ID, U.S.A.
Phytopathology 100:S65
Puccinia triticina, Septoria sp. and powdery mildew are the most harmful
diseases of wheat in Russia. Nine hundred and twenty-two winter and spring
wheat accessions from NSGC, ARIPI, ARINCZ, and CIMMYT were
evaluated for leaf rust, septoriosis and powdery mildew in the infection
nurseries of Central region: All Russian Research Institute of Phytopathology
(ARRIP), Agriculture Research Institute of Non Chernozem Zone (ARINCZ),
and North Caucasian region: Krasnodar Agriculture Research Institute
(KARI). The leaf rust pathotypes and Septoria strains characterized genetic
diversity, high virulence and aggressive were selected for creation artificial
infectious backgrounds. Wheat cultivars were evaluated for resistance to
powdery mildew in natural infection environments. The main resistance types
to septoria and leaf rust in winter and spring wheats were identified based on
field and laboratory data. As a result of a study of partial resistance
components in field nursery (AUDPC) and in an artificial climate chamber
(latent period, pustule/spot density, pustule/spot size) the genotypes with high
level of partial resistance to leaf rust and Septoria were identified. The most of
accessions with race-specific and partial resistance to leaf rust was found out
among synthetic lines of wheat from USDA-ARS and spring wheat cultivars
from CIMMYT. Resistant and moderately resistant to septoriosis cultivars and
group resistant to leaf rust, Septoria and powdery mildew wheat samples were
revealed. Wheat cultivars with complex resistance, partial resistance and
combined different resistance types are represented the greatest interest for
breeding. The juvenile genes of resistance to leaf rust were identified for more
than 100 wheat cultivars by the phytopathologic testing and the STS and SSRmarkers. Resistant wheat cultivars from National Small Grains Collection
(NSGC) with complex of economically valuable features for quality
improvement of wheat cultivars in Central region of Russia were selected. The
following hybrid lines of spring wheat: PI 520375 (Mexico), PI 519425, PI
520528 (U.S.A., N. Dakota) and winter wheat: PI 564430, PI 564291
(Bulgaria), PI 422224, PI 547264, PI 547262 (England), PI 604222 (U.S.A.,
KS) and PI 447407 (China), can be recommended for breeding program.
Field efficacy of Bacillus subtilis MBI 600 (IntegralR) for managing rice
sheath blight caused by Rhizoctonia solani
V. KRISHNA KUMAR (1), M. Reddy (1), J. W. Kloepper (1), K. S.
Lawrence (1), S. Krishnam Raju (2), D. Groth (3), M. Miller (1)
(1) Auburn University, Auburn, AL, U.S.A.; (2) Andhra Pradesh Rice
Research Institute, Acharya NG Ranga Agricultural University, Maruteru,
INDIA; (3) Rice Research Station, Louisiana State University Ag Center,
Rayne, LA, U.S.A.
Phytopathology 100:S65
Rice sheath blight (ShB) caused by Rhizoctonia solani causes significant yield
losses in all rice producing areas of the world. Nursery and field trials were
conducted to assess the PGPR product Integral in India during 2009 against
ShB. Integral was applied as seed treatment (ST), seedling root dip (SD) and
foliar spray (FS) at concentrations of log 8 - 9 cfu per ml. Seedling growth
parameters and ShB severity were measured by adopting Highest Relative
Lesion Height (HRLH) at 90 days after transplanting. Seed bacterization with
Integral resulted in enhanced root (9.3 to 14 cm) and shoot lengths (37 to 45
cm) over the control (8.4 and 36 cm respectively) in nursery. On a
transplanted crop in the field, ShB severity was significantly lower when
Integral was applied as ST + SD + FS at 2.2 × 109 cfu ml–1 (19.2 to 26.5),
followed by at 2.2 × 108 cfu ml–1 (24.5 to 29.4) compared to control (56.2 to
69.7). The ShB severity in carbendazim treated plants ranged from 16.8 to
19.8. In addition, the tiller production/plant was significantly higher in
Integral treated plots at 2.2 × 109 cfu ml–1 (12.3 to 12.9) compared to the
control (10.0 to 10.5). Highest grain yields were recorded in Integral treated
plots at 2.2 × 109 cfu ml–1 (5922 to 6207 kg/ha) compared to the control (3925
to 4199 kg/ha). Overall, Integral significantly reduced the ShB severity, and
increased seedling vigor and grain yields in rice under field conditions.
Multi-scale spatial heterogeneity in disease incidence of Fusarium head
blight of wheat
A. B. KRISS (1), P. A. Paul (1), L. V. Madden (1)
(1) Ohio State University, Wooster, OH, U.S.A.
Phytopathology 100:S65
Vol. 100, No. 6 (Supplement), 2010
S65
A survey for the incidence of Fusarium head blight (caused by Fusarium
graminearum) was conducted in Ohio during the 2002 through 2009 growing
seasons. Sampling was conducted by counting the number of diseased and
healthy wheat spikes per 0.3 m of row at ten sites (about 30 m apart) in each
of 67-159 sampled fields in each of 12-31 sampled counties per year.
Incidence was then determined as the proportion of diseased spikes at each
site. Spatial heterogeneity of incidence among counties, fields within counties,
and sites within fields was characterized by fitting a generalized linear mixed
model (GLMM) to the data, using a complementary log-log link function,
with the assumption that the disease status of spikes was binomially
distributed conditional on the effects of county, field and site. The marginal
model for an individual field corresponds to an overdispersed discrete
distribution. Based on the estimated variance terms, there was highly
significant spatial heterogeneity among counties each year, and also among
fields within counties; magnitude of the estimated variances was similar for
counties and fields. The degree of heterogeneity varied significantly among
years. The lowest level of heterogeneity was among sites within fields, and the
site variance was not significantly greater than 0 in three of the eight years.
Based on the among-site variances, the intra-cluster correlation of disease
status of spikes within sites was low in most fields.
Role of El Niño-Southern Oscillation and atmospheric teleconnection
patterns on variability of Fusarium head blight in Ohio
A. B. KRISS (1), P. A. Paul (1), L. V. Madden (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S66
Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, is a
sporadic disease that is dependent, at least in part, on weather and climatic
conditions. To investigate the effect of climate on FHB, we used crossspectral analysis which is a tool used to partition the variance of time-series
data into temporal scales or periods. The time-series investigated were the
Southern Oscillation Index (SOI), which is a measure of the El Niño-Southern
Oscillation (ENSO), three atmospheric teleconnections [Arctic Oscillation
(AO), Pacific/North American (PNA) pattern, North Atlantic Oscillation
(NAO)] and Fusarium head blight rating in Ohio from 1965 to 2009. Mean
index values for December to February and March to May were used because
winter/spring conditions are important for F. graminearum survival and
development, thereby affecting disease. FHB and winter/spring SOI were
coherent at a periodicity of 5 years (K2 = 0.75/0.70). The phase relationships
showed that the SOI series lead FHB by 0.95 to 1.2 years. Consistent with
other studies, the most significant teleconnection related contributions were
for the winter months. FHB and winter PNA were coherent at a periodicity of
5.6 years (K2 = 0.66), FHB and winter AO were coherent at a periodicity of
2.3 years (K2 = 0.65), and FHB and NAO were coherent at a periodicity of
4.5 to 5 years (K2 = 0.66).
Simple, rapid, and specific DNA-based diagnostics for detection of the
bacterial wilt pathogen Ralstonia solanacearum Race 3 Biovar 2
R. KUBOTA (1), M. A. Schell (2), G. D. Peckham (1), A. M. Alvarez (1), C.
Allen (3), D. M. Jenkins (1)
(1) University of Hawaii, Honolulu, HI, U.S.A.; (2) University of Georgia,
Athens, GA, U.S.A.; (3) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S66
Simple, rapid, and specific diagnostic tools are urgently needed to
discriminate the quarantine pathogen Ralstonia solanacearum (Rs) Race 3
Biovar 2 (R3B2) from other populations of Rs that lack the adaptation to
cause bacterial wilt disease in temperate regions. Isothermal DNA
amplification techniques, such as Loop-mediated isothermal AMPlification
(LAMP), are suitable for rapid detection of bacteria with simple devices due
to the ability to amplify DNA with high specificity, efficiency, and speed at a
constant temperature. Three R3B2-specific LAMP primer sets were designed
by targeting previously identified unique R3B2 DNA sequences, and each
LAMP primer set was evaluated for its specificity, reaction time, and process
simplicity. The specificity of LAMP was assessed against a comprehensive
collection of 264 geographically diverse Rs-complex strains and 4 non-Rs
bacteria, and of these only a single Rs strain (UW348) failed to react with two
of the LAMP primer sets. The speed of LAMP reactions was monitored by
observing the fluorescence of a commercially available intercalating dye, and
the optimal reaction was completed within 20 min. The potential simplicity of
LAMP procedure was evaluated by omitting the denaturing and cooling steps,
and two of the LAMP primer sets amplified under the simplified procedure.
These results illustrate a promising alternative for the development of simple,
rapid, and specific diagnostics for Rs subpopulations.
Comparison of the rainfastness of Revus® and Forum® on lettuce for
control of downy mildew (Bremia lactucae)
P. KUHN (1), A. Hert (1), G. Olaya (1), L. Mantoe (2), G. Knauf-Beiter (2)
S66
PHYTOPATHOLOGY
(1) Syngenta Crop Protection, Vero Beach, FL, U.S.A.; (2) Syngenta Crop
Protection, Stein, SWITZERLAND
Phytopathology 100:S66
Resistance to washoff by rain or overhead irrigation is an important
characteristic of crop protection products including fungicides. We compared
this property for the fungicides Revus (Syngenta) and Forum (BASF) applied
to lettuce for control of downy mildew (B. lactucae). Fungicides were applied
at various rates with or without an adjuvant, followed by artificial rainfall
applied at different times after treatment via either a flat fan nozzle in a
cabinet spray chamber or from a purpose-built rain simulator. Fungicidetreated plants that received no artificial rain, and untreated, inoculated plants
were included as controls. Revus at all rates provided excellent disease
control. In addition, efficacy from Revus was not diminished by water or
simulated rain even when applied at only 30 or 45 mins after fungicide
treatment. These observations are consistent with Revus having a very high
degree of rainfastness. Irrespective of the washoff régime, Forum was
consistently less effective than Revus. Furthermore, the level of disease
control from Forum was markedly reduced after simulated rain, indicating
poor rainfastness over the time intervals evaluated.
Gene expressions of effectors in downy mildew of lima bean pathogen,
Phytophthora phaseoli
S. G. KUNJETI (1), N. M. Donofrio (1), A. G. Marsh (1), B. C. Meyers (1),
T. A. Evans (1)
(1) University of Delaware, Newark, DE, U.S.A.
Phytopathology 100:S66
Lima bean is an important legume crop for the state of Delaware. This crop is
susceptible to two oomycete pathogens, Phytophthora phaseoli and P. capsici,
causing downy mildew and pod blight of lima bean, respectively. In this study
we have identified several genes in P. phaseoli orthologous to effector genes
in P. infestans, a close relative of P. phaseoli. To initially identify these
effector genes, the Illumina next-generation sequencing platform was used for
profiling the transcripts of plant-grown and plate-grown P. phaseoli. Fulllength sequence analysis of three RxLR effector genes showed 97, 94, and 91
percent identity to amino acid sequences of PITG_17063, PITG_15039, and
PITG_04074 genes of P. infestans, respectively. In addition, two elicitors
showed 96 percent identity to the amino acid sequences of the INF1 and INF4
genes of P. infestans. The above five effector genes were validated by performing in-planta RT-PCR. The two elicitors INF1 and INF4 were overexpressed in plant-grown when compared to plate-grown tissue in both P.
phaseoli and P. infestans whereas only INF1 was expressed in plant-grown P.
capsici. A phylogenetic analysis of all five effector genes from other oomycete pathogens confirmed a close relationship of P. phaseoli and P. infestans
for all the corresponding effector genes. Currently, we are performing functional characterization of these effector genes, which will help us gain a better
understanding of this pathosystem and will serve as a basis for future research.
Towards solving ‘inconclusive’ quantitative PCR for the presence of
Huanglongbing (HLB) in orange jasmine leaf samples in Texas
M. KUNTA (1), J. V. da Graça (1), M. Sétamou (1), M. Skaria (1)
(1) Texas A&M University-Kingsville Citrus Center, Weslaco, TX, U.S.A.
Phytopathology 100:S66
As part of an ongoing survey for citrus huanglongbing (HLB) in Texas,
quantitative PCR (qPCR) tests for the casual bacteria, ‘Candidatus’
Liberibacter spp. in citrus and non-citrus species such as orange jasmine and
the vector, the Asian citrus psyllid (ACP) were conducted. Although all citrus
and ACP samples were tested negative so far for Ca. Liberibacter asiaticus, 44
orange jasmine samples yielded Ct values above 32. Some of the Ct values
were confirmed by the Plant Protection and Quarantine Molecular Diagnostic
Lab, Beltsville, MD, and designated as ‘inconclusive.’ The samples that
yielded inconclusive were resampled by collecting leaves and psyllids from
different parts of the plant. A total of 440 samples were collected from 55
plants (8/plant). High Ct values were obtained from 24 of these plants. More
inconclusive results were obtained in the samples collected from the south
side of the plants. All the psyllid samples were tested negative for Ca.
Liberibacter asiaticus. Nested PCR assays on the DNA extracts producing
high qPCR Ct values resulted in amplification of plant chloroplast 16S rDNA
and non-specific amplicons. Since orange jasmine is an alternative host of the
HLB bacterium, a traditional approach is being used to obtain 16S rDNA
sequences of any other bacteria which may be resident in these orange jasmine
plants.
Five years of monitoring foliar diseases of soybean in Minnesota
J. E. KURLE (1), D. K. Malvick (2), C. M. Floyd (2), G. M. Anderson (2)
(1) University of MN Department of Plant Pathology, Minneapolis, MN,
U.S.A.; (2) University of MN Department of Plant Pathology, St. Paul, MN,
U.S.A.
Phytopathology 100:S66
Several important foliar diseases occur on soybean, and soybean rust (SBR)
caused by Phakopsora pachyrhizi can be one of the most destructive to crop
yields. Although SBR has been detected in the southern and central U.S., it
has not been found in Minnesota (MN). For SBR to occur in MN,
urediniospores must be transported to MN and be deposited when
environmental conditions favor infection and disease development. From
2005 through 2009, up to 36 soybean sentinel plots were established each year
in commercial soybean fields or at University of Minnesota research fields as
part of a nationwide USDA Pest Information Platform for Extension and
Education. A minimum of 100 soybean trifoliate leaves from each plot were
analyzed weekly during the growing seasons to determine if SBR was present.
The leaf samples were incubated and then analyzed microscopically for SBR
and other foliar diseases. Spore deposition samplers were also placed in
sentinel plots during the growing seasons to monitor for SBR urediniospores.
Filters from spore samplers were collected weekly and were analyzed for
urediniospores using a nested PCR assay. Soybean rust spores were detected
in 2005, 2006, and 2007. Soybean rust was not found during the five years of
monitoring in MN, however, the sentinel plot effort provided a comprehensive
picture of other foliar diseases of soybean, including brown spot, downy
mildew, and bacterial blight.
Determining the effects of foliar and heading diseases on soft red winter
wheat (Triticum aestivum) yield in Wisconsin
K. LACKERMANN (1), J. Gaska (1), M. Martinka (1), S. Conley (1), P.
Esker (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S67
Foliar and heading diseases both reduce grain yield, but they occur at different
times during the growing season and can be managed independently. To
improve understanding of the effect of foliar and heading diseases on wheat
grain yield, data were collected from winter wheat variety trials at four
locations (Arlington, Chilton, Janesville, Lancaster). Fifty-eight winter wheat
cultivars were planted in a randomized complete block design with four
replications. Focus was on four foliar diseases (powdery mildew (PM),
Septoria leaf blotch (SLB), leaf rust (LR), and stripe rust (SR)) and one
heading disease (Fusarium head blight, FHB). Assessments were made four
times for foliar diseases and one time for FHB. For each foliar disease
assessment, a weighted disease severity value (WS) was calculated for the
upper four leaves. At Zadoks 60 (flowering), the WS ranged from 0 to 418
(PM), 0 to 421 (SLB), 0 to 293 (LR), and 0 to 121 (SR). FHB severity at
Zadoks 85 (soft dough) ranged from 0 to 11% with the highest severity noted
at Lancaster. Grain yields ranged from 2.5 to 7.5 MT/ha. The lowest mean
yield (4.2 MT/ha) was observed at Janesville, which had low levels of FHB (0
to 3.9%), but higher levels of SLB (0 to 421) and LR (0 to 213). There was
high variability in cultivar response to disease and subsequent grain yield. In
2009 foliar diseases may have caused more yield loss than FHB due to the low
incidence of FHB.
Effect of environment, cultivar, and disease on soft red winter wheat
(Triticum aestivum) production in Wisconsin
K. LACKERMANN (1), J. Gaska (1), M. Martinka (1), S. Conley (1), P.
Esker (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S67
Little is known about the impact of foliar diseases on wheat yield in
Wisconsin. Our objective was to compare the yield and disease incidence of
wheat cultivars at several environments in WI. Fifty eight wheat cultivars
were planted in a randomized complete block design at three locations
(Arlington, Chilton, Lancaster). At the fourth location (Janesville), the design
was a split-plot with a Zadoks 45 foliar fungicide application at the whole plot
level. Disease assessments were made at four times during the growing season
for powdery mildew (PM), Septoria leaf blotch (SLB), and leaf rust (LR). A
mixed model was used to study the effects of environment, cultivar, and
disease severity on grain yield, and the effect of environment and cultivar on
disease. Effects were significant at P = 0.05. Overall, SLB and LR were the
most prevalent diseases and were uniformly distributed across environments.
PM was most prevalent at Arlington and Chilton. Yield was affected by
location, cultivar, SLB, LR, and the location × cultivar interaction. SLB and
LR were affected by cultivar while PM was affected by location, cultivar, and
the location × cultivar interaction. At Janesville, the analyses for yield and for
disease revealed an effect of cultivar and PM on yield and an effect of cultivar
on SLB, LR, and PM, but no effect of fungicide on yield or disease. Our
results suggest that wheat growers should use cultivar selection to manage
disease incidence and maintain high yields.
Agrobacterium-mediated transformation of Fusarium oxysporum f. sp.
gladioli to study pathogenesis in gladiolus
D. K. LAKSHMAN (1), R. Pandey (2), K. K. Kamo (3), A. Mitra (4)
(1) Sustainable Agricultural Systems Laboratory, USDA-ARS, Beltsville,
MD, U.S.A.; (2) University of Lucknow, Lucknow, INDIA; (3) Floral and
Nursery Plants Research Unit, USDA-ARS, Beltsville, MD, U.S.A.; (4) Dept.
of Plant Pathology, University of Nebraska-Lincoln, Lincoln, NE, U.S.A.
Phytopathology 100:S67
Fusarium rot caused by F. oxysporum f. sp. gladioli (FOG) is one of the most
serious diseases of gladiolus, both in the field and in storage. Traditionally,
the pathogen had been controlled using methyl bromide soil fumigations that
are now banned, hot water treatments and to a limited extent, using tolerant
cultivars; necessitating further studies into host-pathogen interactions of the
pathogen on gladiolus. In this vein, we have developed an Agrobacteriummediated FOG transformation system using the pBGgHg vector (supplied by
Prof. S. Kang, Penn State) in AGL1 strain of A. tumefaciens. Hygromycin
(100 µg/ml) resistant (HygR) colonies were observed only when
acetosyringone (AS) was added to the co-cultivation medium. Also, the
efficiency of transformation increased when the Agrobacterium culture was
not pre-induced with AS before the cocultivation step and when cellophane
instead of Hybond-N+ membrane was used during cocultivation in medium
containing AS. Transformed isolates were selected by at least four serial
transfers in medium containing Hyg. The transformed mycelia expressed
green fluorescence which was not observed with non-transformed isolates.
PCR with Hyg-specific primers were positive from HygR isolates and not
from untransformed isolates. Transformed FOG isolates will be evaluated for
pathogenicity and behavior in transgenic gladiolus expressing chitinase genes.
Rose rosette and Redbud yellow ringspot are caused by two new
emaraviruses
A. G. LANEY (1), R. Gergerich (1), K. Keller (2), R. Martin (2), I. Tzanetakis
(1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) USDA-ARS,
Corvallis, OR, U.S.A.
Phytopathology 100:S67
Rose rosette (RR) was first described in Canada in the 1940s and Redbud
yellow ringspot (RYRS) in Arkansas in the 1970s. Since then, the only major
breakthroughs on the etiology of RR or RYRS was the discovery of double
membrane-bound bodies in symptomatic plants of both diseases and evidence
that the RR agent is transmitted by eriophyid mites. RR, a widespread disease
east of the Mississippi River and a major threat to the ornamental industry, is
usually associated with witches’ broom, lateral shoot elongation, and
malformation of flowers and leaves, culminating in plant death. RYRS, a
disease with unknown geographic distribution, causes chlorotic ringspots,
oak-leaf, and vein chlorosis in mature leaves. We have acquired data
suggesting that two new negative-stranded RNA viruses, members of the
newly established genus Emaravirus, and provisionally named Rose rosetteassociated virus (RRaV) and Redbud yellow ringspot-associated virus
(RYRSaV), are associated with RR and RYRS respectively. Detection
protocols have been developed and used to survey symptomatic roses and
redbuds for the respective viruses. Both viruses were found in almost all
diseased samples. Potential field alternative hosts were surveyed and several
herbaceous hosts were inoculated mechanically and by grafting. Transmission
studies for RYRSaV using an Aceria species eriophyid mite are under way.
Management of Verticillium wilt of potato with disease-suppressive crop
rotations
R. P. LARKIN (1), W. Honeycutt (1), M. Olanya (1)
(1) USDA ARS, Orono, ME, U.S.A.
Phytopathology 100:S67
The ability of potential disease-suppressive rotation crops to reduce potato
disease problems and increase crop productivity in a field severely infested
with Verticillium wilt was evaluated over three field seasons in Maine.
Rotation treatments consisted of 1) a high glucosinolate mustard blend
(‘Caliente 119’), a mixture of white mustard and oriental mustard with known
biofumigation potential, and 2) a sorghum-sudangrass hybrid, both grown as
green manures. These rotations were compared with a standard barley rotation
and a barley rotation followed by chemical fumigation with Metham sodium
as controls. Both green manure rotations significantly reduced wilt in the
subsequent potato crop compared to the barley control, with average
reductions of 25 and 18%, respectively, but were not as effective as chemical
fumigation (35% reduction). Mustard blend also reduced black scurf and
common scab better than the other rotations. Mustard blend and chemical
fumigation increased tuber yield relative to the barley control by 12 and 18%,
respectively. However, by the second rotation cycle, disease levels were high
in all rotations, and only chemical fumigation substantially reduced disease
(by 35%). Rotations also had significant effects on soil microbiology and
pathogen inoculum levels. This research indicates the potential for using
disease-suppressive rotations for managing Verticillium wilt, but also
demonstrates the limitations of 2-yr rotations regarding the build-up of
soilborne diseases over time.
Vol. 100, No. 6 (Supplement), 2010
S67
First report of Cucurbit leaf crumple virus in snap bean in Georgia
R. LARSEN (1)
(1) USDA ARS, Prosser, WA, U.S.A.
Phytopathology 100:S68
During October and November, 2009, a large commercial field planted with
snap bean (Phaseolus vulgaris) variety ‘Sea Biscuit’ in Tift County, GA was
observed exhibiting virus-like foliar symptoms consisting of interveinal
chlorosis and vein greening, blistering, rugosity and malformation. Pods were
curled, misshapen, and unmarketable. Symptoms were associated with the
presence of Bemesia tabaci and infection rate in the field was estimated at
>75%. Total nucleic acid was extracted from symptomatic leaf samples, and
then amplicons generated by PCR using a set of degenerate primers specific
for begomovirus coat protein (AV1 gene) were cloned and sequenced. The
nucleotide and deduced amino acid sequences of the 533 bp fragment were
97% and 98% identical, respectively, with the Arizona isolate of Cucurbit leaf
crumple virus (CuLCrV; a.k.a. Cucurbit leaf curl virus). Virus-like symptoms
were observed in an adjacent field on variety ‘Eliminator’ where whiteflies
were also present but the disease was determined not to be caused by
CuLCrV. Hence, ‘Eliminator’ may be a potential source of resistance to the
virus. CuLCrV has been restricted to cucurbits in California, Arizona and
Florida until more recently when it was detected in fresh market beans in
southwest Florida. This is the first report of CuLCrV in snap bean in Georgia.
The increased area of detection suggests that the virus may pose a threat to
common bean fields in other regions in the U.S. where B. tabacci are present.
Detection of Pyrenophora teres in infested plant tissues by PCR
R. T. LARTEY (1), T. Caesar-TonThat (1), A. J. Caesar (1), S. Hanson (1), U.
M. Sainju (1)
(1) USDA ARS, Sidney, MT, U.S.A.
Phytopathology 100:S68
Net blotch of barley, a commonly occurring foliar disease is caused by
Pyrenophora teres Drechs. The disease is characterized by small circular
elliptical spots which enlarge to the typical narrow netlike pattern. Lesions in
mature plants appear similar to spot blotch of Cochliobolus sativus, both of
which occur extensively in the Northern Great Plains of the U.S. Conidia and
ascospores from infested straw are probably the most important sources of
primary inoculum. To speed up detection and identification of P. teres in
infected hosts, a rapid PCR technique using Extract-N-Amp Plant PCR Kit
(Sigma-Aldrich) was developed. Bypassing the standard DNA extraction, leaf
disks from diseased tissues, uninfected barley leaves and P. teres pure culture
controls were first homogenized in extraction solution and diluted with the
solution provided in the kit. Aliquots of the homogenate were added to PCR
reaction and subjected to amplification using the newly developed PTACTIN,
a P. teres actin and ITS primers. Sizes of amplicons from diseases leaves
which were resolved on agarose gel, correlated with amplicon from the
control pure cultures. The amplicons were purified from the gel and
sequenced. Sequences alignment confirmed the detection of P. teres. The
technique will enhance rapid detection of P. teres in diseased barley and
alternate hosts, including asymptomatic plants as well as verification of
efficacy of management of net blotch.
Inferring evolutionary relationships of species in the Phytophthora Ic
clade using nuclear and mitochondrial genes
E. S. LASSITER (1), C. Russ (2), C. Nusbaum (2), Q. Zeng (2), C. Hu (3), J.
Thorne (1), J. B. Ristaino (3)
(1) Dept. of Genetics, North Carolina State University, Raleigh, NC, U.S.A.;
(2) Broad Institute, Cambridge; (3) Dept. of Plant Pathology, North Carolina
State University, Raleigh, NC, U.S.A.
Phytopathology 100:S68
Phytophthora infestans the causative agent of potato and tomato late blight is
an important pathogen worldwide and caused the Irish potato famine of the
1840’s. Two sisters species of P. infestans in the Ic clade, P. andina and P.
mirabilis have been described in Ecuador and Mexico, respectively.
Coalescent analysis revealed that P. andina and P. infestans share a common
ancestor in Ecuador on wild Solanum species. We have conducted Bayesian
analysis of several nuclear genes (beta-tubulin, elongation factor Iα, RAS, and
intron 1 of RAS) in isolates from Mexico and Ecuador and document the
shared evolutionary history of the three species, which cluster together and are
distinct from P. phaseoli and P. ipomoeae. Multiple heterozygous sites are
shared among the three species. This is particularly interesting in the light of
suggestion that P. andina may be a hybrid of P. infestans and P. mirabilis. We
have also sequenced, annotated and mapped the entire mitochondrial genomes
of P. andina, P. mirabilis, P. ipomoeae and P. phaseoli and compared then to
the mitochondrial genome of P. infestans. We will report results of the
Bayesian, coalescent and migration analysis using both the nuclear and whole
mitochondrial genomes of these species and resolve the evolutionary histories
of species of this clade.
S68
PHYTOPATHOLOGY
LysM receptor-like kinase 1-mediated chitin signaling and fungal
resistance in Arabidopsis
M. H. LE (1), X. Zhang (1), G. Stacey (1)
(1) University of Missouri, Columbia, MO, U.S.A.
Phytopathology 100:S68
Chitin, a polymer of -1,4-N-acetyl glucosamine is an important component
of the fungal cell wall. Chitin is one of the best studied pathogen-associated
molecular patterns (PAMPs) and is capable of eliciting basal defense
responses against fungal pathogens. Recently, the Arabidopsis receptor for
chitin was identified, theLysM-containing receptor-like kinase 1 (LysM RLK1
(LYK1)/CERK1). However, little is known about the molecular basis of chitin
perception and how chitin elicitation is translated into a cellular signal. To
gain insight into chitin-elicited defense responses, we conducted a yeast twohybrid based screen using the intracellular kinase domain of AtLYK1 as the
bait. We identified 54 putative LYK1-interactors after screening 4.5 × 106
transformants. The annotations of these putative interactors identify them as
both transmembrane and intracellular proteins, including transcription factors
and kinases. T-DNA insertions in some of these genes resulted in a decrease
in the production of reactive oxygen species after chitin treatment and
increased susceptibility to pathogens, including the bacteria Pseudomonas
syringae and the fungus Alternaria brassicicola. Therefore, these interactors
may function as positive regulators in the chitin signaling pathway. We are
currently working on further confirmation of the protein interactions detected
by yeast two hybrid. This study will contribute to our understanding of chitinelicited defense responses and to comparative studies of PAMP action.
Correlation of Erwinia amylovora virulence characteristics to disease
severity in fire blight in apple trees and seedlings
S. A. LEE (1), H. K. Ngugi (2), T. W. McNellis (1)
(1) Dept. Plant Pathology, Penn State University, University Park, PA,
U.S.A.; (2) Dept. Plant Pathology, Fruit Research and Extension Center, Penn
State University, Biglerville, PA, U.S.A.
Phytopathology 100:S68
Bacterial plant pathogens have a major impact on crop production. E.
amylovora, a gram-negative bacterium, is the causal agent of fire blight. Fire
blight is the most significant and destructive bacterial disease of rosaceous
plants, including economically important fruit crops such as apples and pears.
This study quantifies disease severity caused by six E. amylovora isolates
(Ea273, CFBP1367, Ea581a, E2002a, E4001a and Ea06P-1) on apple trees
and seedlings. Isolates produced a range of disease severity, with Ea06P-1
producing the greatest disease severity in every assay. We determined the
variation in virulence characteristic expression, including growth rates in
immature apple fruit, amylovoran production, levansucrase activity, biofilm
formation, carbohydrate utilization, hypersensitive cell death elicitation, and
protein secretion profiles. Multiple regression analysis indicated that
amylovoran production, biofilm formation and growth in immature apple fruit
accounted for over 70% of the variation in disease severity on apple seedlings.
Furthermore, in greenhouse-grown ‘Gala’ trees, over 75% of the variation in
disease severity was accounted for by amylovoran production, biofilm
formation, growth in immature apple fruit, hypersensitive cell death
elicitation, and sorbitol utilization. This study demonstrates that virulence
factor expression levels account for differences in disease severity caused by
wild isolates of E. amylovora on apple trees.
Functional identification of Xylella fastidiosa plasmid replication and
stability factors
M. LEE (1), E. E. Rogers (1), D. C. Stenger (1)
(1) USDA ARS, Parlier, CA, U.S.A.
Phytopathology 100:S68
Xylella fastidiosa (Xf) strain RIV11 harbors a 25 kbp plasmid (pXFRIV11)
belonging to the incP1 incompatibility group. Replication and stability factors
of pXFRIV11 were identified and used to construct plasmids able to
propagate in both Xf and Escherichia coli. Sequences required for replication
in E. coli and conferring antibiotic resistance were derived from the cloning
vector pCR2.1. Replication in Xf required a 1.35 kbp region from pXFRIV11
containing a replication initiation gene (trfA) and the adjacent origin of DNA
replication (oriV). This region also conferred plasmid replication in
Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas
syringae. Constructs containing the trfA gene and oriV derived from
pVEIS01, a similar 31 kbp incP1 plasmid of the earthworm symbiont
Verminephropbacter eiseniae, also were competent for replication in Xf. As
expected, constructs bearing only trfA or oriV from either incP1 plasmid were
unable to replicate in Xf. Although these incP1 replicons could be maintained
in Xf under antibiotic selection, removal of selection resulted in loss of the
plasmid. A novel toxin/antitoxin (pemI/pemK) addiction system of pXFRIV11
was added, improving stability of incP1 replicons in Xf in the absence of
antibiotic selection. The resulting 6 kbp Xf shuttle vector (pXF20-PEMIK)
also contains 10 unique endonuclease recognition sites for insertion of foreign
DNA.
Finer differentiation of phytoplasma strains based on phylogenetic
analysis of the secY gene
I. LEE (1), K. D. Bottner-Parker (1), Y. Zhao (1), R. E. Davis (1), N. A.
Harrison (2)
(1) USDA ARS MPPL, Beltsville, MD, U.S.A.; (2) FLREC, University of
Florida, Ft. Lauderdale, FL, U.S.A.
Phytopathology 100:S69
The 16S rRNA gene has been widely used as a molecular marker for
differentiation of phytoplasmas. Earlier studies indicated that 16S rRNA genebased phylogenetic analysis was sufficient to classify phytoplasmas into 16Sr
groups and subgroups, but its efficacy for differentiation of closely related
strains within a subgroup was relatively limited. Additional markers are
needed to overcome this limitation. The protein translocase gene, secY, is
more variable than the 16S rRNA gene and may represent a potential marker.
Comparative phylogenetic analyses with 16S rRNA and secY gene sequences
from representative phytoplasma strains were performed to assess the efficacy
for delineating phytoplasma strains within each 16Sr group and subgroup. The
phylogenetic interrelatedness among phytoplasma taxa inferred by secY genebased phylogeny was nearly congruent with that inferred by 16S rRNA genebased phylogeny. However, the secY gene-based phylogeny not only readily
resolved 16Sr subgroups within a given 16Sr group, but also delineated
distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high
resolving power makes the secY gene a more useful genetic marker than the
16S rRNA gene for finer differentiation of closely related phytoplasma strains
based on collective RFLP patterns using select restriction enzymes. The
genetic interrelationships among these strains thus determined coincided with
delineations by phylogenetic analysis.
Characterization of four Mitogen-activated protein kinase genes
(CsMPS1, CsHOG1, CsCHK1 and CsSte11) in the fungal cereal pathogen,
Cochliobolus sativus
Y. LENG (1), S. Zhong (1)
(1) Department of Plant Pathology, North Dakota State University, Fargo,
ND, U.S.A.
Phytopathology 100:S69
Mitogen-activated protein kinase (MAPK) pathways have been demonstrated
to be involved in fungal development, sexual reproduction and/or virulence in
several filamentous plant pathogenic fungi, including Cochliobolus
heterostrophus and Magnaporthe grisea, but genes for MAPKs in
Cochliobolus sativus, the causal agent of spot blotch, common root rot and
black point in barley and wheat, have not been characterized. We identified
four MAPK genes (CsMPS1, CsHOG1, CsCHK1 and CsSte11) in the whole
genome sequence of the C. sativus isolate ND93-1 based on their homology to
MAPK genes characterized in other fungi. Gene knockout mutants were
generated for CsSte11 and CsCHK1 and are being generated for the other two
genes (CsMPS1 and CsHOG1) using the split marker system. The knockout
mutants of CsSte11 and CsCHK1 were defected in conidiation, indicating that
they are involved in the asexual development of C. sativus, as ChSte11 and
CHK1 of C. heterostrophus do, respectively. However, CsCHK1 appears not
to be involved in melanization because no difference was observed between
the CsCHK1 mutant and the wild type in colony pigmentation on PDA plates.
Thus, CsCHK1 is different from the CHK1 of C. heterostrophus in that the
latter is involved in regulation of the melanin synthesis. The results of
pathogenicity tests and other phenotypic changes of the knockout mutants in
comparison with the wild type isolate will be presented.
A multiplex TaqMan assay for detection and differentiation of
Leptosphaeria maculans and L. biglobosa, causal agents of canola blackleg
W. Leonard (1), X. LI (1), D. S. Smith (1), J. Nie (1), T. Dumonceaux (2)
(1) Canadian Food Inspection Agency, Charlottetown, PE, CANADA; (2)
Agriculture and Agri-Food Canada, Saskatoon, SK, CANADA
Phytopathology 100:S69
Blackleg of canola is caused by two related fungal species, Leptosphaeria
maculans, a highly virulent species causing severe cankers at the base of
stems, and L. biglobosa, a weakly virulent species causing mild stem lesions.
For rapid detection and differentiation in canola seed, we designed a multiplex
TaqMan assay that targeted two genomic loci of L. maculans, LopB, a
pathogenicity gene and LMR1, a repetitive element linked to a virulence
region, and the mating protein gene, Mat1-2, of L. biglobosa. The primers
specifically amplified 110, 145, and 135 bp products from the three loci,
respectively, and TaqMan probes were labelled with Quasar 670, Cal-FluorRed 610, or Cal-Fluor-Orange 560 fluorescent dyes and compatible
quenchers, respectively. To validate negative results in the mutiplex PCR, a
FAM-labelled probe to a 200-bp internal control, designed from a rice
sequence bordered by LopB primer sequences, was included in the assay. The
final assay involved a 48-hr enrichment of 4-g samples in a minimal medium
with antibiotics, sample treatment in a beadbeater, and DNA extraction with
magnesil paramagnetic beads prior to assay by multiplex PCR. Under
optimized conditions the assay detected single infected seeds in a sample and
differentiated between L. maculans and L. biglobosa. Positive detection and
diagnosis was based on cycle threshold cutoff values and confirmation of
amplicon identity by capillary electrophoresis analyses.
An Indiana survey of Phytophthora species in nurseries, greenhouses, and
landscape plantings
A. J. LEONBERGER (1), C. Speers (1), G. Ruhl (1), T. Creswell (1), J.
Beckerman (1)
(1) Purdue University, West Lafayette, IN, U.S.A.
Phytopathology 100:S69
From 2006 to 2008, samples with symptoms consistent with Phytophthora
blight and crown rot were collected as part of the USDA-APHIS
Phytophthora ramorum nursery survey and submitted by additional outside
sources to the Purdue Plant and Pest Diagnostic Laboratory. From 30 sites,
121 Phytophthora isolates were obtained from 1657 host samples containing
32 genera. Comparison of the internal transcribed spacer (ITS) sequence of
the ribosomal DNA identified 9 Phytophthora sp. A majority of the isolates
were either P. citricola (44.4%) or P. citrophthora (27.2%). P. citricola
isolates were collected at 10 sites from 5 host genera, and included Forsythia,
Juglans, Pieris, Rhododendron, and Syringa. P. citrophthora was isolated
from 8 sites on 6 host genera: Abies, Forsythia, Ilex, Pieris, Rhododendron,
and Syringa. The other identified Phytophthora sp. consisted of P. cactorum,
P. capsici, P. cryptogea, P. drechsleri, P. nicotianae, P. palmivora, and P.
syringae. Sixteen isolates showed signs of possible species hybridization.
Four isolates were found to be hybrids of P. cactorum and P. hedraiandra as
verified by cloning and sequencing the ITS sequences. Three of the P.
cactorum x hedraiandra isolates came from Rhododendron plants at the same
site. The other hybrid isolate was recovered from Dicentra, which is not a
known host of either of the parental species, P. cactorum or P. hedraiandra,
and suggests an increase of host range due to species hybridization.
Reduced sensitivity and complete resistance of Michigan Venturia
inaequalis populations to sterol- and quinone-outside inhibitor fungicides
K. E. LESNIAK (1), T. J. Proffer (1), A. Irish-Brown (1), G. W. Sundin (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S69
Control strategies for Venturia inaequalis rely heavily on chemical
applications. Single-site fungicides such as the quinone-outside inhibitors
(QoI) and the sterol-inhibitors (SI) have been used in Michigan apple orchards
for 11 and more than 20 years, respectively. In 2008, we detected the G143A
mutation in the cytochrome b gene, conferring complete resistance to QoI’s,
in V. inaequalis isolates from 12 orchards located in two apple producing
regions in MI. In 2009, >1,500 single monoconidial isolates were established
from 66 orchards throughout Michigan and five non-orchard sites from
Michigan and Ohio. These 2009 isolates were subjected to two assays;
molecular detection of the G143A mutation using PCR, and a relative growth
(RG) assay on SI-amended media using discriminatory dosages for SI
sensitivity. To date, the G143A mutation has been detected in 63% of isolates
from 59 Michigan orchards. Previous studies indicate that practical resistance
to SI’s occurs in an orchard when the mean RG of all isolates is >67%. In 50
of 54 orchards currently tested, the average RG was above that level. The
mean RG’s of isolates from three non-SI treated sites was 43%. These
combined results indicate the immediate need for altered management
strategies of V. inaequalis in Michigan orchards.
Biological and molecular characterization of a cucumber isolate of Melon
necrotic spot virus from Ohio
D. LEWANDOWSKI (1)
(1) The Ohio State University, Columbus, OH, U.S.A.
Phytopathology 100:S69
An isolate of Melon necrotic spot virus (MNSV), designated MNSVcucumber was collected from symptomatic cucumbers (Cucumis sativus)
within a commercial greenhouse in Ohio in 2008. MNSV-cucumber was
mechanically transmitted to C. sativus, Cucumis melo, and Citrullus lanatus,
producing both local and systemic symptoms on some cultivars. MNSVcucumber also produced local lesions on mechanically inoculated cotyledons
of Cucurbita moschata and Cucurbita pepo. MNSV-cucumber was
transmitted by an isolate of Olpidium bornovanus collected from the MNSVinfected cucumbers to cucumber, melon, and watermelon. The ITS region of
the Ohio cucumber isolate of O. bornovanus was identical to a cucumber
isolate collected in Spain. Total RNA extracted from MNSV-infected
cucumber leaf tissue using the RNeasy Plant Mini Kit (Qiagen, Inc.) was
amplified by RT-PCR using MNSV-specific primers. The genome of MNSVcucumber was most similar to MNSV-Al, a melon isolate from Spain. The
Vol. 100, No. 6 (Supplement), 2010
S69
MNSV-cucumber genome was 4,267 nts in length and had a typical MNSV
genome organization, encoding five proteins: p29, p88, p7A, p7B, and p42
(coat protein). To the best or our knowledge, this is the first MNSV isolate
from the United States to be sequenced and characterized, and only the second
isolate from cucumber to be characterized.
Dual use research in the life sciences
P. LEWIS (1)
(1) Natl Inst of Health, Bethesda, MD, U.S.A.
Phytopathology 100:S70
Life sciences research is vital to improving public health, agriculture and the
environment, while strengthening our national security and economy.
However, the very same research may also yield information or technologies
that could be misused for harmful purposes. For instance, information from
certain life sciences research can be misapplied to create dangerous pathogens
to threaten humans, animals, and plants. The development of new
technologies and the generation of information with the potential for both
benevolent and malevolent purposes is referred to as “dual use research.”
Scientists have a professional responsibility to be aware of dual use research
issues and the potential ways in which information and products of their
research could be misused, and to take steps to minimize misuse of their work.
The National Science Advisory Board for Biosecurity (NSABB) was
established by the U.S. Government to advise on strategies for dealing with
potential dual use research. The NSABB has produced a series of reports on
strategies for overseeing and educating about dual use research. In the future,
the Board will continue to address topics of relevance to the plant pathology
community, including: (1) enhancing the culture of responsibility that already
exists among life sciences researchers and (2) advising on the HHS and
USDA Select Agent Program, as requested.
Disruption of the salicylate and jasmonate signaling pathways by the
cucumber mosaic virus 2b RNA silencing suppressor
M. G. Lewsey (1), A. M. Murphy (1), D. MacLean (2), N. Dalchau (3), J.
Westwood (1), K. Macaulay (1), M. H. Bennett (4), M. Moulin (1), D. E.
Hanke (1), G. Powell (4), A. G. Smith (1), H. Ziebell (5), J. CARR (1)
(1) University of Cambridge, Cambridge, UNITED KINGDOM; (2)
Sainsbury Laboratory, Norwich, UNITED KINGDOM; (3) Microsoft
Research, Cambridge, UNITED KINGDOM; (4) Imperial College, London,
UNITED KINGDOM; (5) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S70
The cucumber mosaic virus (CMV) 2b counter-defense protein disrupts plant
antiviral mechanisms mediated by RNA silencing and salicylic acid (SA). We
used microarrays to investigate defensive gene expression in 2b-transgenic
Arabidopsis thaliana plants. Surprisingly, 2b inhibited expression of few SAregulated genes and in some instances enhanced the effect of SA on certain
genes. Strikingly, the 2b protein inhibited changes in the expression of 90% of
genes regulated by jasmonic acid (JA). Consistent with this, infection of
plants with CMV, but not the 2b gene deletion mutant CMV∆2b, strongly
inhibited JA inducible gene expression. JA levels were unaffected by infection
with either CMV or CMV∆2b. Although the CMV-Arabidopsis interaction is
a compatible one, SA accumulation, usually considered to be an indicator of
plant resistance, was increased in CMV-infected plants but not in CMV∆2binfected plants. Thus, the 2b protein inhibits JA signaling at a step
downstream of JA biosynthesis but it primes induction of SA biosynthesis by
another CMV gene product or by the process of infection itself. JA is
important in plant defense against insects and CMV, like many plant viruses,
is aphid-transmitted. This raises the possibility that disruption of JA-mediated
gene expression by the 2b protein may influence CMV transmission. Funded
by grants from the BBSRC and Leverhulme Trust.
Detection and identification of pectobacteria associated with potato
blackleg reveals the presence of mixed populations
X. LI (1), L. Ward (1), J. Nie (1), J. Nickerson (1), A. MacDonald (1), J.
Gourley (1), S. H. De Boer (1)
(1) Canadian Food Inspection Agency, Charlottetown, PE, CANADA
Phytopathology 100:S70
The causal agents of the potato stem rots, blackleg and aerial stem rot, were
recently reclassified on their phenotypic and phylogenetic characteristics as
Pectobacterium atrosepticum (PA) and P. carotovorum (PC). Moreover, P.
brasiliensis (PB) and Dickeya spp. (DI) are now also recognized as causal
agents of blackleg-like symptoms in potato in Brazil and Europe, respectively.
In a preliminary survey of pectobacteria associated with blackleg symptoms
on potato in Canada, not only were PA and PC detected using PCR with taxon
specific primers and direct isolation, but P. wasabiae (PW), originally
described as the causal agent of soft rot of horse radish, was also detected. Our
study revealed that the most common pectobacteria associated with potato
blackleg in Canada was PA (85% of the total stem samples with blackleg
symptom). However, PW was also frequently present (36%, often in the same
S70
PHYTOPATHOLOGY
stems as PA), while PB (3%) and DI (2%) were found occasionally. To detect
and identify pectobacteria directly in diseased stems, strategies were
developed including a multiplex PCR assay targeting various genomic regions
of PA, PB, PC, PW and DI, and a rapid LAMP protocol for detecting and
identifying PA. The LAMP protocol targets the genomic region encoding the
pathogenicity-related polyketide synthase gene. The assay was specific for all
strains of the species tested and showed no cross-amplification of related
pectobacteria, dickeya strains, or other selected plant pathogenic bacteria.
Isolation and application of a Bacillus subtilis strain to control gray mold
on tomato and powdery mildew on cucumber
S. LI (1), X. Lu (1), B. Li (1), Q. Guo (1), Z. Li (1), P. Ma (1)
(1) Plant Protection Institute, Hebei Academy of Agricultural and Forestry
Sciences, Baoding, Hebei, PRC PEOPLES REP OF CHINA
Phytopathology 100:S70
Gray mold and powdery mildew are important diseases on tomato and
cucumber respectively causing a great damage in China. The control of both
diseases primarily depends on the application of chemical pesticides.
However, it becomes a popular and important problem that many chemicals
show the potential toxic effect on humans, wildlife, foods, pathogen resistance
and environment. So, it is a hot research point nowadays to find and use
natural product to control these diseases. Microbial fungicides show such a
more potential efficacy of control on plant diseases in greenhouse that the
studies thereof become more popular in China. In this study, a bacterial isolate
Bacillus subtilis BAB-1 were screened against the pathogen Botrytis cinerea
by dual culture and showed a significant efficacy to control gray mold on
tomato in pot test. A preparation, 5 × 108 cfu/ml spores AS, was made with
spores of B. subtilis BAB-1 and applied in greenhouse. Naturally infested, the
tomato and cucumber at flowering stage were sprayed 4 times (interval 7
days) with 50-fold diluted solution of this preparation. 45 days after first
spraying, the diseases were rated. This experiment was designed randomly
and conducted in two years, 4 replicates for each year. The treatment could
significantly reduced both diseases at p = 0.05 with a control efficacy 81.6%–
93.7% on gray mold of tomato and 81.1%–98.3% on powdery mildew of
cucumber. This study will provide a new and environment-friendly fungicide
to control both diseases in China.
Screening germplasm for resistance to Phomopsis seed decay in soybean
S. LI (1), J. C. Rupe (2), P. Chen (2), A. Wrather (3)
(1) USDA ARS CGRU, Stoneville, MS, U.S.A.; (2) University of Arkansas,
Fayetteville, AR, U.S.A.; (3) University of Missouri, Portageville, MO,
U.S.A.
Phytopathology 100:S70
Phomopsis longicolla is the primarily cause of soybean Phomopsis seed decay
(PSD), a major cause of poor seed quality in the United States. To identify
new sources of soybean lines resistant to PSD, field screening of 135 selected
soybean germplasm lines representing 28 worldwide origins and maturity
groups 3-5 along with PSD resistant and susceptible checks were tested in
Arkansas, Missouri, and Mississippi. Each entry was grown in a single 3-m
row plot in a randomized complete block design with four replications.
Frequent rainfall during seed maturation led to high levels of seed infection by
a number of fungi. Significant differences in seed infection by P. longicolla
occurred among soybean lines with some lines having no PSD while others
had levels as high as 90%. These differences between lines were reflected in
visual seed quality and in seed germination. Several lines with low disease
incidence, good visual quality, and high germination rate at all locations will
be tested for resistance in 2010 field trials.
Insights into common functional domains of tospovirus NSm proteins
W. Li (1), D. J. Lewandowski (2), M. E. Hilf (3), S. ADKINS (3)
(1) University of Florida, Lake Alfred, FL, U.S.A.; (2) The Ohio State
University, Columbus, OH, U.S.A.; (3) USDA ARS USHRL, Ft. Pierce, FL,
U.S.A.
Phytopathology 100:S70
Direct demonstration of tospovirus gene function has been impeded by the
absence of reliable reverse genetics systems for this virus genus. Use of a
Tobacco mosaic virus (TMV)-based expression system has demonstrated that
the Tomato spotted wilt virus (TSWV) NSm protein supports cell-to-cell
movement in the absence of other TSWV proteins. Essential TSWV NSm
domains required for tubule formation, movement and symptoms were
identified previously by deletion-mapping and alanine-substitution
mutagenesis using the TMV-based system. Our mutagenesis studies of TSWV
NSm amino acids that are conserved in other tospovirus NSm proteins suggest
that functional domains may be conserved across the genus, especially for
species in the ‘New World’ group. To that end, we initiated studies of the
Impatiens necrotic spot virus (INSV) NSm protein using similar TMV-based
mutagenesis approaches and we report here new insights into the tospovirus
NSm protein based on our functional analysis of the INSV NSm protein.
Evaluation of resistance to Pucciniastrum hydrangeae in Hydrangea
arborescens
Y. LI (1), M. Windham (2), R. Trigiano (2), A. Windham (3), S. Reed (4), T.
Rinehart (5), J. Spiers (5)
(1) Connecticut Agric Experiment Station, New Haven, CT, U.S.A.; (2)
University of Tennessee, Knoxville, TN, U.S.A.; (3) University of Tennessee,
Nashville, TN, U.S.A.; (4) USDA/ARS, McMinnville, TN, U.S.A.; (5)
USDA/ARS, Poplarville, MS, U.S.A.
Phytopathology 100:S71
Smooth hydrangea, Hydrangea arborescens L., is a native shrub in the eastern
North America. Rust caused by Pucciniastrum hydrangeae (B. & C.) Arth. is
an important disease on smooth hydrangeas in nurseries, gardens and
landscapes. In order to understand resistance to the rust disease in smooth
hydrangea, pustule formation and sporulation of the fungus on H. arborescens
‘Annabelle’, ‘Frosty’, ‘Green Dragon’, ‘Mayes Starburst’, ‘Pink Pincushion’,
‘Ryan Gainey’, and ‘White Dome’ were evaluated using a leaf disk bioassay.
Variation in resistance to rust in P. arborescens was observed although
formation of uredia and urediniospores was present on all seven cultivars. The
cultivar ‘Frosty’ displayed the highest resistance and had a relatively small
amount of uredia and urediniospore production. ‘Green Dragon’ was highly
susceptible and permitted the formation of the largest number of uredia per
disk. Intermediate reactions were detected with the other five cultivars, in
which ‘Mayes Starbust’ and ‘Annabelle’ had significantly larger numbers of
uredia than ‘Ryan Gainey’, ‘Pink Pincusion’ and ‘White Dome’. These results
provide important information for breeders to develop smooth hydrangea
cultivars with rust resistance and for landscape designers and nurserymen to
select smooth hydrangea for gardens and nursery production.
Biofilm formation and virulence associated with lipopolysaccharide
biosynthetic genes in Xanthomonas axonopodis pv. citri
J. LI (1)
(1) Citrus Research and Education Center, University of Florida IFAS, Lake
Alfred, FL, U.S.A.
Phytopathology 100:S71
Xanthomonas axonopodis pv. citri (Xac) is the bacterial pathogen of citrus
canker which is one of the most disastrous diseases of citrus worldwide. The
formation of biofilms in planta plays an important role in the epiphytic
survival of Xac prior to development of canker disease. Lipopolysaccharide
(LPS) has been indicated to be involved in biofilm formation and an important
virulence factor in various animal and plant-associated bacteria. LPS is also
being increasingly recognized as a major pathogen associated molecular
pattern (PAMP) for plants inducing plant defense responses. Very little is
known about the relationship between LPS and biofilm formation and
virulence of Xac. In this study, six Tn5 insertion mutants of Xac strain306
with disrupted putative LPS biosynthetic genes of were examined. The LPS
profiles of the six mutants were determined by SDS-PAGE analysis. The
ability to form biofilm on plastic and glass surface was impaired in all the six
mutants. In planta tests, the grapefruit seedlings inoculated with LPS-defect
mutants by spraying showed reduced symptoms on leaves compared with
those treated with the wild type strain306. The affected phenotypes tested
could be restored fully or partly to those of the wild type by introducing the
intact genes into the respective mutants. These findings suggest that LPS
production is vital to biofilm formation and virulence of Xac.
Colletotrichum acutatum forms a novel internal infection cushion-like
structure in the cuticle layer of chili pepper
C. LIAO (1), K. Kuo (2), J. Wang (3), M. Lee (1)
(1) Department of Plant Pathology, National Chung Hsing University,
Taichung, TAIWAN; (2) Department of Plant Protection, Bureau of Animal
and Plant Health Inspection and Quarantine, Council of Agriculture,
Executive Yuan, Taipei, TAIWAN; (3) The World Vegetable Center, P.O.
Box 42, Shanhua, Tainan, TAIWAN
Phytopathology 100:S71
Fruit anthracnose of Capsicum spp. caused by Colletotrichum acutatum is a
severe disease in chili pepper in Taiwan. To understand how the pathogen
infects the plant, the infection process of C. acutatum on resistant and
susceptible fruits were studied using three isolates with various virulences on
chili pepper. Surface attachment, germination, appressorium formation and
turgor pressure accumulation of appressorium were analyzed in vitro. The
isolate with the lowest virulence showed less attachment ability and turgor
pressure accumulation compared to the other two isolates. However, the three
isolates formed penetration hyphae in the epidermal cells of susceptible hosts
at 72 hours post-inoculation (hpi). Interestingly, this fungus forms a novel
internal infection cushion-like structure on resistant and susceptible chili
pepper fruits before penetrating the epidermal cell. The internal infection
structure was located in the cuticle layer and was demonstrated by light and
flurorecent microscopy with GFP-tagged transformants. The internal infection
cushion-like structure formed within 24 hpi and kept growing in the cuticle
layer and subsequently penetrated into the epidermal cell at 72 hpi. Formation
of this internal structure by C. acutatum appears to be a response to the thick
cuticle layer of pepper fruits. This fungus also formed similar internal
structures on resistant hosts. However, the structure turned dark at 13 days
post-inoculation and no further infection was observed.
A novel bipartite launch system for a potexviral vector suitable for either
protein expression or virus-induced gene silencing (VIGS)
H. Lim (1), A. Vaira (2), L. L. Domier (3), J. HAMMOND (1)
(1) USDA ARS MPPL, Beltsville, MD, U.S.A.; (2) CNR, IVV, Torino,
ITALY; (3) USDA ARS, Urbana, IL, U.S.A.
Phytopathology 100:S71
We have developed plant virus-based vectors for virus-induced gene silencing
(VIGS) and protein expression, based on Alternanthera mosaic virus
(AltMV), for infection of a wide range of host plants including Nicotiana
benthamiana, Arabidopsis thaliana, and Glycine max (soybean). Infection
may be established by either mechanical inoculation of in vitro transcripts or
via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of
bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection
from a binary vector in the absence of the Cauliflower mosaic virus 35S
promoter. An artificial bipartite viral vector delivery system was created by
separating the AltMV RNA-dependent RNA polymerase and Triple Gene
Block (TGB)123-Coat protein (CP) coding regions into two constructs each
bearing the AltMV 5′ and 3′ non-coding regions, which recombined in planta
to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and
equivalent changes in other potexvirus TGB1 proteins, affected RNA
silencing suppression efficacy and suitability of the vectors from protein
expression to VIGS.
A single amino acid substitution in PthA of Xanthomonas axonopodis pv.
citri altering canker formation on grapefruit leaves
H. LIN (1), S. Hsu (2), K. Tzeng (2)
(1) Chungchou Institute of Technology, Changhua County, TAIWAN; (2)
National Chung-Hsing University, Taichung, TAIWAN
Phytopathology 100:S71
Three novel atypical symptom-producing variants of Xanthomonas
axonopodis pv. citri (Xac) were described recently in Taiwan. Only the
variant designated as Af type produces typical erumpent canker lesions on
Mexican lime (Citrus aurantifolia) but induces flat necrotic with water-soaked
margin lesions on grapefruit leaves (C. paradisi). Two homologous pthA were
cloned and characterized from strains XW19 (a typical canker lesion
producing strain) and XW47 (a strain of Af type). The pthA homolog from
XW19 was transformed into XW47. The transformant induced typical
erumpent canker lesions on grapefruit leaves. Sequence analyses revealed
over 99% homology in nucleotide and deduced amino acid sequences
compared with pthA homologs deposited in GenBank. The amino acid
residues located at positions 49, 286, 742 and 767 of PthA were different
between XW47 and XW19. The PthA mutants with a single amino acid
substitution at each of these four positions were constructed by site-directed
mutagenesis. Modified PthA (S286P) from XW47 in transformant 47SP
induced erumpent canker lesions on grapefruit leaves, whereas another
modified PthA (P286S) from XW19 in transformant 47PS only induced flat
necrotic lesions. These results suggested that a single amino acid substitution
from either serine to proline or proline to serine at position 286 of PthA can
alter canker formation by Xac on grapefruit leaves.
Development of a multiplex real-time RT-PCR assay for simultaneous
detection of three pome fruit viroids
L. LIN (1), R. Li (1), R. Mock (1), G. Kinard (1)
(1) USDA ARS, Beltsville, MD, U.S.A.
Phytopathology 100:S71
Several viroids infect pome fruit trees and their control is based primarily on
quarantine and certification programs to distribute clean stock material. A
multiplex, single tube TaqMan RT-PCR assay was developed to
simultaneously detect Apple scar skin (ASSVd), Pear blister canker
(PBCVd), and Apple fruit crinkle (AFCVd) viroids. Total nucleic acid extracts
were prepared from healthy and infected pome fruit trees using a CTAB
method. For each of the three viroids, a pair of primers and a probe labeled
with a specific fluorescent reporter dye were designed and evaluated in both
simplex and multiplex Taqman RT-PCR assays. The optimum primer
combinations and concentrations were determined for the multiplex TaqMan
RT-PCR assay using extracts, including mixed extracts, from trees infected
with one of the viroids. The extract dilution end points for detecting each
viroid were 10–5, 10–4, and 10–2 for AFCVd, ASSVd, and PBCVd,
respectively, in the multiplex format. The method was further validated using
samples from trees inoculated with all of these viroids. The results indicate
multiplex TaqMan RT-PCR is capable of specifically detecting the presence
of and differentiating each viroid in pome fruit trees with mixed viroid
Vol. 100, No. 6 (Supplement), 2010
S71
infections. This assay could be very useful as a fast and sensitive complement
to existing diagnostic methods for pome fruit viroids. This technique is
particularly applicable to quarantine and certification programs where many
samples need to be tested.
Bioinformatic analysis of genome sequence data for Ca. Liberibacter
asiaticus
M. LINDEBERG (1), S. Saha (1)
(1) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S72
One-step multiplex RT-PCR for simultaneous detection of four viroids
affecting pome fruit trees
L. LIN (1), R. Li (1), R. Mock (1), G. Kinard (1)
(1) USDA ARS, Beltsville, MD, U.S.A.
Phytopathology 100:S72
Ca. Liberibacter asiaticus (Las) is a bacterium vectored by psyllid insects and
believed to be the causal agent of citrus greening. However, given the
difficulty in culturing this organism, much about its biology remains a
mystery. Availability of genome sequence data for a single isolate of Las
(Duan et al, 2009), provides the opportunity to use sequence analysis to gain
insights into its mechanisms of adaptation to host and vector. Approaches
include characterization of metabolic pathways and comparison with those of
bacteria either phylogenetically related or inhabiting analogous environmental
niches. Mapping of extragenic elements such as promoters can additionally
pinpoint genes for which coordinate regulation mediates adaptation to the host
environment. Regulatory proteins of particular interest include RpoH,
associated with response to elevated temperature and RirA, mediating cell
uptake of iron. Knowledge of regulatory binding sites and other repetitive
sequences is also useful when designed primers for strain detection. Results of
genomic analysis of Ca. Liberibacter asiaticus, guides for genome viewing,
and links to various online resources and genome analysis tools can be
found at the Citrus Greening-HLB Genome Resources Website (http://
citrusgreening.org)
Apple scar skin (ASSVd), Apple dimple fruit (ADFVd), Apple fruit crinkle
(AFCVd), and Pear blister canker (PBCVd) viroids naturally infect pome
fruit trees. These viroids are distributed worldwide and are important
quarantine pathogens for the international movement of germplasm. A singlestep multiplex reverse transcription polymerase chain reaction assay (mRTPCR) was developed for the simultaneous detection of these viroids. Total
nucleic acids were prepared from fruit trees infected with individual viroids
and used as templates for mRT-PCR for either single or mixed viroid
detections. Four pairs of viroid-specific primers were designed to amplify
products of different sizes that were discernible by gel electrophoresis. The
expected products of 371 bp for AFCVd, 270 bp for ADFVd, 186 bp for
ASSVd and 120 bp for PBCVd were obtained in the both simplex and
multiplex RT-PCRs. The sensitivities, specificities and efficiencies of mRTPCR for detecting all four viroids were very similar to the individual simplex
RT-PCR. The method was validated using samples from pome trees
inoculated with all four of viroids. All viroids could be detected from infected
pear trees and up to two viroids could be detected from apples. These
techniques proved to be a simple, rapid and cost-effective means to detect
these viroids in fruit trees. The procedure is especially applicable to
certification and quarantine programs, where numerous samples need to be
tested for all four viroids.
Extracellular Xylella fastidiosa genomic DNA enhances biofilm formation
in vitro
H. LIN (1), D. M. Cheng (1), E. L. Civerolo (1)
(1) USDA-ARS PWA, Parlier, CA, U.S.A.
Phytopathology 100:S72
Xylella fastidiosa (Xf) is a Gram negative, xylem-limited bacterium that
causes Pierce’s Disease (PD) of grapevine, as well as other diseases of
economically important crops and landscape plants. Many bacteria produce
large amounts of extracellular DNA, which may function as a matrix
component in biofilms. Biofilm formation is essential for Xf establishment in
planta. However, factors affecting Xf biofilm biogenesis in planta are not
completely understood. The objective of this study was to determine if
extracellular genomic DNA is involved in the Xf biofilm formation in vitro.
The relative amounts of extracellular DNA were positively correlated with
planktonic growth and biofilm formation in vitro, but were negatively
correlated with cell viability. DNase I treatment of actively growing Xf
cultures in culture medium decreased or inhibited biofilm formation. In
contrast, addition of Xf genomic DNA promoted biofilm formation. These
results suggest that biogenesis of extracellular DNA may play a role for Xf
biofilm formation and could be a critical step in establishment of hostbacterium interaction.
A new molecular diagnostic tool for quantitatively detecting and
genotyping “Candidatus Liberibacter species”
H. LIN (1), H. Liao (2), Y. Bai (2), E. L. Civerolo (1)
(1) USDA ARS PWA, Parlier, CA, U.S.A.; (2) Guangxi Academy of
Agricultural Sciences, Nanning, PRC PEOPLES REP OF CHINA
Phytopathology 100:S72
A new molecular diagnostic method was developed for quantitative detection
of “Candidatus Liberibacter” species associated with citrus Huanglongbing
(“Ca. Liberibacter asiaticus”, “Ca. Liberibacter africanus” and “Ca.
Liberibacter americanus”) and potato zebra chip disorder (“Ca. Liberibacter
solanaceraum”). This detection system employs a pair of universal primers
designed in sequences conserved among the four Liberibacter species.
Polymorphism due to deletions, insertions and nucleotide substitutions in the
amplicons among the four Liberibacter species can be distinguished based on
high resolution melting curve analyses. In contrast to multiplex PCR or
multiple sets of primers or primers/probe commonly used for detection of
different Liberibacter species, this diagnostic system, using only one pair of
primers, greatly simplifies detection and eliminates competition between
primer/primer and / or primer/probe combinations that may occur in multiplex
systems. The assay is robust and cost-effective for reliable detection,
quantification and identification of Liberibacter species in plants and insect
vectors. This new diagnostic system is suitable for high throughput screening
for regulatory and epidemiological studies in locations where multiple
Liberibacter species may be present.
S72
PHYTOPATHOLOGY
Development and application of a single-tube immunocapture real-time
PCR technology for sensitive detection of a panel of viruses in crop
plants
K. LING (1), C. Feng (2), J. Q. Xia (3)
(1) USDA ARS, Charleston, SC, U.S.A.; (2) University of Arkansas,
Fayetteville, AR, U.S.A.; (3) AC Diagnostics, Inc., Fayetteville, AR, U.S.A.
Phytopathology 100:S72
Enzyme-linked immunosorbent assay (ELISA) is the most widely used
technology for plant virus detection. Its sensitivity however may not be
satisfactory in detecting viruses in tissues with early infection, seeds or woody
plants. Recently, real-time PCR has been introduced for plant virus detection
with higher sensitivity. Immunocapture (IC) real-time PCR is a combination
of these two technologies which traps virus particles by immunocapture and
washes away PCR inhibitors, followed by real-time PCR in the same tube for
virus detection. The objectives of this study are to evaluate the factors
affecting the immunocapture capabilities in PCR tubes and to develop
sensitive real-time PCR to a panel of viruses. Sixteen PCR tube types were
evaluated for their ability to immunocapture. Computer-assisted sequence
analysis was used to select the most conserved genomic regions for primer
and probe design. The target viruses captured on the PCR tubes were
comparatively analyzed with ELISA, PCR and real-time PCR. PCR tubes for
the most effective immunocapturing of target viruses were identified. The IC
real-time PCR could effectively detect 14 target viruses of tomato or pepper in
crude tissue extract diluted up to 10–5 or 10–7, which is approximately 1000
times higher than ELISA. Furthermore, multiplex IC real-time RT-PCR was
developed for a simultaneous detection of 2–3 viruses. This technology has a
potential to supplement ELISA for plant virus detection.
Transmission of cucumber green mottle mosaic virus by cucumber
pollen
H. LIU (1), L. Luo (1), J. Hao (2), J. Li (1)
(1) China Agricultural University, Beijing, PRC PEOPLES REP OF CHINA;
(2) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S72
It has been postulated that pollen may transmit cucumber green mottle mosaic
virus (CGMMV) in cucumber. To confirm this hypothesis, laboratory and
greenhouse experiments were conducted. Cucumber seeds free of CGMMV
were grown in the greenhouse. Plants were covered with screens to exclude
insects, and divided into two blocks, with 22 plants in block one, and 109
plants in block two. When cucumber plants in block one had three true leaves,
they were mechanically inoculated with CGMMV. At blooming stage, leaves
from inoculated plants were collected and tested for CGMMV using reverse
transcription polymerase chain reaction (RT-PCR). All leave samples tested
positive for CGMMV. Pollen grains from the inoculated plants in block one
were collected and used to artificially pollinate 109 emasculated cucumber
plants in block two. Fruit were harvested from 45 of these plants. One
cucumber fruit was selected from each plant, and the seeds were removed.
RT-PCR test indicated that 24 of the 45 seeds sets (54%) were CGMMV
positive. The 24 sets of CGMMV-positive seeds were planted in 24 groups of
pots. Six seedlings from each group were tested for CGMMV using RT-PCR.
Seedlings from nineteen groups (80%) were CGMMV positive. Therefore,
CGMMV can be transmitted from plant to plant via cucumber pollen and is
seed-borne disease.
Comparison of fumigation, mustard meal (MM) amendments and
grafting on Fusarium and Pythium communities in tomato fields
B. LIU (1), J. Sun (2), F. Louws (2)
(1) North Carolina State University, Raleigh, NC, U.S.A.; (2) PLPA, NCSU,
Raleigh, NC, U.S.A.
Phytopathology 100:S73
Community analysis of soil Pythium and Fusarium is essential for
understanding pathogen ecology and the impact of fumigation, MM and
grafting on pathogen communities. The dynamics in microbial communities
was monitored in the following treatments at the NC Mountain Horticulture
Research
Station:
inactive-MM
with
no-grafting
(a);
methyl
bromide:chloropicrin (MC) fumigation combined with grafting on Maxifort
rootstock (b) or with no-grafting (c); MM with (d) or with no grafting (e); nofumigation with (f) or with no grafting (g) and no-fumigation with selfgrafting (h). Soil samples were assessed in July (before planting), September
(full bloom) and October (full fruiting). Based on dilution plating, Fusarium
populations were not significantly different in soils from the first collection
(before treatment) and third sampling (Oct). At the second sampling (Sept),
Fusarium populations were reduced significantly in soils with treatment d (62
CFU/gds) and increased in soils with treatment b (3.0 × 104 CFU/gds) and c
(4.6 × 104 CFU/gds) compared with no fumigation baseline f, g and h (7 ×
102 to 2.5 × 103 CFU/gds). Moreover, at the second (Sept) and third sampling
(Oct), Pythium populations were decreased significantly in treatment b (21
and 43 CFU/gds) and c (16 and 12 CFU/gds) but not by other treatments
compared with baseline level f, g, and h [1.5 × 102 to 3.1 × 102 (Sept) and 6.2
× 102 to 8.7 × 102 CFU/gds (Oct)]. The differential impact of MM and
fumigation on Fusarium and Pythium populations is a productive avenue for
future research.
Comparison of fumigation, mustard meal amendments and grafting on
bulk soil microbial communities in tomato fields
B. LIU (1), J. Sun (2), C. Rivard (2), R. Welker (2), F. J. Louws (2)
(1) North Carolina State University, Raleigh, NC, U.S.A.; (2) PLPA, NCSU,
Raleigh, NC, U.S.A.
Phytopathology 100:S73
Plant health benefits associated with soil fumigation, mustard meal (MM)
amendments and tomato grafting may be mediated by microbial communities.
The dynamics and shifts in microbial communities was monitored in the
following treatments at the NC Mountain Horticulture Research Station:
inactive-MM with no-grafting (a); methyl bromide:chloropicrin (MC)
fumigation combined with grafting on Maxifort rootstock (b) or with nografting (c); MM with (d) or with no grafting (e); no-fumigation with (f) or
with no grafting (g) and no-fumigation with self-grafting (h). Soil samples
were assessed in July (before planting), September (full bloom) and October
(full fruiting). Based on dilution plating, fungal populations did not differ
significantly in soils from the first collection (before soil treatment) and third
sampling (Oct). At the second sampling (Sept), fungal populations were
significantly increased in soils with treatment b (3.3 × 105 CFU/ gram dry
soil) and c (3.9 × 105 CFU/gds) compared with no-fumigation baseline f, g
and h (81 to 2.4 × 102 CFU/gds). Moreover, total bacterial populations were
decreased in treatment b and d but not by other treatments compared to
baseline levels in f, g, and h. Denaturing gradient gel electrophoresis is in
progress to characterize fungal and bacteria species diversity in soils with
different treatments. Knowledge about the biology and ecology of microbial
communities could provide a more informed framework to advance plant
health and disease management programs.
Variation of prophage frequency in “Canidatus Liberibacter asiaticus”
strains from two geographical distinct citrus growing Provinces in China
R. Liu (1), P. Zhang (1), J. Chen (2), X. DENG (1)
(1) South China Agricultural University, Guangzhou, PRC PEOPLES REP
OF CHINA; (2) USDA ARS PWA, Parlier, CA, U.S.A.
Phytopathology 100:S73
Prophages are important genetic elements of bacterial genomes and are
involved in lateral gene transfer, pathogenicity, environmental adaptations and
interstrain genetic variability. In this study, the sequence of a phage terminase
gene of “Candidatus Liberibacter asiaticus”, a bacterium associated with citrus
Huanglongbing (HLB), was identified and used to represent the corresponding
prophage. Based on the DNA sequence, a set of primers was designed and
used for PCR detection of prophage in HLB citrus samples collected from two
geographically distinct provinces, Guangdong with an average altitude of
<500 m, and Yunnan with an average altitude of >2,000 m. The frequency of
prophage detection was 15.8% (19/120) in Guangdong and 97.4% (38/39) in
Yunnan. Chi-square analysis showed that the prophage frequencies between
the two regions were significantly different (P < 0.0001). However, the
prophage gene sequences obtained from 10 Guangdong strains and 8 Yunnan
strains shared 100% similarity, suggesting identity or high similarity of the
corresponding prophage. The absence of the terminase gene sequence in some
“Ca. L. asiaticus” strains suggests that the prophage or putative phage has
both lytic and lysogenic cycles. To our knowledge, this is the first observation
on phage lytic activity in “Ca. L. asiaticus”.
Molecular characterization of the mitochondrial cytochrome b gene in
Podosphaera clandestina, the causal agent of powdery mildew on sweet
cherry
Q. LIU (1), M. E. Nelson (1), G. G. Grove (1)
(1) Washington State University, Prosser, WA, U.S.A.
Phytopathology 100:S73
The mitochondrial cytochrome b gene (cytb) encodes a protein that is targeted
by QoI (strobilurin) fungicides, which cause energy deficiency in fungal cells
during spore germination. Mutations in the cytb gene, most commonly at
positions 143 and 129, confer resistance to QoI fungicides in various
phytopathogens including powdery mildews. The objective of current study
was to develop a procedure for amplifying and characterizing the cytb gene of
Podosphaera clandestina, the causal agent of cherry powdery mildew, so that
putative mechanism for QoI fungicide resistance can be promptly determined
once it arises. Using degenerate primers and nested polymerase chain reaction,
products of expected size amplified from two greenhouse isolates were cloned
and sequenced. The two amplicons were 641 bp and 638 bp in size,
respectively. There were no introns within either amplicons. Among amino
acids they encoded, 69–71% and 86–95% were identical to the corresponding
regions of powdery mildew cytb genes deposited in GeneBank. Most
variations between two amplicons occurred within those 100 amino acids at 3′
end. Amino acids at positions of 129 and 143 were phenylanlanine (F) and
glycine (G), respectively, for both amplicons. These results provide sequence
information for the P. clandestina cytb gene. This information will be useful
to further investigation of QoI resistant P. clandestina isolates if that
resistance is due to mutations in the cytb gene as has been reported in other
fungi.
CANARY biosensors for rapid detection of Ralstonia, Potyvirus and
Phytophthora
Z. LIU (1), K. Rappaport (1), E. Twieg (1), V. Mavrodieva (1), L. Levy (1)
(1) USDA-APHIS-PPQ-CPHST, National Plant Germplasm and
Biotechnology Laboratory, Beltsville, MD, U.S.A.
Phytopathology 100:S73
CANARY (Cellular Analysis and Notification of Antigen Risk and Yield) is a
cell-based technology that is capable of rapidly identifying low levels of
pathogens through detection of photons emitted by bioluminescent proteins
upon crosslinking of antigens to engineered antibodies expressed and
anchored on the outer membrane of the cell. The method was invented by
scientists at the Massachusetts Institute of Technology and has been applied
for medical diagnostic assays and monitoring of select agents. MIT and
USDA APHIS developed plant-pathogen-specific-cell lines for detection of
Ralstonia solanacearum, and to the genus-level for Potyvirus and
Phytophthora. The eventual goal is to implement the CANARY system in
plant diagnostic labs. For Ralstonia detection, we sliced a small piece of
infected plant tissue and soaked it in assay buffer for a few minutes before
CANARY testing. As few as 3 CFUs of Ralstonia can be detected in a single
test. For Potyvirus detection, we used polystyrene beads to capture viruses
from plant extract for CANARY. For both the Potyvirus and Ralstonia assays,
only 10 minutes or less are required for sample preparation and sample
testing. For Phytophthora detection, a second antibody, which is different
from cell membrane antibodies, was used to coat magnetic beads that were
applied to capture mycelia in plant extract in preparation for CANARY. The
protocols and data will be discussed to demonstrate the speed and sensitivity
of this technology.
Involvement of rice endogenous peptide elicitors in defense signaling and
disease resistance
W. LIU (1), Q. Wang (1), Y. Yang (1)
(1) Department of Plant Pathology and Huck Institutes of Life Sciences,
Pennsylvania State University, University Park, PA, U.S.A.
Phytopathology 100:S73
Plant disease resistance is generally activated by perception of pathogenassociated molecular patterns or effectors and frequently mediated by salicylic
acid, jasmonic acid, ethylene and/or abscisic acid signaling pathways.
However, relatively little is known about the role of endogenous plant peptide
elicitors in defense signaling and disease resistance. Recently, a small family
of Arabidopsis peptide elicitors (AtPeps) was shown to mediate defense gene
activation and basal resistance. Here we have identified a 7-member family of
rice peptide elicitor precursor genes (OsPROPEP1-7), which encodes a class
of 25–42 amino-acid mature peptides. Based on publicly available rice
microarray data and our quantitative RT-PCR analysis, at least three
OsPROPEPs were significantly induced by fungal infection, wounding,
jasmonic acid and/or ethylene treatments. A 25 amino-acid peptide (OsPep7)
Vol. 100, No. 6 (Supplement), 2010
S73
was synthesized and is being tested for its activation of rice defense genes and
resistance responses. In addition, transgenic rice lines with overexpression of
OsPROPEP7 have been generated and will be characterized for altered
defense gene expression and disease resistance against rice blast
(Magnaporthe oryzae) and sheath blight (Rhizoctonia solani).
Identification of a conserved rice protein that interacts with a Nep1-like
toxin from Magnaporthe oryzae
Z. LIU (1), Q. Wang (1), Y. Yang (1)
(1) Department of Plant Pathology and Huck Institutes of Life Sciences,
Pennsylvania State University, University Park, PA, U.S.A.
Phytopathology 100:S74
Necrosis and ethylene-inducing peptide1 (Nep1)-like proteins (NLPs) are
conserved in a diverse array of microorganisms including bacteria, fungi and
oomycetes and act as virulent toxins to elicit necrotic cell death in
dicotyledonous plants. Recently, we have identified a family of four
Magnaporthe oryzae NLPs (MoNLPs) that are capable of eliciting necrotic
cell death in both dicots and monocots. To better understand the mode of
action and potential host cellular target(s) of NLP toxins, MoNLP1 was used
as a bait to screen for interacting rice proteins by the yeast two-hybrid system.
Three interacting rice protein were identified, including OsNPI1 (Oryza sativa
NLP Interactor1), which is highly conserved in eukaryotes. Quantitative RTPCR revealed that OsNPI1 is constitutively expressed in rice plants during the
infection of M. oryzae. Interestingly, knockout of the NPI1 orthologue in
Arabidopsis T-DNA mutants led to retarded growth and lethal phenotype. In
addition, suppression of OsNPI1 by RNA interference in transgenic rice
appears to negatively affect shoot regeneration and plant growth. Functional
characterization of OsNPI1 at the protein level and further analysis of
suppression and overexpression transgenic lines should shed light on the role
of OsNPI1 as a potential MoNLP1 target which mediates necrotic cell death
and disease development.
Study on the influence on germination of resting spores of
Plasmodiophora brassicae on pakchoi
Y. LIU (1), X. Huang (1), H. Liu (1)
(1) Sichuan Academy of Agricultural Sciences, Institute of Plant Protection,
Chengdu, PRC PEOPLES REP OF CHINA
Phytopathology 100:S74
The germination of resting spores of Plasmodiophora brassicae on club root
pathogen will cause different infection degree on crucifer. Understanding the
restricting factor on germination of resting spores of P. brassicae become the
key points to control the club root efficiently. The germination of resting
spores of P. brassicae from pakchoi under different conditions were tested
through liquid culture method, such as temperature, pH, nutrient condition,
visible light and different rotted degrees of clubs. The result shown that the
optimum temperature for germination of resting spores of P. brassicae was
24°C with suitable range from 20–28°C and the lethal temperature was 48°C;
the optimum pH was 6.3 with suitable range from 6.0–6.7, the germination of
resting spores was restrained by the visible light but motivated observably
after the treatment of rotted clubs. The highest germination rate for the resting
spores was 71.07% in the filtering sterilized root exudates, which shown that
the germination of resting spores was motivated observably by tissue
secretion.
Suppression of zinnia powdery mildew in the greenhouse with siliconcontaining media amendments
J. C. LOCKE (1), J. E. Altland (2), J. M. Frantz (1)
(1) USDA-ARS-ATRU, Toledo, OH, U.S.A.; (2) USDA-ARS-ATRU,
Wooster, OH, U.S.A.
Phytopathology 100:S74
We have previously reported that supplemental silicon supplied in hydroponic
solution or soilless media can reduce the incidence and severity of powdery
mildew (PM) on zinnia. This research reports the use of 1) silicon-containing
organic amendments, 2) mineral compositions high in silicon content, and 3)
drenched potassium silicate solution as delivery systems for silicon to zinnia
grown in a peat-based growing medium. Efficacy varied with silicon
concentration and/or availability in the amendments, amount of amendment
supplied, and the disease pressure. Three of the amendments; rice (Oryza
sativa) hulls (RH), chopped miscanthus grass (Miscanthus x giganteum) straw
(MS), and chopped switchgrass (Panicum virgatum) straw (SG) reduced
severity of PM ratings after three weeks but the degree of reduction decreased
with increasing exposure time for all three. After 6 and 8 weeks, only RH and
MS provided substantial reduction in disease. Declining protection correlates
with a declining level of silicon in the zinnia leaf tissue and may correspond
with a depleted silicon supply in the medium. Constant application of
potassium silicate solution provided the best protection suggesting the need
for a continuous supply of silicon to the leaf tissue and negating the potential
of a slow-release supply of silicon from the amendments or an accumulated
S74
PHYTOPATHOLOGY
reserve of silicon in the zinnia tissue. Additional approaches are being
explored to extend the usefulness of silicon supplementation in suppressing
mildew.
Incidence of Candidatus Liberibacter americanus and Ca. L. asiaticus in
orange jasmine and citrus trees in urban areas in Sao Paulo State, Brazil
S. LOPES (1), G. F. Frare (2), L. E. Camargo (2), N. A. Wulff (3), D. C.
Teixeira (3), R. B. Bassanezi (3), A. C. Beattie (4), A. J. Ayres (3)
(1) FUNDECITRUS, Araraquara, BRAZIL; (2) Piracicaba, BRAZIL; (3)
Araraquara, BRAZIL; (4) Sydney, AUSTRALIA
Phytopathology 100:S74
Liberibacters are phloem-limited bacteria associated with huanglongbing
(HLB), a destructive citrus disease. Two species occur in Brazil, Candidatus
Liberibacter asiaticus (Lam) and Ca. L. americanus (Las), both vectored by
Diaphorina citri. Orange jasmine is a widespread ornamental tree and host of
both liberibacters and D. citri. We determined liberibacter incidence in cities
and villages within the most important Brazilian citrus belt, and their grafttransmissibility. To assess inoculum source potentials, quantitative PCR was
applied to all PCR-positive trees. Liberibacter was detected in 91 of 786 trees
distributed in 10 of 76 locations, all within the region of high HLB incidence.
PCR-positive trees exhibited yellow shoots and/or dieback indistinguishable
from those on PCR-negative trees. Among the PCR-positive trees, Lam was
proportionally more frequent in the 2005/6 and Las in the 2009 survey.
Transmission succeeded only in homologous host combinations, including
Lam (2/10) from/to orange jasmine and Lam (5/18) and Las (5/9) from/to
citrus. Symptoms were mild and progressed less in orange jasmine probably
due to lower liberibacter multiplication. In this host, Lam and Las titers
averaged 4.3 and 3.0 log cells/g tissue compared to 5.5 and 7.3 in citrus.
Orange jasmine is not as suitable for liberibacter multiplication as citrus.
However, its importance to the HLB epidemics should not be underestimated.
It is a preferred host of D. citri and is not under any insect- or strict tree
eradication-control program.
Distribution and diversity of Pratylenchus spp. associated with biofuel
crops and species identification in a multiplex PCR assay
H. LOPEZ NICORA (1), T. Mekete (1), K. Reynolds (1), M. E. Gray (1), T.
L. Niblack (1)
(1) University of Illinois, Urbana, IL, U.S.A.
Phytopathology 100:S74
The distribution of Pratylenchus spp. was surveyed in field plots of bioenergy
crops in six states: Illinois, Iowa, South Dakota, Kentucky, Tennessee, and
Georgia. The populations were identified based on morphology and
morphometrics and further characterized based on sequences of the rDNA
D2D3 region. The region revealed variations in sequence information that
supported the morphological identification. In this work, 6 Pratylenchus spp.
were detected: P. scribneri, P. penetrans, P. crenatus, P. hexincisus, P.
neglectus, and P. brachyurus. P. scribneri, P. crenatus, and P. penetrans were
distributed most widely with detection rates up to 34%, 29% and 15%,
respectively. P. hexincisus, P. brachyurus and P. neglectus were distributed
sporadically, with detection rates up to 10%, 3%, and 2%, respectively. A
single-step multiplex PCR was developed for the simultaneous detection of P.
scribneri, P. crenatus, and P. penetrans. Sequence data from this research and
NCBI were used to generate different primer sets that are species-specific. We
have designed 24 and selected 3 sets of primers that discriminate P. scribneri,
P. crenatus, and P. penetrans in multiplex PCR. All the tested primers showed
specificity and had no cross-reaction with non-tagret species. When used in a
uniplex, duplex, and triplex PCR, the 3 selected primers gave a unique
electrophoretic DNA banding pattern characterized by a single DNA fragment
for P. scribneri (ca. 750), P. crenatus (ca. 690), and P. penetrans (ca. 520).
The method could be used for routine diagnostic programs.
Competitive ability of iprovalicarb-resistant mutants of Phytophthora
capsici
X. H. LU (1), J. Hao (2), X. Liu (1)
(1) China Agricultural University, Beijing, PRC PEOPLES REP OF CHINA;
(2) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S74
Iprovalicarb is a compound that inhibits plant pathogenic oomycetes, but its
application may be limited by resistance in the pathogen. Iprovalicarbresistant mutants of Phytophthora capsici were developed in the laboratory,
and four of them (R2-1, R2-2, R1-3, and R1-7) were used to evaluate their
competitiveness. Zoospore suspensions of resistant and sensitive isolates were
mixed at ratios of 1:9, 3:7, 5:5, 7:3 and 9:1, respectively, inoculated on Petri
plates containing carrot agar (CA), and incubated at 25°C. After production of
sporangia, zoospore suspensions were prepared and spread on CA medium
amended with or without iprovalicarb (5 µg/ml), to determine the frequency
of iprovalicarb-resistant subpopulation. Zoospores were concomitantly
transferred to CA plates to initiate a new cycle of growth. After five cycles,
R2-1 and R2-2 were nearly non-detectable, but R1-3 and R1-7 were dominant.
In the greenhouse, sweet pepper (Capsicum frutescens) seedlings were
inoculated by pouring zoospore suspensions in the rhizosphere. After three
days, P. capsici was isolated from the stem tissues of 20 infected seedlings,
assayed for competitiveness, and inoculated on plants for five cycles of
infection. Regardless of initial ratios of the resistant to sensitive isolate, R2-1
and R2-2 were dominant, and R1-3 and R1-7 were less competitive than the
sensitive isolates at last. Thus, some iprovalicarb-resistant isolates may
survive competitively under contain conditions.
Immunodiagnostic assays targeted to urediniospore wall proteins of
Asian soybean rust
D. G. LUSTER (1), M. B. McMahon (1)
(1) USDA ARS Foreign Disease Weed Science Research Unit, Fort Detrick,
MD, U.S.A.
Phytopathology 100:S75
Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR),
continues to expand across the southeast and mid-south regions of the U.S.,
resulting in increased fungicide applications for producers. Our objectives in
this research were to identify ASR protein targets for development of immunodiagnostic assays, preferably expressed in planta and early in infection. We
identified and characterized a small family of extracellular proteins in the P.
pachyrhizi urediniospore wall, termed PHEPs (for PHakopsora Extracellular
Protein). One protein family member, PHEP 369, was selected as an ideal
immunodiagnostic target after localization studies confirmed its extracellular
location and Western blot analysis detected PHEP 369 in plants as early as 3
DPI. Monoclonal antibodies (MAbs 2E8E5-1 and 3G6H7-3) generated against
recombinant PHEP 369 were tested for sensitivity against the recombinant
protein and extracts from ASR-infected plants, and for specificity against a set
of common soybean pathogens (from cultures and infected soybeans). MAb
3G6H7-3 was highly specific for the target protein, detected as little as 10 ng
protein, and did not react with any of the soybean pathogens or urediniospores
of related rust fungi. Immunolocalization studies with MAb 3G6H7-3
confirmed the urediniospore wall location for PHEP 369. These antibodies
will prove applicable in immunodiagnostic assays with infected soybeans and
to identify ASR spores from sentinel surveillance plots.
Quantify changes in root architecture caused by Meloidogyne incognita,
Thielaviopsis basicola or their combination on cotton
J. MA (1), J. Jaraba-Navas (1), T. Kirkpatrick (2), C. Rothrock (1)
(1) University of Arkansas, Fayetteville, Fayetteville, AR, U.S.A.; (2)
Southwest Research & Extension Center (SWREC), University of Arkansas,
Hope, AR, U.S.A.
Phytopathology 100:S75
Cotton root system morphology was evaluated in relation to infection by two
soilborne pathogens in controlled environmental studies. Meloidogyne
incognita, the root-knot nematode, and Thielaviopsis basicola, a fungus that
causes black root rot, are important cotton pathogens in Arkansas. Both
pathogens cause distinct symptoms on affected roots and a synergistic
interaction between these two pathogens increases disease losses dramatically.
This study characterized the changes in seedling root architecture caused by
these pathogens individually and in combination. Cotton was planted in soil
either not infested or infested with each of these pathogens or with both
pathogens, and placed in growth chambers for six weeks. WinRhizo software
was used to determine the morphometric and topological changes including
magnitude (number of exterior links), altitude (the longest individual path
length) and total exterior path length. Root volumes per plant were 0.631 cm3,
0.411 cm3 and 0.219 cm3 for soil infested with M. incognita, T. basicola or
both pathogens, respectively. Changes in root system morphological and
topological parameters caused by either of these pathogens included
reductions in root system magnitude, altitude, number of root links and total
exterior path length compared to healthy root systems. Analysis of topological
parameters to evaluate root system architecture of cotton should enable the
quantitative assessment of the effect of root pathogens.
Exploring lineage-specific chromosomes in F. oxysporum species complex
L. MA (1), S. Zhou (2), L. R. Gale (3), A. Breakspear (3), A. Chakrabarti (4),
D. Gardiner (5), W. Jonkers (3), K. Kazan (5), J. Manners (5), P. Dodds (6),
D. Schwartz (2), J. White (1), M. Koehrsen (1), Q. Zeng (1), J. Galagan (1), C.
Cuomo (1), J. Ellis (6), C. Kistler (3)
(1) Broad Institute, Cambridge, MA, U.S.A.; (2) Madison, WI, U.S.A.; (3) St.
Paul, MN, U.S.A.; (4) Canberra, AUSTRALIA; (5) Brisbane, AUSTRALIA;
(6) Black Mountain, AUSTRALIA
Phytopathology 100:S75
The Fusarium comparative genomes of F. graminearum (Fg), F.
verticillioides (Fv) and F. oxysporum (Fo) revealed greatly expanded lineagespecific (LS) chromosomes in Fo. These mobile LS chromosomes contribute
to fungal pathognicity and host–especificity, providing an explanation for the
polyphyletic origin of host specificity and the emergence of new pathogenic
lineages in the F. oxysporum species complex (FOSC). Following this
discovery, a comparative study focusing on the members of FOSC was
developed to: 1) examine genome structural variation and confirm the
presence of LS chromosomes among different isolates using optical mapping;
2) determine gene content variation among these selected isolates using nextgeneration sequencing (NGS); 3) identify all lineage-specific genes using
targeted sequencing of the LS chromosomes and RNA sequencing via whole
transcriptome approaches. One human isolate and 11 plant pathogenic isolates
that represent eight formae speciales were included in the study. Preliminary
results from the optical mapping confirm the existence of LS chromosomes in
different isolates. Genomic data generated using NGS detects genome-wide
patterns of mutation among isolates during their brief time of evolutionary
divergence. RNA-seq data shows great promise in detecting novel genes
encoded in the LS chromosomes and for determining gene expression profiles
under different conditions.
Development, registration and commercialization of a microbial fungicide
for controlling cotton verticillium wilt in China
P. MA (1), S. Li (1), X. Lu (1), Q. Guo (1), B. Li (1)
(1) Institute of Plant Protection, Hebei Academy of Agricultural and Forestry
Sciences, Baoding, Hebei, PRC PEOPLES REP OF CHINA
Phytopathology 100:S75
Cotton verticillium wilt (CVW), causing by Verticillium dahliae, is a
constraint factor in cotton production around the world. Antagonistic Bacillus
subtilis strain NCD-2 against V. dahliae was isolated from the cotton rhizosphere in Hebei province of China. The efficacy bioassays of strain NCD-2
for controlling CVW were performed in greenhouse and field conditions. A
preparation of the microbial fungicide, 109 cfu/g B. subtilis WP, was
formulated with strain NCD-2. Field trials showed that the preparation could
reduce 60%–80% severity of CVW by seed treatment in different regions of
China. It was no-pathogenic to 8 crops including cotton, wheat, corn, cotton,
potato, eggplant, cucumber and soybean. The survival rate of strain NCD-2
was over 90% after 18 months storage under normal condition. This preparation was registered for controlling CVW in China in 2006. The mass
production technology of strain NCD-2 was optimized in 500L, 5000L and
15000L fermentation tanks, respectively. Control spectrum studies revealed
that the microbial fungicide significantly controlled some other soil born
diseases such as eggplant verticillium wilt, cotton fusarium wilt, watermelon
fusarium wilt, yam root rot. Preliminary study indicated that the supposed
action mechanisms of strain NCD-2 included inhibition (antifungal compounds
production), colonization on the surface of cotton root and transportation in
the cotton plant to block the pathogen extension, and growth promotion.
Demethylation inhibitor (DMI) fungicide resistance mechanism in
Sclerotinia homoeocarpa causing dollar spot in turfgrasses
B. MA (1), L. P. Tredway (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S75
The cytochrome P450 sterol 14α-demethylase gene (ShCYP51) in Sclerotinia
homoeocarpa was cloned and sequenced. ShCYP51 gene was 1680 bp in
length and contained two introns of 53 bp and 57 bp located after nucleotide
positions in 247 and 498, respectively. The deduced amino acid sequence had
a similarity of 73.0, 70.5 and 54.5% to the CYP51 genes from Monilinia
fructicola, Botryotinia fuckeliana, and Venturia inaequalis, respectively. An
~1700 bp fragment in the upstream region of the gene was also amplified and
sequenced. It was predicted to have two promoters located at the base pair
position of -122 to -172 and -791 to -841 of the gene, respectively. The
sensitivities of 43 S. homoeocarpa isolates to DMI fungicide were determined
in vitro using a discriminatory dose of 0.02 µg/ml of propiconazole. The
ShCYP51 genes and the promoter regions from those isolates were sequenced
and compared. Three types of point mutation were detected among 12
isolates; however, none of them were associated with DMI sensitivity. No
inserts or repeats was found in the promoter region from any of the isolates.
Real-time PCR was used to quantify ShCYP51 gene expression in six
sensitive and six resistant isolates, and no difference was found between the
two types of isolates. Results from this study show that point mutation and
over-expression of the ShCYP51 gene and modifications of the gene’s
promoter region are not the mechanisms operating in S. homoeocarpa leading
to the resistance to DMI fungicide.
Molecular characterization of Colletotrichum populations causing crown
rot of strawberry in Australia
S. Mackenzie (1), A. Gomez (2), D. Hutton (2), N. A. PERES (1)
(1) University of Florida, Gulf Coast Research and Educaion Center,
Wimauma, FL, U.S.A.; (2) Queensland Primary Industries and Fisheries,
Maroochy Research Station, Nambour, AUSTRALIA
Phytopathology 100:S75
Vol. 100, No. 6 (Supplement), 2010
S75
Colletotrichum crown rot of strawberry is caused by two closely related
species, C. gloeosporioides and C. fragariae. Within the C. gloeosporioiodes
species designation, two subpopulations exist in the U.S. One heterothallic
and highly variable that is commonly found on strawberry and other hosts,
and one homothallic that is often referred to as Glomerella cingulata. C.
fragariae was believed to occur only on strawberry, but we recently reported
affecting other hosts. Colletotrichum isolates from crown rot-affected plants in
Australia were compared to isolates from the U.S. using sequence data from
three areas of the genome. Among the C. gloeosporioides isolates, only two
different genotypes were observed in Australia, in contrast to the highly
variable population from the U.S. Most isolates sampled were from a
homothallic subpopulation that more appropriately should be described as G.
cingulata. This subpopulation was not found on strawberry in the U.S. Only a
couple of C. gloeosporioides isolates was found in Australia that are probably
from the same subpopulation commonly isolated from diseased crowns in the
southeastern U.S. C. fragariae isolates from Australia were genetically the
same as an isolate found on date palm, but not strawberry, in the U.S. The
lack of genetic diversity of the populations in Australia may indicate that the
fungus is spread mainly on transplants and does not come primarily from
alternate hosts.
Morphological and pathogenic variability of Cochliobolus sativus from
Nepal
B. N. MAHTO (1), S. Gurung (1), T. B. Adhikari (1)
(1) North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S76
Spot blotch, caused by Cochliobolus sativus, is a major disease of wheat in
Nepal. A total of 48 monoconidial cultures of C. sativus, collected from
different locations in Nepal, were analyzed for morphological characteristics
and variation in virulence on wheat genotypes. Among them, 11 isolates (CS
2, 9, 10, 12, 15, 17, 19, 24, 33, 37, and 45) varied from each other in colony
appearance such as color of the substrate, margin, zonation, sectoring, and
exudation. The eleven isolates plus isolates CS 32 and 49 were further tested
for virulence on eight wheat genotypes (Chirya 1, Chirya 7, Milan/Shanghai 7,
SW 89-5422, PBW 343, BL 1473, BL 3036, and RR 21) at the seedling stage
in a greenhouse at NDSU. Based on infection responses, wheat genotypes
varied in resistance and susceptibility. The wheat cultivar Chirya 7 was
resistant to all isolates tested. C. sativus isolates also differed significantly
from each other in virulence. For example, isolate CS 45 from Bhairahwa was
highly virulent followed by CS 49, 33, and 37, while isolate CS 12 from
Bhaktapur was the least virulent. In general, isolates from the Tarai (plain)
region were more virulent than isolates from higher elevation. The interaction
between wheat genotypes and isolates was significant, indicating the
possibility of race specificity.
Spatial heterogeneity of leaf wetness duration in winter wheat canopy and
its influence on plant disease epidemiology
A. Mahtour (1), M. El Jarroudi (1), F. Giraud (2), P. Delfosse (3), L. Huber
(4), L. Hoffmann (3), B. TYCHON (1)
(1) Arlon, BELGIUM; (2) Martillac, FRANCE; (3) Belvaux,
LUXEMBOURG; (4) Grignon, FRANCE
Phytopathology 100:S76
Leaf wetness duration (LWD) is an important factor influencing the
occurrence of plant disease epidemiology. Despite considerable efforts to
determine LWD, little attention has been given to study its variability within
the canopy. The objective of this study was to evaluate its spatiotemporal
variability in wheat fields in a heterogeneous landscape. The spatiotemporal
variability of LWD was evaluated in a site close to Arlon (Belgium) during
the period May to July 2006 and 2007. LWD measurements were made using
a set of flat plate sensors deployed at five different distances from a 18 m high
hedge (5, 10, 20, 50, 100 m). Each set of two sensors was placed horizontally
close the flag leaf. In addition, we collected the amount of dew water that
deposited on rigid epoxy plates placed next to each sensors. Experimental
results showed that LWD measurements revealed substantial heterogeneity
among sensor positions. LWD is longer for sensors closer to the hedge
mainly because of its shadowing effect. 3 to 4 hours of difference was
observed between sensors located at 5 m and those located at 100 m, and
besides, a significant quantitative difference (p < 0.0001) of dew deposit was
observed between area beside hedge and those placed at 100 m. In summary,
this study provides new information on how wetness is distributed on wheat
leaves according to the distance from a hedge. This leads to local
microclimate conditions that will contribute to the disease spatial
heterogeneity.
Development of prototype Pathogen Detection Lab-On-a-Chip
(PADLOC) system for real-time on-field plant disease diagnostics
M. M. MALAPI-NELSON (1), V. L. Vaughn (1), B. Ma (1), A. Han (1), D.
Gross (1), W. Shim (1)
S76
PHYTOPATHOLOGY
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S76
Crop production in the U.S. is highly vulnerable to new and emerging
diseases. The introduction of exotic plant pathogens is continually challenging
U.S. agriculture and today threatens a variety of production fields.
Consequently, there is a critical need in developing new technologies for rapid
and accurate detection and identification of these pathogens. In particular,
implementation of portable and rapid diagnostics tool can facilitate effective
on-field plant pathogen detection. To achieve this aim, we are developing a
real-time PCR microchip that will engine the portable plant pathogen
detection lab-on-a-chip (PADLOC) system. We are constructing a
microsystem composed of a microfluidic chamber, where PCR occurs, and
integrated microfabricated electrodes as heater and temperature sensor. PCR
volume (2 l) in this chamber enables rapid heating and cooling. A computer
controlled instrument controls PCR, and a compact fluorescent detection unit
allows real-time PCR monitoring. Also, we are currently optimizing sample
preparation and micro-scale real-time PCR protocol methodologies necessary
to achieve high diagnostic specificity and sensitivity in PADLOC system.
Specifically, we are using Pseudomonas syringae pv. syringae B728a and
Fusarium oxysporum f. sp. lycopersici as models that represent plant bacterial
and fungal pathogens, respectively, to develop on-field pathogen DNA
extraction protocols as well as specific PCR diagnostic assays.
Intraspecific analysis of Phytophthora nicotianae from diverse hosts and
geographic locations using mitochondrial and nuclear markers
M. A. MAMMELLA (1), L. Schena (2), M. D. Coffey (3), S. O. Cacciola (4),
F. N. Martin (5)
(1) Dipartimento Gestione dei Sistemi Agrari e Forestali, Università
Mediterranea di Reggio Calabria, Italy; USDA-ARS, Salinas, CA, U.S.A.; (2)
Dipartimento Gestione dei Sistemi Agrari e Forestali, Università Mediterranea
di Reggio Calabria, ITALY; (3) Department of Plant Pathology and
Microbiology, University of California, Riverside, CA, U.S.A.; (4)
Dipartimento di Chimica biologica, Chimica medica e Biologia molecolare,
Università degli Studi di Catania, ITALY; (5) USDA-ARS, Salinas, CA,
U.S.A.
Phytopathology 100:S76
Phytophthora nicotianae is an economically important pathogen with a
worldwide distribution that causes disease in hundreds of plant species. In
Italy it is a particular problem on citrus, where it primarily causes root rot. In
an effort to better understand the population structure of isolates recovered
from citrus in Italy and how these relate to those recovered from different
hosts and geographic regions we have been examining the mitochondrial
haplotype for a collection of over 90 isolates. Four regions of the
mitochondrial genome (totaling 3 kb) have been sequenced for this purpose
with a total of 49 haplotypes identified thus far. Interestingly 17 isolates from
citrus recovered from Italy, California and Philippines represent 9 haplotypes
that cluster closely together (these are differentiated by a total of 9 SNPs and 5
deletions occurring in two homopolymeric thymine regions). Single citrus
isolates from Tunisia, Trinidad and Italy have distinctly different haplotypes.
Some isolates from other host species exhibited a similar type of grouping,
with many isolates recovered from tobacco and ornamentals clustering in their
individual host associated clades. Investigations classifying nuclear genotypes
for the same group of isolates are currently under way and their correlation
with mitochondrial haplotypes will be discussed.
Direct delivery of viral vectors into plant suspension cells
S. A. MANABAYEVA (1), H. B. Scholthof (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S76
Plant cell suspension cultures are of great interest for the biotechnology
industry to synchronously produce valuable proteins on a large scale. For this
purpose we developed Tomato bushy stunt virus (TBSV) DNA- based
expression vectors for direct delivery into plant suspension cells. As
expression platforms we used a previously established Nicotiana tabacum
(NT1) suspension cell culture and generated a new culture from N.
benthamiana (NB). NT1 and NB suspension cells were transformed using
biolistic and Agrobacterium-mediated gene delivery with TBSV DNAcassettes expressing GFP. Biolistic delivery was performed at 9 cm from
stopping screen to target tissue using DNA coated gold particles, at 1350 psi
helium pressure. Agrobacterium-mediated transformation parameters that
continue to be optimized include different strains, bacterial concentrations,
incubation and co-cultivation periods, and acetosyringone concentrations.
Initial results showed that with both delivery systems, transient GFP
expression is observed within a few days post-transformation. In conclusion,
we established a new cell suspension culture of N. benthamiana, and adapted
biolistic and Agrobacterium–mediated delivery systems for newly developed
TBSV-based DNA vectors into cell suspension cultures.
Alternative method for rapid processing, shipping and testing of a large
number of psyllids for the presence of “Candidatus Liberibacter spp.”
K. L. MANJUNATH (1), C. Ramadugu (2), J. Drake (1), H. A. Arevalo (3),
S. E. Halbert (4), P. A. Stansly (3), R. Lee (1)
(1) USDA ARS National Clonal Germplasm Repository for Citrus and Dates,
Riverside, CA, U.S.A.; (2) Dept. of Botany and Plant Sciences, University of
California, Riverside, Riverside, CA, U.S.A.; (3) University of Florida
SWFREC, Immokalee, FL, U.S.A.; (4) Florida Division of Plant Industry,
Gainesville, FL, U.S.A.
Phytopathology 100:S77
Huanglongbing is a devastating disease of citrus in several countries. HLB
associated “Candidatus Liberibacter spp.” can be detected in psyllids
(Diaphorina citri) long before the development of symptoms in trees.
Currently, psyllids are being monitored for the presence of Liberibacters in
several citrus industries for both prevention and management of HLB. Handcollected psyllids are preserved in ethanol, shipped to testing laboratories,
stored in freezers, DNA is then extracted by using standard methods and the
presence of Liberibacters is tested by real time PCR. A simple alternative
method for rapid processing of a large number of samples was developed in
this study initially using another psyllid, Bactericera cockerelli carrying “Ca.
L. psyllaurous” associated with psyllid yellows of tomatoes. The procedure
was then successfully applied to the Asian citrus psyllids. DNA is fixed by
pressing the frozen insects between two layers of a cellulose membrane. The
psyllid remains are then removed, and the membranes are shipped to testing
laboratories at ambient temperature. DNA was released from the membranes
by boiling them in an extraction buffer before testing by real time PCR. This
alternative method was found to be comparable in the sensitivity of detection
to standard protocols currently being followed, but allows rapid processing of
a large number of samples at significantly lower cost.
Identification of Burkholderia sp. genes related to biological control of
phytopathogens
E. T. Mano (1), A. A. Neves (1), V. C. Santos (2), A. FERREIRA (2), W. L.
Araújo (1)
(1) Laboratory of Molecular Biology and Microbial Ecology, NIB, University
of Mogi das Cruzes, Mogi das Cruzes, SP, BRAZIL; (2) Department of
Genetics, Escola Superior de Agricultura “Luiz de Queiroz”, University of
São Paulo, Piracicaba, SP, BRAZIL
Phytopathology 100:S77
The species from Burkholderia genus have been considered important target
for molecular studies mainly due the potential for biological control of
phytopathogens. These species are able to produce a huge variety of
antimicrobial compounds, which have been biochemically characterized.
However, the genes associated to the synthesis of these molecules are poorly
described, mainly due the low number of genetic studies in this area. In this
context, molecular techniques have been applied to identification and genetic
characterization of this pathway. Therefore, the aim of this work was the
identification of genes from endophytic bacterium Burkholderia sp. associated
to biocontrol of Pectobacterium carotovora in Orchids and biocontrol in vitro
of fungal Fusarium oxysporum, Fusarium verticillioides, Ceratocystis
paradoxa, Colletotrichum sp., and the Phytophthora parasitica. For this, a
library with 1788 clone was obtained by random mutagenesis based on Tn5
transposon insertion. Mutants were confirmed by specific PCR for Tn5
transposon and hybridization with specific probes confirmed the number of
Tn5 insertion. Clones defectives to control of these pathogens or showing a
variation in this phenotype were selected. Some mutants showed different
growth rate and colony pigmentation. The analysis of partial sequence of 15
DNA sequences showed that 40% is hypothetical protein, allowing the association of some phenotypes to these putative genes. The identification and cloning
of such genes will allow a better understanding of the production of these
antimicrobial compounds and further applying in a biotechnological view.
Methyl esterase 1 (StMES1) is required for systemic acquired resistance
against Phytophthora infestans in potato
P. MANOSALVA (1), S. Park (2), F. Forouhar (3), L. Tong (4), W. Fry (5),
D. Klessig (1)
(1) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (2)
Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State
University, Blacksburg, VA, U.S.A.; (3) Department of Biological Sciences,
Northeast Structural Genomics Consortium, Columbia University, New York,
NY, U.S.A.; (4) Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, New York, NY, U.S.A.; (5) Department of Plant
Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S77
In tobacco and Arabidopsis, methyl salicylate (MeSA) serves as a longdistance phloem-mobile signal that must be converted into its biologically
active form, salicylic acid (SA), in the distal uninfected tissue by the esterase
activity of salicylic acid-binding protein 2 (SABP2) in tobacco or members of
the AtMES family in Arabidopsis. In contrast to tobacco and Arabidopsis,
which have very low levels of endogenous SA, potato contains a high basal
level of SA and its role in the development of systemic acquired resistance
(SAR) has been controversial. In this study we identified the potato ortholog
of tobacco SABP2 (StMES1) and showed that the recombinant protein shares
similar biochemical properties with NtSABP2. Recombinant StMES1 converts
MeSA to SA and its esterase activity is feedback inhibited by SA or its
synthetic analog, 2,2,2,2’-tetra-fluoroacetophenone (tetraFA). Moreover, potato
in which the distal uninfected tissue was treated with tetraFA was compromised for the development of SAR induced by arachidonic acid (AA) against
Phytophthora infestans. Similar results were obtained via a genetic approach;
StMES1-silenced potato displayed a defective SAR phenotype that correlated
with elevated MeSA levels in the uninfected distal tissue. In addition, AAinduced pathogenesis-related (PR) gene induction was attenuated in these
plants as compared with the wild type. Together these findings argue that
StMES1, and MeSA are critical components for SAR in potato.
Using confocal microscopy to study the infection of Mentha longifolia by a
GFP strain of the verticillium wilt pathogen
M. E. MANSFIELD (1), K. J. Vining (2), M. A. Townley (3), T. M. Davis (4)
(1) Microbiology B.S. Program, University of New Hampshire, Durham, NH,
U.S.A.; (2) Oregon State University, Dept. of Forest Ecosystems and Society;
also Oregon State University Center for Genome Research and Biocomputing,
Corvallis, OR, U.S.A.; (3) Department of Zoology, University of New
Hampshire, Durham, NH, U.S.A.; (4) Department of Biological Sciences,
University of New Hampshire, Durham, NH, U.S.A.
Phytopathology 100:S77
The threat of verticillium wilt, caused by Verticillium dahliae, is a serious
concern of the U.S. mint industry. Peppermint (Mentha × piperita) is
susceptible to the wilt fungus, while spearmints (M. spicata, M. gracilis) are
less susceptible to infection. Commercial mint species are polyploid,
presenting difficulties as genetic research subjects. Therefore we are studying
the related diploid species, Mentha longifolia, with the aim of identifying
genes that may act to confer tolerance to Verticillium infection in mint. A GFP
strain of V. dahliae obtained from Lynda Ciuffetti at Oregon State University
was used to infect tolerant and susceptible M. longifolia accessions. Confocal
microscopy was used to track the fungal invasion and dissemination within
the host plant. The progression of the infection in root and vascular tissues
was examined at two day intervals post-inoculation. Equally extensive root
infection was observed in both the susceptible and the resistant accessions of
mint. By eight days post-inoculation, the fungus was observed in stem tissue
of susceptible plants. Guided by these findings, we targeted infected stem
tissue for total RNA isolation, the sequencing of which will provide an
opportunity to examine patterns of plant and fungal gene expression during
the infection process. This knowledge may help identify plant genes that
confer tolerance to V. dahliae infection.
Multiplex PCR for simultaneous detection of eight major onion bacterial
pathogens
M. Mansfield (1), B. GUGINO (1)
(1) The Pennsylvania State University, University Park, PA, U.S.A.
Phytopathology 100:S77
Bacterial diseases of onion cause significant pre- and post-harvest yield losses
in Pennsylvania and other onion growing regions in the U.S. Correlating
symptoms to specific pathogens using traditional morphological and
physiological techniques is both labor and time intensive and often
confounded by isolate variation. To facilitate rapid detection of these bacterial
pathogens from onion tissue, we have developed a multiplex PCR method to
simultaneously detect Burkholderia cepacia, B. gladioli, Pectobacterium
caratovora, Pantoea ananatis, P. agglomerans, Pseudomonas marginalis, P.
viridiflora, and Xanthomonas axonopodis. Eight sets of species specific
primers, based on the single copy gyrase B gene, were used in a multiplex
PCR reaction and the resulting PCR products resolved using standard gel
electrophoresis. Pathogen detection from symptomatic bulb and leaf tissue
using the multiplex PCR was consistently more sensitive and reliable than
culturing with selective media, thus the sample size and statistical power of
research trials can be increased and bacterial diseases in commercial onion
fields diagnosed more rapidly and accurately. This method will augment
current efforts to study onion bacterial pathogens and assist future work to
develop integrated pest management practices for onion in Pennsylvania.
Screening of plant introduction materials from Brassica species for
resistance against PG3 and PG4 isolates of blackleg
D. A. MARINO (1), L. E. del Rio (1)
(1) Dept. Plant Pathology North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S77
Blackleg, caused by Leptosphaeria maculans, is the most destructive
pathogen of canola (Brassica napus and B. rapa) in North America. Strains of
Vol. 100, No. 6 (Supplement), 2010
S77
this pathogen have been classified in pathogenicity groups (PG) depending on
their virulence profile on three differential genotypes. The discovery of strains
with new virulence profiles (PG3 and PG4) in North Dakota and Western
Canada highlights the need to characterize the reaction of commercial
cultivars to such strains as well as to identify sources of resistance against
them. Efforts are in place to close this gap. Greenhouse trials are being
conducted to evaluate the reaction of cultivars and plant introduction materials
to multiple strains of PG3 and PG4 using the cotyledon test. In these
replicated trials, seedlings were inoculated with a blend of 3–4 isolates of each
PG (107 spores ml–1) and incubated for 24 hours in a misting chamber. Plants
were returned to greenhouse room and evaluated for disease reaction ten days
later using a 0–9 scale. Only two of the 72 commercial cultivars were
considered moderately susceptible while the rest were susceptible to strains of
both PGs. All 450 B. rapa accessions were considered susceptible, while
seven of 220 B. juncea accessions evaluated so far were classified as
moderately resistant to both PGs. Seedlings of these materials have been taken
to seed production to evaluate their reaction to blackleg as adult plants.
Additional B. juncea materials are being evaluated.
Xanthomonas sacchari – a pathogen or an endophyte?
C. J. MAROON-LANGO (1), K. L. Schneider (2), R. S. Turner (1), G. G.
Presting (2), A. M. Alvarez (3)
(1) USDA APHIS PPQ PHP Plant Germplasm Quarantine Program,
Beltsville, MD, U.S.A.; (2) Molecular Biosciences and Bioengineering,
University of Hawaii, Manoa, HI, U.S.A.; (3) Plant Pathology Department,
University of Hawaii, Manoa, HI, U.S.A.
Phytopathology 100:S78
During routine indexing of sugarcane at the Plant Germplasm Quarantine
Program of the USDA-APHIS, colonies typical of xanthomonads were
isolated from symptomless sugarcane introductions on selective media
routinely used for isolating Xanthomonas albilineans, the cause of bacterial
leaf scald of sugarcane. Tissue blot assay of cane setts from the same plants
using the antiserum to the leaf scald bacterium was negative, indicating that
the xanthomonads were distinct from X. albilineans. From six of the seven
xanthomonad isolates, PCR using the ITS primers for Xanthomonas resulted
in an amplicon with a 450-bp stretch that shared 100% identities with X.
sacchari. RIF primers were used to confirm the identity obtained with the ITS.
The six isolates grouped basal to X. albilineans, however no reference match
existed in the database. Because of the rarity by which we encounter X.
sacchari and the lack of information on its pathogenicity, we are inoculating a
few sugarcane introductions, sorghum and Miscanthus. Results of the
bioassay will be presented.
Biological traits of Pectobacterium clades
M. Marquez-Villavicencio (1), A. CHARKOWSKI (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S78
The reported host range of Pectobacterium species includes over 30% of dicot
plant families and 60% of monocot plant families. It is often difficult to
determine from published reports which Pectobacterium species or subspecies
was isolated and characterized, thus the host range of the Pectobacterium taxa
remains obscure. Recently, multi-locus sequence analysis allowed us to place
Pectobacterium strains into well-supported clades. To test the hypothesis that
Pectobacterium clades vary in host range, we inoculated previously reported
Pectobacterium hosts with representative strains from three Pectobacterium
clades. These clades include the narrow host range pathogen P. atrosepticum
(Pa), the broad host range pathogen P. carotovorum subsp. carotovorum
(Pcc), and P. carotovorum subsp. brasiliensis (Pcb), for which the host range
is unknown. Our results did not support the hypothesis that Pa is a narrow host
range pathogen; some Pa strains infected hosts such as carrot, onion, radish,
celery, and rapini. Similarly, Pcc and Pcb strains varied and most were
similarly limited in host range as Pa. We were unable to infect corn, spinach,
beet, or asparagus with any of the strains tested. We also inoculated tubers and
stems of commercial potato varieties to determine if these three bacterial taxa
vary in aggressiveness on potato and found that Pcc and Pcb did not differ
from each other, but that they both caused significantly more tuber decay, but
not more stem decay, on all potato varieties than Pa.
Insights into the introduction of bacterial heart rot of pineapple to
Hawaiian plantations on the basis of molecular and biochemical analyses
G. MARRERO (1), W. Kaneshiro Sueno (1), A. S. De Silva (1), A. M.
Alvarez (1)
(1) University of Hawaii at Manoa, Honolulu, HI, U.S.A.
Phytopathology 100:S78
Bacterial heart rot disease of pineapple caused by Dickeya sp. is a major
constraint to pineapple production throughout the tropics and subtropics. In
2003, the disease was reported in Hawaiian fields planted with stocks that had
originated from Costa Rica and Honduras. Regulatory action prevented further
S78
PHYTOPATHOLOGY
importation of stocks from these locations and fields with diseased plants
were subsequently destroyed. In 2006, an outbreak of the disease was again
seen in Hawaiian pineapple fields. Since Dickeya sp. can cause latent
infections and vegetative materials are used for successive crops, our study
aimed at determining the source of the 2006 outbreak. Rep-PCR fingerprint
analysis distinguished three groups observed from the 2003 outbreak; these
groups were different from those observed from the 2006 strains. Molecular
phylogenies produced from dnaA, dnaX, dnaJ, and recN sequences confirmed
the separation of strains isolated in 2003 and 2006 and supported the
hypothesis that two separate introductions of the pathogen gave rise to these
outbreaks. Strains isolated in 2006 were more closely related to Dickeya sp.
reference strains isolated from Malaysia. Although the strains isolated from
infected pineapple were grouped together in a single cluster sister to D. zeae,
these strains differ biochemically and genetically from this species. This
group is sufficiently different to warrant classification as a new species.
Mitochondrial haplotype analysis as a tool for differentiating populations
of Verticillium dahliae
F. N. MARTIN (1)
(1) USDA ARS, Salinas, CA, U.S.A.
Phytopathology 100:S78
The ability to monitor mitochondrial background in Verticillium dahliae may
provide an additional tool for population studies and monitoring clonal
populations. Published mitochondrial genome sequences of V. dahliae
(DQ351941) were used to design primers for amplification of 5 regions
representing 19% of the genome (5.2 kb) for assessment of mitochondrial
haplotype. Observed differences among isolates representing a range in VCG,
host, and geographic origin were due to single nucleotide polymorphisms,
different numbers of bases in specific homopolymeric regions, and copies of
subrepeated sequences. For 30 isolates a total of 15 mitochondrial haplotypes
were identified. Some of the observed grouping correlated with VCG. For
example, five VCG-1A and –1B isolates from California, Spain and Greece
had identical haplotypes. While a single haplotype predominated among a
group containing VCG-2A isolates, there were 8 haplotypes within a group
containing VCG-2B isolates. Likewise, five VCG-4 isolates fell into 4
mitochondrial haplotypes, one of which was identical to the VCG-2A
grouping. Phylogenetic analysis with these five regions revealed the
mitochondrial background of VCG-1 to be monophyletic but VCG-2 and
VCG-4 were polyphyletic. The results obtained indicate that variation in
mitochondrial haplotypes may be a useful for characterization of isolates,
particularly those which are heterokaryon self-incompatible.
Turfgrass disease identification and management aided by “smart”
phone technology
A. MARTINEZ-ESPINOZA (1), C. F. Waltz (1), W. Hudson (2), P.
McCullough (1)
(1) University of Georgia, Griffin, GA, U.S.A.; (2) University of Georgia,
Tifton, GA, U.S.A.
Phytopathology 100:S78
Turfgrass managers, extension professionals, crop consultants and sod producers often require real-time, in-situ disease management recommendations
and disease diagnosis. Taking advantage of universal mobile telecommunications systems, remote server technology and downloadable programs, we have
developed an application (app) for Iphone® and Blackberry® “smart phones”
which allows access to a library of resources in the field. The “Turfgrass
Management” application contains a full suite of disease etiology, symptomatology, epidemiology, and disease and pathogen resources. Included are image
galleries, cultural and chemical management strategies for forty-three foliar,
root, crown, abiotic and non-infectious diseases, and descriptions of more than
eighty fungicide products. The interface allows easy searching and crossreferencing by active ingredients or common name of fungicides, or by disease.
Information is kept current and readily can be updated for new diseases,
cultural strategies or new fungicide products. Additionally, the program contains
a comprehensive description of turfgrass broadleaf, grassy, post emergent and
pre-emergent weeds, herbicides, growth regulators, insects and insecticides as
well as turfgrass taxonomy. More than 1600 subscriptions from over thirty countries have been downloaded in the nine weeks since the program was released.
Influence of temperature and leaf wetness duration on orange rust of
sugarcane
T. D. MARTINS (1), R. N. Raid (2), W. L. Burnquist (3), A. S. Urashima (4),
A. Bergamin Filho (1), J. Comstock (5)
(1) Escola Superior de Agricultura Luiz de Queiroz / USP, Piracicaba,
BRAZIL; (2) Everglades Research and Education Center/ UF, Belle Glade,
FL, U.S.A.; (3) Centro de Tecnologia Canavieira, Piracicaba, BRAZIL; (4)
Centro de Ciências Agrárias/UFSCar, Araras, BRAZIL; (5) USDA/Sugarcane
Field Station, Canal Point, FL, U.S.A.
Phytopathology 100:S78
Sugarcane orange rust, caused by Puccinia kuehnii, is a disease of increasing
concern. New to the Western Hemisphere since 2007, yield losses in excess of
40% have been reported on susceptible varieties in Florida. Epidemiological
studies were performed to investigate the influence of temperature and leaf
wetness duration on mean lesion density and disease severity. Tests were
carried out at temperatures 10, 15, 20, 25, 30 and 35°C, and leaf wetness
durations of 0, 4, 8, 12, 18 and 24 h (moist-chamber conditions), in a
combined manner. Inoculations were performed on sugarcane plants, in pots,
using the orange rust susceptible variety CL85-1040. All experiments were
repeated twice. Spore suspensions were spread on the leaf surfaces and the
pots were maintained inside growth chambers. Disease incidence and severity
were recorded during every assessment. Significant orange rust symptoms
developed over a rather limited temperature range of 20–25°C, and lesions
were notably larger at 25°C than at 20°C. An incubation and latent period of
11–17 days were observed at these temperatures. The pathogen required a
minimum of 8 h of leaf wetness for successful infection and rust was
predictably most severe at leaf wetness durations in excess of 12 h. The
prevalence of favorable temperature conditions (20–25°C) and leaf wetness
durations (> 8 h) throughout much of the growing season in Florida may help
to explain the importance of this newly introduced disease. Research
supported by CNPq.
Agrobacterium-mediated transformation is an efficient tool for insertional
mutagenesis of the vascular wilt fungus, V. dahliae
K. MARUTHACHALAM (1), S. Kang (2), S. J. Klosterman (3), K. V.
Subbarao (4)
(1) University of California, Salinas, CA, U.S.A.; (2) Pennsylvania State
University, University Park, PA, U.S.A.; (3) USDA ARS, Salinas, CA,
U.S.A.; (4) University of California, Davis, CA, U.S.A.
Phytopathology 100:S79
Agrobacterium tumefaciens-mediated transformation (ATMT) is a highly
efficient tool for both the targeted and random mutagenesis of filamentous
fungi. In this study, ATMT was optimized for Verticillium dahliae, the causal
agent of vascular wilt on many economically important crops worldwide. A.
tumefaciens strain EHA105, carrying a hygromycin phosphotransferase gene
(hph) and a green fluorescent protein (GFP) gene, was used to transform
conidia of V. dahliae. The transformation efficiency was correlated with cocultivation time. Southern blot analysis indicated that T-DNA was inserted
randomly into the V. dahliae genome and about 80% of the transformants had
a single copy of T-DNA integration. DNA sequences flanking T-DNA
insertion were identified through inverse PCR followed by sequencing.
Several mutants with altered phenotypes were obtained during the
development of mutant library, including those that lost the ability to form
microsclerotia. Based on preliminary virulence assay of 130 transformants, we
identified about 10 mutants that did not cause any symptoms on lettuce plants.
In two of these mutants, T-DNA was inserted in the endoglucase I (VdEg-I)
and hydroxyl-methyl glutary-CoA synthase (VdHMGS1) genes, which may
be required for plant cell wall degradation and parasitic growth of V. dahliae
in the susceptible plants. These results suggest that ATMT can be used to
identify genes involved in pathogenicity and other biological functions in V.
dahliae.
Variation in the number of sub-repeat sequence in the IGS region
identifies isolates of Verticillium dahliae from crucifer hosts
K. MARUTHACHALAM (1), Z. K. Atallah (1), K. V. Subbarao (2)
(1) University of California, Salinas, CA, U.S.A.; (2) University of California,
Davis, CA, U.S.A.
Phytopathology 100:S79
A large number of fungal species including Verticillium dahliae possess subrepeat sequences in the intergenic spacer region (IGS). We sequenced the IGS
rDNA of 111 isolates of V. dahliae from different hosts and analyzed for subrepeat motif pattern. A Verticillium-specific sub-repeat motif sequence of 17
nucleotides was found and the number of sub-repeats detected ranged from
one to eleven arranged at random. All long and short-spored V. dahliae
isolates from cruciferous hosts had an only one sub-repeat motif. The highest
number of repeats was found in isolates from marigold, cotton and olive,
which included 11 sub-repeat motifs. Additionally, a pair of PCR primers
designated VdCr1 and VdCr2 were designed from the IGS rDNA to
specifically amplify V. dahliae isolates from crucifers. There was no
amplification in V. dahliae isolates from non-crucifer hosts with this primer
pair. The difference in number of sub-repeats and specific-primer developed
in the IGS rDNA region is thus diagnostic and can be used for rapid
identification of V. dahliae isolates from crucifer and non-crucifer hosts.
Comparative efficacy of fungicide application programs alternating
among different products for management of powdery mildew on
cantaloupe
M. E. MATHERON (1), M. Porchas (1)
(1) University of Arizona, Yuma, AZ, U.S.A.
Phytopathology 100:S79
Powdery mildew on cantaloupe and other melon crops, caused by the fungus
Podosphaera xanthii, can result in significant yield loss. A field trial was
conducted in 2009 to evaluate fungicide application programs that alternate
between a highly efficacious fungicide and a product with moderate to low
activity. Foliar applications of products were made on 18 May and 2, 9 and 16
June. Powdery mildew was first observed in plots on 28 May and a high level
of disease developed on nontreated plants by crop maturity. When applied
exclusively throughout the trial, reduction of powdery mildew on the
underside of leaves, compared to nontreated plants, was 98% for Procure
(triflumizole) and Quintec (quinoxyfen), 74% for Flint (trifloxystrobin), 67%
for Cabrio (pyraclostrobin), 58% for Quadris (azoxystrobin), 39% for
Kaligreen (potassium bicarbonate), 30% for Topsin-M (thiophanate-methyl)
and Sovran (kresoxim-methyl), and 21% for Serenade MAX (Bacillus
subtilis). In comparison, disease reductions ranging from 81 to 100% were
recorded in plots when a highly efficacious fungicide (Procure or Quintec)
was alternated with one of the tested products with less activity. The high
level of disease control achieved at crop maturity when utilizing these
treatment programs demonstrates that lower performing fungicides can be part
of treatment programs that expose Podosphaera xanthii to more fungicidal
modes of action as part of a resistance management program without
sacrificing acceptable levels of disease control.
Interactions between Fusarium virguliforme and Phialophora gregata in
soybean using greenhouse studies
C. MATTUPALLI (1), P. D. Esker (1)
(1) University of Wisconsin-Madison, Madison, WI, U.S.A.
Phytopathology 100:S79
Sudden death syndrome (SDS) and Brown stem rot (BSR) are two yieldlimiting diseases of soybean. SDS and BSR are caused by the fungal
pathogens Fusarium virguliforme (Fv) and Phialophora gregata genotypes A
(PgA) and B (PgB) respectively. As soil-borne pathogens, we hypothesize
that there is a potential for interactions between them. Two greenhouse trials
were conducted to study these potential interactions. The experimental design
was a multi-factor randomized complete block with soybean variety (Jack and
Williams82) and inoculum (untreated control, Fv, PgA, PgB, Fv+PgA,
Fv+PgB, PgA+PgB, Fv+PgA+PgB) as the factors. Foliar symptoms were
assessed at weekly intervals beginning at first foliar symptoms as an estimate
of total infected canopy (0–100%) from which an area under disease progress
curve (AUDPC) was calculated. AUDPC was affected by variety and
inoculum in both trials. Marginal variety × inoculum interactions (P = 0.07)
were observed during the first trial only. Jack, a variety supposed to be
resistant to Fv showed increased disease symptoms when inoculated with Fv
alone or in combination with PgA or PgB compared to Williams82. A varietal
difference (P = 0.03) was observed for seed yield during the first trial whereas
there was an interaction of variety x inoculum (P = 0.01) in the second trial.
Results from the two trials suggest some interactions between the pathogens
to cause yield losses with a differential effect between the two varieties.
Irrigation water is an unlikely source of inoculum of Pseudomonas
cannabina pv. alisalensis
S. J. MAUZEY (1), C. T. Bull (2)
(1) California State University Monterey Bay, Seaside, CA, U.S.A.; (2)
USDA ARS, Salinas, CA, U.S.A.
Phytopathology 100:S79
Pseudomonas cannabina pv. alisalensis causes severe bacterial blight on
crucifers across the United States. These experiments examined the potential
of irrigation water as a source of inoculum for P. cannabina pv. alisalensis.
Water samples were collected from multiple irrigation reservoirs and
sprinklers near infected fields. The samples were tested for the presence of P.
cannabina pv. alisalensis using PCR for detection of coronatine and ethylene
biosynthesis genes. Pseudomonas cannabina pv. alisalensis was not detected
in irrigation water samples, however the limit of detection using these
methods was high. Survival in water was modeled using a rifampicin resistant
strain of P. cannabina pv. alisalensis and enumeration on antibiotic
containing media. Population levels of P. cannabina pv. alisalensis added to
irrigation water dropped significantly within the first few days after
inoculation. On average P. cannabina pv. alisalensis populations dropped
from 5.5 log (CFU/ml) at inoculation to below 2.0 log (CFU/ml) after one
week. Additionally, the relationship between population levels of P.
cannabina pv. alisalensis spray inoculated onto broccoli raab (Brassica rapa
subsp. rapa) and the resulting level of disease was evaluated. Under
greenhouse conditions populations of P. cannabina pv. alisalensis as low as
2.0 log (CFU/ml) resulted in disease. These results indicate that irrigation
water is an unlikely source of inoculum for P. cannabina pv. alisalensis on
crucifers.
Vol. 100, No. 6 (Supplement), 2010
S79
Quantifying Fusarium virguliforme in soil using SYBR green and
Taqman assays
G. C. Mbofung (1), M. Vincent (2), A. Fessehaie (3), M. K. Bhattacharyya
(4), L. F. LEANDRO (1)
(1) Dept. of Plant Pathology, Iowa State University, Ames, IA, U.S.A.; (2)
Food Science, Iowa State University, Ames, IA, U.S.A.; (3) Seed Science
Center, Iowa State University, Ames, IA, U.S.A.; (4) Dept. of Agronomy,
Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S80
Fusarium virguliforme is a soilborne fungus that causes sudden death
syndrome in soybean. An effective method to specifically quantify the
pathogen in soil is needed for epidemiological studies on this disease. Our
objective was to develop and compare two real-time PCR assays to detect and
quantify F. virguliforme inoculum density in soil. SYBR green and TaqMan
assays were developed based on sequences of the locus 96 and the FvTox1
gene of F. virguliforme. Assay specificity was tested on 58 taxonomically
closely related and distant fungal isolates. The sensitivity of the assays was
appraised on tenfold serial dilutions of genomic DNA, spore suspensions, and
soil spiked with conidia. Applicability was evaluated on soil samples from
soybean fields and the greenhouse. Real-time PCR assays detected speciesspecific DNA sequences only. The detection sensitivity for genomic DNA,
spores suspensions, and soil spiked of conidia was 5 pg/ l, 500 spores/ml and
50 spores/g soil, respectively, for the SYBR green assay, and 1 pg/ l, 1000
spores/ml and 1000 spores/g soil, respectively, for the TaqMan assay. F.
virguliforme inoculum density in the soil samples ranged between 0 and 106
conidia per g soil. Both assays were equally sensitive and specific for
diagnostic and quantification purposes. The assays can be applied in
diagnostic surveys of F. virguliforme in soil and for quantification of the
pathogen in roots.
Nozzle selection for maximizing brown patch control with fungicides
D. S. MCCALL (1), B. J. Horvath (2), M. A. Cutulle (1), A. E. Nichols (1), V.
R. Sykes (2)
(1) Virginia Tech, Blacksburg, VA, U.S.A.; (2) University of Tennessee,
Knoxville, TN, U.S.A.
Phytopathology 100:S80
Brown patch (Rhicoctonia solani) is the most common and destructive disease
on tall fescue (Festuca arundinacea). Because of time and labor constraints,
turf managers seek ways to maximize the longevity of fungicide applications.
Field studies were conducted in 2008 and 2009 to assess the influence of
nozzle type on longevity of control with common fungicides. Plots were 1.8 ×
1.8 m and were arranged as a randomized complete block design with four
replications. Fungicides evaluated included azoxystrobin (0.305 kg ai/ha),
polyoxin-D (0.308 kg ai/ha, and a pre-formulation of triadimefon (1.53 kg
ai/ha) and trifloxystrobin (0.305 kg ai/ha). Nozzles tested were XR TeeJet,
TurfJet, Air Induction TeeJet, and Turbo TwinJet. All fungicides were applied
at spray volume of 435 liters per ha. Disease was assessed weekly by visual
estimation of brown patch percentage within plots for at least six weeks.
In three of five field trials, there were no significant differences
between nozzle type. In two trials, TurfJet nozzles had significantly more
disease than all other nozzles at three weeks after application. Results indicate
that control may be extended slightly using a flat-fan type nozzle instead of
flood-jet type.
The σ factor rpoN is required by Brenneria rubrifaciens for HR elicitation
in tobacco and virulence on walnut plants
A. MCCLEAN (1)
(1) CPGRU USDA-ARS, Davis, CA, U.S.A.
Phytopathology 100:S80
Deep bark canker (DBC) of walnut is caused by the bacterium, Brenneria
rubrifaciens. The disease leads to a chronic reduction in nut yield and tree
vigor. DBC symptoms are characterized by deep vertical cracks in trunks and
scaffold branches which exude a discolored bacteria laden sap. The disease
develops on trees at least 10 years old but is never observed on seedlings. A
collection of 650 B. rubrifaciens 6D370 transposon mutants was screened for
production of the unique red pigment rubrifacine hypothesized to be
associated with virulence. Three hyper pigment producing mutants and 81
other pigment producing and pigment deficient mutants were screened in a
tobacco leaf bioassay to evaluate hypersensitive response (HR) elicitation.
Three of the 84 mutants, including one of the enhanced pigment producers,
failed to elicit a HR in tobacco leaves. Two of the three HR minus mutants,
Br-212 and Br-415, were attenuated in their ability to cause necrosis on tissue
cultured walnut plants with Br-415 being severely attenuated. Genetic analysis
revealed Br-415 contained a transposon insertion in an open reading frame
(ORF) with homology to rpoN-like σ54 factors for RNA II polymerase.
Mutant Br-415 also grew slower in minimal medium and was impaired in
movement on motility agar relative to wildtype. These findings indicate rpoN
S80
PHYTOPATHOLOGY
σ54 dependent transcription is required by B. rubrifaciens for HR elicitation
in tobacco and virulence on walnuts.
The Fungal Genetics Stock Center at UMKC is an international
Biological Resource Center
K. MCCLUSKEY (1), A. Wiest (1), M. Plamann (1)
(1) University of Missouri-KC, Kansas City, MO, U.S.A.
Phytopathology 100:S80
Founded in 1960, the Fungal Genetics Stock Center enters it’s fiftieth year of
operation during a period of tremendous growth. The collection has more than
doubled since moving to UMKC in 2004 and has added new materials that
reach out beyond it’s traditional constiuancy. Joining our extensive set of
genetically engineered Magnaporthe strains are deletion sets for Neurospora,
Cryptococcus and Candida as well as molecular genetic tools for working
with plant pathogens, industrial fungi, model organisms, and human
pathogens. With distribution growing every year, the FGSC sends materials to
scientists in over 35 countries every year; approximately half of our orders are
from within the U.S. In addition to being part of an NIH funded multiinstituition Functional Genomics Program for Neurospora, the FGSC is
involved in cutting edge genomics research with collaborators at the US DOE
Joint Genome Institute. The FGSC and its staff are actively involved in
national and international societies and ad hoc working groups fostering the
development of collection resources.
Management of powdery mildew in cucurbit crops with biopesticides and
integrated programs
M. MCGRATH (1)
(1) Cornell University, Riverhead, NY, U.S.A.
Phytopathology 100:S80
Two biopesticides, Organocide (5% sesame oil) and Milstop (85% potassium
bicarbonate), were evaluated for powdery mildew in adjacent field
experiments with pumpkin, muskmelon, and butternut squash conducted in
2007 and 2009. They were tested alone or combined with host plant resistance
and/or conventional, mobile fungicides (Quintec, Procure, and/or Pristine) in
various programs with these fungicides applied in alternation a total of 2 to 6
times. Applications were made with a tractor-sprayer on a 7-day interval
beginning when symptoms were first found. Both biopesticides usually were
effective, especially when used in an integrated program. For example,
severity of powdery mildew on upper leaf surfaces was reduced 39% with
Milstop and 57% with Organocide applied to a susceptible pumpkin cultivar
based on AUDPC values relative to the nontreated susceptible cultivar;
severity was reduced 93% and 73% with Milstop and Organocide,
respectively applied to a resistant cultivar; and 63–95% with Organocide
applied with mobile fungicides to the susceptible and resistant cultivars. All
treatments were equally, highly effective (no significant differences) applied
to butternut squash (64–100% control) and to muskmelon (96–100% control)
based on severity on 8 Sep 2009. In 2007, severity on upper leaf surfaces was
reduced 23% with Milstop applied to a susceptible pumpkin cultivar and 61%
with winter squash. The reduction was 52% and 89% with Organocide,
respectively.
Microarray characterization of the HrpL regulon of the fire blight
pathogen Erwinia amylovora
R. R. MCNALLY (1), Y. Zhao (2), I. K. Toth (3), P. J. Cock (3), P. E. Hedley
(3), G. W. Sundin (4)
(1) Michigan State University, East Lansing, MI, U.S.A.; (2) University of
Illinois, Urbana, IL, U.S.A.; (3) Scottish Crop Research Institute, Invergowrie,
SCOTLAND; (4) Scottish Crop Research Institute, East Lansing, MI, U.S.A.
Phytopathology 100:S80
The bacterium Erwinia amylovora causes fire blight of apple, pear, and other
rosaceous plants. HrpL, an alternate sigma factor recognizing hypersensitive
and pathogenicity (hrp) promoters, is required for pathogenesis. Hrp
promoters regulate the expression of both structural and translocated
components of the type III secretion system (T3SS) in E. amylovora. While
HrpL plays a vital role in T3SS regulation, HrpL may also influence
additional signaling networks controlling other virulence factors. To explore
the HrpL regulon of E. amylovora Ea1189, an open reading frame-specific
whole-genome microarray was constructed. Microarray analysis compared
mRNA extracted from wild-type and ∆hrpL strains incubated in hrp-inducing
minimal medium. Twenty-four genes were found to exhibit fold-change
expression ratios greater than 1.5 in Ea1189∆hrpL. Five genes were upregulated while 19 genes were found to be down-regulated, suggesting
positive control by HrpL. Many genes identified have known roles in T3SS
while others encode proteins of unknown function. Knockout mutants of
genes of interest identified in the microarray experiment were phenotypically
analyzed revealing attenuated virulence in an immature pear assay and altered
motility. Continued characterization is expected to reveal novel insights into
the pathogenesis of fire blight by E. amylovora.
Probability distributions for disease severity and time-to-event data
N. MCROBERTS (1), L. V. Madden (2)
(1) Scottish Agric College, Edinburgh, UNITED KINGDOM; (2) OARDC
Ohio State University, Wooster, OH, U.S.A.
Phytopathology 100:S81
When disease severity is measured either as the proportion (or percentage) of
tissue diseased, or by using a continuous rating scale, the resulting data are
continuous and bounded, and may often be skewed. Until now the fact that
such data do not have strictly Gaussian (normal) properties has usually been
ignored in plant pathology, and statistical analyses founded on assumptions of
normality are applied routinely. With advances in generalized nonlinear and
mixed modeling algorithms it is now straightforward to fit probability
distributions with appropriate statistical properties to severity data. We
illustrate two examples of such distribution: the beta distribution and
Johnson’s SB distribution. Changes in the parameters of these distributions
over time characterize changes in the spatial heterogeneity of severity during
disease progress; preliminary analysis of several diverse data sets indicates a
common relationship between the skewness and kurtosis of observed data
irrespective of measurement scale of visual symptoms. In the case of disease
incidence data, we show that the Laplace distribution provides an alternative
means to describe disease progress over time. The cumulative distribution
function serves as a reasonable time-to-event model for disease progress, with
the corresponding probability density function describing the instantaneous
rate of disease progress.
Characterization of nepoviruses by an integrated approach
E. MEEKES (1), M. Botermans (2), R. Hooftman (1), T. van Schadewijk (3),
R. Miglino (3), A. Roenhorst (2)
(1) Naktuinbouw, Roelofarendsveen, NETHERLANDS; (2) Plant Protection
Service, Wageningen, NETHERLANDS; (3) Dutch Flower Bulb Inspection
Service, Lisse, NETHERLANDS
Phytopathology 100:S81
In 2006 a five year program started to reinforce plant health infrastructure in
the Netherlands. This program focused on regulated organisms, aims to update
existing plant-pathogen collections, enables the development of new detection
methods and to share data in an innovative way. Included isolates are
characterized biologically, serologically and by sequence analysis. Within the
project isolates of various pathogens were collected, including viruses
belonging to the genus Nepovirus s.l. (including Sadwavirus and Cheravirus).
Generally these viruses are nematodes-transmitted (Xiphinema spp. /
Longidorus spp.) and can be seed transmitted to various extends. Many
species have an economic impact and are regulated for European countries
(under EU Directive 2000/29/EC). Research focuses on Arabis mosaic virus
(ArMV), Strawberry latent ringspot virus (SLRSV, Sadwavirus), Tomato
black ring virus (TBRV), Tomato ringspot virus (ToRSV) and Tobacco
ringspot virus (TRSV). Over 50 isolates were characterized using
indicator-plant research, ELISA and sequencing of important genome
fragments (proteinase-polymerase gene region, polymerase and coat protein
genes). Experimental data on the various nepovirus species and techniques, will be presented. The characteristics of the virus isolates are
publicly accessible via Q-bank databases for regulated plant pests (www.qbank.eu), an initiative of the Dutch Ministry of Agriculture, Nature and Food
Quality. Physical collections are maintained, and can be obtained via the
website.
Host and life strategy adaptations mediate competition among isolates of
Aspergillus flavus
H. L. MEHL (1), P. J. Cotty (1)
(1) USDA-ARS, Tucson, AZ, U.S.A.
Phytopathology 100:S81
Communities of Aspergillus flavus, the primary causal agent of aflatoxin
contamination in crops, are composed of diverse isolates that collectively
cause aflatoxin contamination. Isolates vary in competitive ability on maize,
but it is unclear the extent to which host-specific interactions determine
success of individual isolates during competition. Seed from maize, cotton,
sorghum, and soybean were surface-sterilized (80°C, 45 sec) and coinoculated
with pairs of isolates previously identified as the most and least competitive
on maize kernels. Czapek’s agar was seeded with the same isolate mixtures.
After 7 days at 31°C, isolate-specific SNPs were quantified by
pyrosequencing. DNA from mycelia was used to compare colonizing abilities
and DNA from conidia was used to quantify sporulation during competition.
Isolates most competitive on maize were not always most competitive on
other hosts. Relative colonizing ability was equal to relative ability to
sporulate when isolates were grown on Czapek’s agar. However, during
competition on plant hosts, some isolates were better colonizers than
sporulators and the extent to which an isolate competed during either
colonization or sporulation varied by host. In general, less competitive
colonizers were more competitive as sporulators, suggesting adaptation to
different life strategies. Host and life strategy adaptations may modulate
diversity within A. flavus communities.
Sensitive detection and discrimination of different Pepino mosaic virus
genotypes
N. MEHLE (1), I. Gutierrez-Aguirre (1), N. Prezelj (1), D. Delic (2), U. Vidic
(1), P. Kramberger (3), M. Ravnikar (1)
(1) National Institute of Biology, Ljubljana, SLOVENIA; (2) University of
Banjaluka, Faculty of Agriculture, Banjaluka, BOSNIA-HERZEGOVINA; (3)
BIA Separations d.o.o, Ljubljana, SLOVENIA
Phytopathology 100:S81
We developed quantitative real time PCR (RT-qPCR) assays for the detection
and discremination of presently circulating PepMV (Pepino mosaic virus)
genotypes. The following genotype combinations, European tomato-Peruvian,
Ch2, and US1, were successfully distinguished within the tested isolates. Onestep RT-qPCR detected PepMV European tomato genotype particles at least
two orders of magnitude more sensitively than ELISA. The method detected
as little as one naturally infected seed among 5000 uninfected seeds. The
developed RT-qPCR assay will enable detailed studies on the demographic
distribution of the circulating PepMV genotypes. At the same time, the
disposability of more than one gene target for the detection of PepMV RNA,
will increase the reliability of the virus detection in samples with low expected
virus concentration, such as latent infections or irrigation waters. It is
suspected that PepMV can be present in the environment at concentrations,
which despite being too low to be detected by conventional detection
methods, they can still constitute a risk of potential infection. We have
experimentally confirmed that PepMV remained infective in water for up to
three weeks. We are investigating the potential of monolithic chromatographic
supports (CIM Convective Interaction Media) for concentration of PepMV.
The combination of both technologies (CIM for concentration and qPCR for
detection) will allow detection of extremely low concentrations of PepMV
from environmental waters.
Fruit flesh type and harvest method affect postharvest decay of southern
highbush blueberry
L. MEHRA (1), H. Scherm (1)
(1) University of Georgia, Athens, GA, U.S.A.
Phytopathology 100:S81
Postharvest decay is a major problem in all blueberry growing areas of the
U.S. Since the risk of infection is increased by fruit bruising, which in turn is
increased by machine-harvest, it has not been possible to harvest southern
highbush blueberry (SHB) fruit mechanically for the fresh-market. This may
change with the advent of SHB genotypes with crispy berries, i.e., fruit with
qualitatively firmer flesh and/or skin. Four SHB genotypes having crispy
berries and four with conventional berries were either hand-picked or
machine-harvested in April 2009. Fruit were sorted, packed, and placed in
cold-storage (2°C) for 0, 7, 14, or 21 days. Counts of total aerobic bacteria,
yeasts and molds, coliforms, and E. coli on fruit samples from the 0-day
storage period were below commercial tolerance levels. Natural decay
incidence following cold-storage was lowest for hand-harvested crispy fruit
and highest for machine-harvested conventional fruit. Interestingly, machineharvested crispy fruit had the same decay incidence as hand-picked
conventional fruit. Across all treatments, fruit firmness was a good indicator
of natural decay incidence. In a separate experiment, samples from the 0-day
storage period were inoculated with Alternaria, Botrytis, and Colletotrichum
spp, and fruit decay was assessed after 7 days in the cold room. Artificial
inoculation caused less decay on crispy and hand-harvested berries, but the
magnitude and significance level varied by pathogen species.
Genetically diverse isolates of Grapevine virus A are present in
Washington vineyards
T. MEKURIA (1), R. A. Naidu (2)
(1) Washington State University, IAREC, Prosser, WA, U.S.A.; (2)
Washington State University, Irrigated Agriculture Research and Exten Ctr,
Prosser, WA, U.S.A.
Phytopathology 100:S81
Grapevine virus A (GVA; genus Vitivirus, family Flexiviridae) is the putative
agent of grapevine kober stem grooving disorder of the Rugose Wood
complex. The virus has a worldwide distribution and known to be transmitted
to grapevine by some species of mealybugs and soft scale insects. Since kober
stem grooving symptoms usually develop in grapevines grafted onto a
rootstock but remain latent in ungrafted vines, the presence of GVA was
investigated in own-rooted grapevines in Washington vineyards. Petiole
extracts from six wine grape cultivars (Cabernet Sauvignon, Chardonnay,
Lemberger, Merlot, Pinot Noir and Zinfandel) were tested by one step-single
tube reverse transcription (RT)-PCR using primers specific to the coat protein
(CP) and the RNA-dependent RNA polymerase (RdRp) domain of the
replicase gene. The amplicons were cloned in pCR2.1 plasmid vector and the
Vol. 100, No. 6 (Supplement), 2010
S81
sequence of gene-specific clones determined in both orientations. Pairwise
comparison of the CP nucleotide sequences showed 77 to 100% identity
between Washington isolates and 75 to 93% identities with corresponding
sequences available in GenBank. Pairwise comparison of RdRp nucleotide
sequences showed 74 to 100% identity between Washington isolates and 74 to
87% identity with other isolates from GenBank. These results indicated the
presence of genetically diverse isolates of GVA in Washington vineyards.
Additional data will be presented on phylogenetic relationships of GVA
isolates.
The relationship between endophytic Bacillus cereus and Bacillus cereus
causing foodborne illness
R. MELNICK (1)
(1) Penn State University, University Park, PA, U.S.A.
Phytopathology 100:S82
Bacillus cereus is a common inhabitant of soil as well as the endophytic
portions of plant tissue. Although common in the environment, some isolates
cause diarrheal foodborne illness through the production of enterotoxins. A
collection of endophytic B. cereus isolates were obtained from several crops;
including apple, cacao, tomato, and potato. PCR was conducted using 11
primer sets to amplify the genes for B. cereus enterotoxin T and the
complexes that encode for operons of hemolysin BL (HBL) and the
nonhemolytic enterotoxin (NHE). Of the 35 endophytic B. cereus isolates
tested, the vast majority had one or more of the genes required for enterotoxin
production. Approximately 14% of isolates tested lacked any enterotoxin
genes. The endophytic host had no impact on the presence of enterotoxin
genes, as they were detected in isolates from all tested plants. The enterotoxin
genes from endophytic isolates were sequenced and compared to B. cereus
isolates known to cause foodborne illness. Data will be presented on the
presence of enterotoxin genes in environmental isolates of B. cereus,
comparisons of sequence data between environmental and clinical samples,
and whether endophytic isolates of B. cereus express the enterotoxin genes
under differing environmental conditions.
Preliminary characterization of Tomato yellow leaf curl virus in Hawaii
M. J. Melzer (1), D. Y. Ogata (1), S. K. Fukuda (1), R. Shimabuku (2), W. B.
Borth (1), D. M. Sether (1), J. S. HU (3)
(1) University of Hawaii at Manoa, Honolulu, HI, U.S.A.; (2) University of
Hawaii at Manoa, Maui, HI, U.S.A.; (3) University of Hawaii, Honolulu, HI,
U.S.A.
Phytopathology 100:S82
Tomato yellow leaf curl disease, caused by the begomovirusTomato yellow
leaf curl virus (TYLCV; family Geminiviridae), is a destructive disease of
tomato (Solanum lycopersicum L.), particularly in tropical and sub-tropical
regions. In the fall of 2009, tomato plants showing stunted new growth,
interveinal chlorosis, and upward curling of leaf margins were reported in
Wailuku, Maui, and in Poamoho, Oahu. A ~1.5 kbp PCR product was
amplified from symptomatic tomato plants using degenerate geminivirus
primers, but not healthy controls. Sequence analysis revealed these products
had a 92–99% nucleotide identity to TYLCV sequences in GenBank. The
Wailuku isolate (GenBank accession # GU322424) appears to be more closely
related to TYLCV isolates from Japan and China, whereas the Poamoho
isolate (GU322423) appears to be more closely related to TYLCV isolates
from the mainland U.S.A. and Mexico, suggesting the virus has been
introduced into Hawaii on multiple occasions. The identification of reservoir
hosts of TYLCV in Hawaii’s flora is critical for disease management. Several
common agricultural weed species from the Poamoho site were evaluated for
the presence of TYLCV using a squash-blot hybridization assay with a
digoxigenin-labeled probe derived from the ~1.5 kbp PCR amplicon.
Cheeseweed (Malva parviflora L.), a reported host of TYLCV, tested positive
for TYLCV using this assay as did the solanaceous weed, Apple of Peru
(Nicandra physalodes L.).
Identification of Streptomyces stelliscabiei causing potato common scab in
Michigan
Q. MENG (1), J. Hao (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S82
Four Streptomyces isolates were obtained from potato (Solanum tuberosum)
tubers with common scab symptoms from a field in Michigan in 2009.
Isolates were purified by single colony transfer. Genomic DNA was extracted
from the isolates. Result of polymerase chain reaction (PCR), using speciesspecific primers, for all the isolates was positive for Streptomyces
stelliscabiei, but negative for any of the other known pathogenic Streptomyces
spp. 16S ribosomal RNA from the isolates was sequenced, and analyzed using
the BLAST algorhythm against the NCBI Genbank. The results showed 99 (2
isolates) to 100% (2 isolates) identity to S. stelliscabiei depending on the
isolate. The isolates were all confirmed by PCR to have txtAB, nec1, and tomA
S82
PHYTOPATHOLOGY
genes, which are associated with pathogenicity of scab-causing Streptomyces
spp. Thaxtomin production was detected when the isolates were cultured in
oat broth media. Culture plugs of the isolates were placed on sterile potato
tuber slices, and necrotic lesion was observed after 3 days of incubation.
Radish seeds were grown in water agar or soil, infested with one of the
isolates. After one week, radish roots of plants inoculated with bacteria were
darkened or showed suppressed growth. The pathogen was re-isolated from
the lesion from radish roots and confirmed as identical to the original
inoculum by using PCR and morphological characterization. This is the first
report of S. stelliscabiei in Michigan.
Visual and quantitative characterization of ironwood tree (Casuarina
equisetifolia) decline on Guam
Z. MERSHA (1), R. L. Schlub (1), P. O. Spaine (2), J. A. Smith (3), S. C.
Nelson (4)
(1) UOG Cooperative Extension, University of Guam, Mangilao, GUAM; (2)
USDA Forest Service, Athens, GA, U.S.A.; (3) University of Florida’s
Institute of Food and Agricultural Sciences, Gainesville, FL, U.S.A.; (4)
Cooperative Extension Service, University of Hawaii At Manoa, Hilo, HI,
U.S.A.
Phytopathology 100:S82
To assess the level of ironwood tree decline on Guam, photographs of 44
randomly selected trees with varying levels of decline were categorized into
small (CBH ≤ 100 cm) or large (CBH >100 cm) based on their circumference
at breast height (CBH) and visually catalogued into a five-scale decline
severity (DS) rating. On subsequent surveys, trees with different DS ratings
were characterized visually for branch thinning and quantitatively for
branchlet (“needle”) biomass. As DS increased from 0 (healthy) to 4 (nearly
dead) branch thinning, progressively increased from 0 to 95.0% and 0 to
92.5% for small and large trees, respectively. There was no significant
difference between branchlet biomass for DS 0 and DS 1 nor between DS 2
and DS 3 trees. The greatest branchlet weight loss, at 95.3%, occurred to DS 4
trees. Internal symptoms included various patterns of discolorations in trunks
and a white soft-rot in roots. Discoloration was consistently traced into
branches through cross-sectioning at the branch trunk interface. In branches
the presence of discoloration was only 100% reliable for DS 3 and 4 trees.
External symptoms starts at the top of trees and progresses downward;
whereas, internal discoloration starts at the tree’s base and diminishes
acropetally This gradient of discoloration was well described by the
exponential decay function (P = 0.0001) with R2 values of 0.85 and 0.73 for
small and large trees, respectively. Ironwood tree decline has expanded into
areas such as Cocos Island, which were virtually decline free a year earlier.
Enhancing Guam’s agriculture professionals’ knowledge of ecological
disease management
Z. MERSHA (1), R. W. Brown (1), R. L. Schlub (1)
(1) UOG Cooperative Extension, University of Guam, Mangilao, GUAM
Phytopathology 100:S82
In 2009, a plot was set up at the Yigo Experiment Station to collect data for an
agriculture professional training manual on detection of soil nutrient
deficiencies and the role of soil nutrients in plant health and disease
suppression. Crops included tomato (Solanum lycopersicum cv. Season Red),
eggplant (Solanum melongena cv. Pingtung Long), and cucumber (Cucumis
sativus cv. Joy). Nutrient treatments were NPK, NPK plus micronutrients,
deficient N plus PK, deficient P plus N K, and deficient K plus NP.
Deficiency levels were at 25 percent of the current UOG Cooperative
Extension recommendation. Data collected included information on diseases,
sugar content of fruits, soil pH, salinity, soil nutrients content, and plant
biomass, height, yield, leaf count, tissue nutrient content, and chlorophyll
levels. A variety of equipments was used for data collection and for training
evaluation purposes: soil salinity meter, pH meter, YSI9500 photometer
nutrient analyzer, brix meter, SPAD502 chlorophyll meter, and Cardy meters
(NO3- and K+). Partial analysis of the data indicates that a 75% reduction in
recommended amounts of N, P, or K was sufficient to impact yield but not the
development of diseases or visible distinct symptoms. Cardy meters, YSI9500
photometer, and SPAD502 chlorophyll meter emerged as potential tools that
can be used to detect nutrient deficiencies in the field.
Seasonal dynamics of black leaf mold (Pseudocercospora fuligena) on
tomato (Solanum lycopersicum) grown under protected cultivation in
Thailand
Z. MERSHA (1), B. Hau (2)
(1) University of Florida, Tropical Research and Education Centre,
Homestead, FL, U.S.A.; (2) Leibniz Universitaet Hannover, Hannover,
GERMANY
Phytopathology 100:S82
Epidemics of black leaf mold (BLM), caused by Psudocercospora fuligena,
were studied in 24 plantings of tomato (Solanum lycopersicum cv. FMTT260)
under naturally ventilated greenhouse condition in central Thailand from May
2005 to March 2007. Three BLM peak-epidemics periods were identified:
August-September in 2005 (I) and 2006 (II), and plantings in December 2005January 2006 (III). Mean BLM severity (DS*) of 0.30, 0.14 and 0.20 were
recorded from I, II and III, respectively. Favorability indices of temperature
(FIT) and relative humidity (FIRH) of the respective peak periods were 0.90,
0.61 and 0.66 for FIT and 0.51, 0.35 and 0.44 for FIRH. Linear, multiple and
stepwise regressions were performed to detect weather-disease-host
relationships. FIRH was highly correlated with DS* (r2 = 0.71) and the multiple
regression of FIRH and FIT in the model DS*=a+(b*FIT)+(d*FIT*FIRH)
improved R2 value to 0.74 with significant parameter estimates. Maximum
plant height (MPH) of all experimental plants was fairly correlated (r2 = 0.32,
p < 0.0001) with marketable yield (MY). Despite the poor linear correlation
between MY and DS*, integration of MPH in the model MY = (a+b*MPH)*(1c*DS*) resulted in R2 value of 0.35 and high significance of the parameters b
and c. A 1% DS*, according to this model, reduced MY by ≅ 1.2%. The actual
yield losses recorded from comparison of sprayed and non-sprayed treatments
of I, II and III were 34.1, 39.2 and 20.6%, respectively. The last model
explained the actual yield losses of I and III but the loss in II was higher than
the prediction.
Anthracnose fruit rot, caused by the fungus Colletotrichum acutatum, is an
important disease of blueberries. The objectives of this study were to evaluate
a rapid screening technique for anthracnose fruit rot resistance and to
determine if certain fruit characteristics are correlated with resistance. Cut
surfaces of ripe fruit were inoculated with a C. acutatum suspension (106
conidia/ mL) in two replicated experiments with 24 and 26 blueberry
cultivars, respectively. Resistance was evaluated by quantifying sporulation
on the cut fruit surface after 3 days of incubation at 22–24°C. Resistance in
this bioassay correlated well with published resistance ratings (Polashock et
al. 2005). Fruit pH, titratable acids, sugar content, and firmness of the
cultivars were assessed from 2005 to 2008. A negative linear correlation was
found between fruit rot incidence and sugar content (P = 0.001, R2 = 0.36).
The direct effect of sugar concentration (1%, 2%, 4%, 8%, and 16% glucose
or fructose) and pH (2.0, 2.5, 3.0, 3.5, 4.0, 5.5, 7.0) on hyphal growth of C.
acutatum on defined media was evaluated. A reduction in hyphal growth was
observed at pH values below 3.5 and sugar concentrations above 8%. The
roles of pH and sugar concentration in fruit rot resistance require further
investigation. The cut-fruit inoculation method provides a rapid means for
fruit rot resistance screening in blueberries and requires less fruit than
conventional whole fruit inoculation methods.
Efficacy of acibenzolar-S-methyl and copper fungicides for the control of
angular leaf spot of strawberry
J. C. MERTELY (1)
(1) University of Florida-GCREC, Wimauma, FL, U.S.A.
Phytopathology 100:S83
Identifying anthracnose disease resistant strawberry clones using
traditional techniques and molecular markers for screening
M. MILLER-BUTLER (1), B. Krieser (1), K. J. Curry (1), B. J. Smith (2)
(1) University of Southern Mississippi, Department of Biological Sciences,
Hattiesburg, MS, U.S.A.; (2) USDA ARS Southern Horticultural Laboratory,
Small Fruit Research Unit, Poplarville, MS, U.S.A.
Phytopathology 100:S83
Angular leaf spot (ALS), caused by Xanthomonas fragariae, is an
occasionally severe disease of strawberry The most common symptoms are
spotting and blighting of the leaves, although the calyx may also be infected,
rendering the fruit unsightly and unmarketable. Products for controlling ALS
have been tested in replicated field trials at the Gulf Coast Research and
Education Center in Wimauma, Florida since 2005–06. Test products are
typically applied as foliar sprays on a 7-day calendar schedule throughout the
winter growing season. Over the past five seasons, representative
biopesticides, copper fungicides, and organic fungicides have been tested, as
well as acibenzolar-S-methyl, hydrogen peroxide, and kasugamycin. To date,
acibenzolar-S-methyl applied at 26 g a.i./ha and copper fungicides applied at
ca. 300 g metallic copper/ha have given the greatest and most consistent
disease suppression. Higher rates often improved disease suppression, but did
not increase yield due to copper phytotoxicity or the metabolic costs
associated with acibenzolar-S-methyl. During the 2008–09 season, when up to
40% of the foliage was killed or damaged by ALS, weekly applications of
acibenzolar-S-methyl at 26 g a.i./ha or copper hydroxide at 294 g metallic
copper/ha significantly increased marketable yield. Yield improvements were
not demonstrated under conditions of lower disease pressure in the other four
trials.
Pomegranate decay caused by Pilidiella granati in California
T. J. MICHAILIDES (1), R. Puckett (1), D. Morgan (1)
(1) University of California Davis, Parlier, CA, U.S.A.
Phytopathology 100:S83
During the last decade and after the recent discoveries of the high antioxidant
content of pomegranate fruit and juice, the pomegranate industry has
increased to >20,000 acres in California. Pomegranates are marketed as intact
fresh fruit, extracted arils, or juice. Fruit diseases such as black heart caused
by Alternaria spp., Aspergillus and Penicillium rots, are considered the most
important diseases of pomegranate fruit. However, in the last two years,
another rot caused by a pycnidial fungus Pilidiella granati has been
commonly observed in fruit from fields or packinghouses. Pilidiella rot is very
different from black heart rot in that it decays the arils and the fruit rind while
black heart decays only the arils. On APDA, P. granati produces white to
light yellow, leathery mycelia with abundant black, solitary pycnidia of
various sizes. Optimum temperature of growth on APDA ranges from 25 to
30°C; the fungus grows at 15°C, but not at 35°C. Although infection needs a
wound, infection moved from infected fruit to intact fruits in contact. The
pathogen overwinters as pycnidia and mycelia in rotten fruit in the field. It has
been isolated also from dying shoots of young pomegranate trees. P. granati is
characterized by the hyaline to pale brown conidia (length:width >1.5) in
contrast to Coniella spp. which have dark brown conidia (length:width <1.5).
To our knowledge, this is the first report of P. granati causing significant fruit
rots of pomegranate in California.
Anthracnose fruit rot resistance in blueberries: Evaluation of a rapid
screening technique and correlation with fruit characteristics
T. D. MILES (1), J. F. Hancock (1), P. W. Callow (1), A. M. C. Schilder (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S83
Anthracnose of commercially grown strawberry, Fragaria × ananassa, may
be caused by any of three Colletotrichum species: C. acutatum, C. fragariae,
or C. gloeosporioides. These destructive pathogens may cause fruit rot, leaf
spot, petiole lesions, crown rot, wilt, and death of the plant. Traditional and
molecular approaches were used to identify anthracnose resistant strawberry
clones. We used a traditional disease screening method, i.e., inoculating
detached leaves of 52 strawberry clones with conidial suspensions of two C.
fragariae isolates and one C. gloeosporioides isolate and determined that 48%
were susceptible, 23% were intermediate, and 29% were resistant to
anthracnose based on their disease score. Researchers have identified two
molecular markers linked to the Rca2 gene found in strawberry germplasm
resistant to C. acutatum. These two markers were used to screen the 52
strawberry clones for the presence/absence of this gene. Thirty-seven percent
of the clones had both markers, 50% had one of the markers, and 4% had
neither marker. More strawberry clones will be screened for the presence of
these two markers and for anthracnose resistance using traditional techniques.
Correlation of the presence or absence of the gene markers with resistance or
susceptibility to anthracnose caused by any of the three Colletotrichum
species should decrease the time it takes to screen selections for anthracnoseresistant genotypes in strawberry breeding programs.
Occurance of Prothallonema asymmetricum Siddiqi, 1986 in Arak
Province of Iran
M. MIRZAEE-QOMI (1), F. Khozeini (2), S. Barooti (3), S. Rezaee (1)
(1) Department of Plant Pathology, College of Agriculture and Natural
Resources, Science and Research Branch, Islamic Azad University, Tehran,
IRAN; (2) Plant Protection Ministry, Iranian Research Institute of Plant
Protection, Tehran, IRAN; (3) Nematology Research Department, Iranian
Research Institute of Plant Protection, Tehran, IRAN
Phytopathology 100:S83
During a survey in Arak county of Iran a species belonging to the genus
Prothallonema was isolated. Based on morphological and morphometrics
studies, it was indentified as Styctylus asymmetricus Thorne,1941 with new
scientific name as Prothallonema asymmetricum siddiqi,1986. Iranian
specimen extracted from alfalfa soil of Marzijaran region in Arak porovince
of Iran. Females contained a thin layer of culticul over their head with a three
partite 10 m stylet with asymmetric knobs. Dorsal knobs of spear much
smaller than the others crops of esophagus fusiform. Basal bulb esophagus
ovoid to pyriform with elongated stem extending into intestine. A
morphometric characteristic feature of this nematode is esophagus in which
the terminal bulb was envaginated with intestine. Ovary outstretched with one
or two flexures. Posterior uterine branch absent. Nematodes contained 4
laterals in which the width was quarter to third of the body width. Esophagal
duct was located on the end of the stylet and D.G.O was exactly located on the
beneath of the knobs. Females were Mono-Pro- Delphic and vulva was near to
the annus. In against of young nematodes with long mono-prodelphic ovary,
older contains a amphi-delphic ovary with one or two inverts. V% was also
about 90% in females. Except for sexual organs all other parts of male bodies
were same as what has observed in females. Compared to females with 1000
µm length, males were shorter. This is the first report of this nematode in Iran.
Vol. 100, No. 6 (Supplement), 2010
S83
The powder formulation survey of strain Bacillus subtilis M36 for control
of bean root rot caused by Fusarium solani f. sp. phaseoli
P. MIRZAEE-QOMI (1), H. Zamanizadeh (1), R. Saberi-Riseh (2), M. Lak
(3)
(1) Department of Plant Pathology, College of Agriculture and Natural
Resources, Science and Research Branch, Islamic Azad University, Tehran,
IRAN; (2) Department of Plant Pathology & Entomology, College of
Agriculture, University of Tehran, Rafsanjan, IRAN; (3) Central Province
Agricultural Research and Natural Resources Center, Arak, IRAN
Phytopathology 100:S84
In the survey 194 gram posetive bacteria isolated from the rhizosphere of bean
from Markazi province and also 41 isolates were prepared from the collection
of Research Center of Biological Control at Tehran Universitiy were
evaluated on the basis of inhibitionry zone of fungal growth. Finaly eight the
better strain of B. subtilis were selected for greenhouse trials. Result showed
that antagonistic bacterium B. s M36, isolated from the rhizosphere of bean
fields Markazi province exhibited the greatest effect on reducing the Fusarium
root rot bean. In greenhouse trials, after six weeks, between nine formulations
of strain B. s M36, the one which obtained from M1 culture medium with
Arabic Gum as the sticker with 68.42% control, depicts the greatest effect on
reducing Fusarium root rot was more effective than two fungicides Rovral-TS
and Trichomix-HV with 56.14% and 54.38% control respectively. The result
showed that the population of B. s M36 grown from in powder formulations
has decreased during the storage at 4 and 25°C over a 150-day period. In
addition, formulation of strain B. s M36 stored at 4°C had longer shelf life
than those stored at 25°C. Long-term stability of formulation (M1-AG) of
strain B. s M36 was the best. Study of media on population of effective strain,
exhibited that the M1 medium had the most effect in this study.
Addressing the relationship between Pseudoperonospora cubensis and P.
humuli using multigenic and host specificity assays
M. N. MITCHELL (1), D. H. Gent (2)
(1) Oregon State University, Corvallis, OR, U.S.A.; (2) USDA ARS
NFSPRC/ OSU BPP, Corvallis, OR, U.S.A.
Phytopathology 100:S84
The pathogens Pseudoperonospora cubensis and P. humuli, the causal agents
of the downy mildews on cucurbits and hops respectively, have been
demonstrated to be sister taxa. Species concepts in downy mildews are in flux
and include morphology, host range, reproductive isolation and molecular
evidence. A recent study that examined sequence data from the internal
transcribed spacer (ITS) and morphological characteristics of both pathogens
suggested that the species are synonymous. As nomenclature has implications
for pathogen identification, disease management tactics, and plant quarantine
regulations, it would be significant to resolve whether the synonymy of P.
cubensis and P. humuli is correct. To increase resolution, the mitochondrial
cytochrome oxidase (cox) gene cluster, and two nuclear loci, ITS and tubulin, were analyzed with parsimony and Bayesian approaches. For nuclear
loci, all but two isolates of P. humuli were moderately to strongly resolved as
a clade within P. cubensis. However, the cox cluster showed the opposite
relationship. Host specificity experiments performed on universally
susceptible cucurbit hosts and hop cultivars indicate that the pathogens have
discrete pathogenic capabilities. Thus, there is evidence that there exist
biologically relevant characteristics that differentiate the two organisms with
implications for the detection and management of both that may be concealed
by the reduction of P. humuli to a taxonomic synonym of P. cubensis.
Cercospora and Corynespora leaf spots in Hydrangea macrophylla
M. T. MMBAGA (1)
(1) Tennessee State University School of Agriculture and Consumer Sciences,
McMinnville, TN, U.S.A.
Phytopathology 100:S84
Leaf spots and leaf blight symptoms observed in garden hydrangea in midTennessee were identical to those described for both Cercospora and
Corynespora leaf spot. Pathogenicity tests with isolated organisms indicated
that the leaf spots/ blight symptom was caused by several pathogens. Spores
of Corynespora cassiicola and Cercospora hydrangeae were observed on leaf
samples that showed identical symptoms. In some samples, spores of both C.
cassiicola and C. hydrangeae were observed in the same lesions, but only C.
cassiicola was isolated in culture. On other samples, lesions were infected
with only C. cassiicola or C. hydrangeae. Single spore isolation from leaf
lesions resulted in pure cultures of both C. cassiicola and C. hydrangeae.
These fungi did not sporulate well when cultured on Potato Dextrose Agar.
Morphological characteristics and DNA sequence analysis were used to
identify the pathogens and specific primers for C. cassiicola and C.
hydrangeae were designed to identify the two pathogens on infected leaves.
Differences in the virulence and aggressiveness of C. cassiicola and C.
hydrangeae on 77 hydrangea cultivars showed that C. cassiicola is a highly
S84
PHYTOPATHOLOGY
aggressive pathogen and demonstrated the role of each pathogen in the leaf
spot/blight disease complex.
Leaf spot disease in ornamental flowering cherry nursery production
M. T. MMBAGA (1), L. Mackasmiel (2), J. Joshua (3)
(1) Tennessee State University School of Agriculture and Consumer Sciences,
McMinnville, TN, U.S.A.; (2) Tennessee State University, McMinnville, TN,
U.S.A.; (3) Tennessee State University, Nashville, TN, U.S.A.
Phytopathology 100:S84
Ornamental flowering cherry trees are grown primarily for their floral display
in early spring, but they are closely related to fruit cherry trees that are grown
for their fruits. A leaf spot disease emerged recently in a Tennessee nursery
causing great concern to growers. The disease is characterized by small redbrown spots and shot holes that often merge to form large irregular lesions
and shot holes. Fungicide applications were initiated soon after disease
symptoms were observed, but the grower had no guidance on effective
fungicides or the timing of spray program and disease control failed. The
disease caused severe defoliation; by June, only a few leaves remained on
infected trees. Persistent wet weather throughout 2009 favored disease
development and plant vigor was significantly reduced. Infected trees had
severe winter injury and reduced market value. Disease symptoms were
consistent with that of cherry leaf spot, a well known problem in stone fruit
trees. Source of infection was traced back to the liners and cuttings. Although
some cultivars appeared to be resistant to the disease, the resistance has not
been confirmed. The integration of copper based fungicide (Captan), with a
Demethylation inhibitor (Tebuconazole) and a QoI fungicide (Trifloxystrobin)
have been effective in fruit trees and were evaluated on ornamental cherry
following a spray program based on local weather.
A chemo-thermotherapy technique for eliminating viruses from rose
plants
A. MODARRESI CHAHARDEHI (1), J. Mozafari (2), F. Rakhshandehroo
(3), D. Ibrahim (1)
(1) School of Biological Sciences, Universiti Sains Malaysia (USM), Penang,
MALAYSIA; (2) Department of Genetics and National Plant Gene-Bank,
Seed and Plant Improvement Institute, Karaj, IRAN; (3) Department of Plant
Pathology, Islamic Azad University, Science and Research Branch, Tehran,
IRAN
Phytopathology 100:S84
Arabis mosaic virus (ArMV) and Prunus necrotic ringspot virus (PNRSV) are
among the most commonly found viral agents on roses. Conventional method
of thermotherapy is not an efficient technique in regenerating virus-free
plants. Using a combination of thermotherapy, chemotherapy and in vitro
culture we have developed a procedure for eliminating ArMV and PNRSV
from rose plants. Viral infected rose stem nodal segments were surface
sterilized and cultured on MS media containing 0.4 mg/l NAA, 0.4 mg/l BAP
(pH 5.8) and 10, 20 or 30 mg/ml of ribavirin. After 20 to 40 days, regenerated
segments were excised and transferred to new media and were tested for virus
titer by enzyme linked immune-sorbent assay (ELISA). In addition to
ribavirin treatment plants were also subjected to combination of ribavirin and
thermotherapy treatments where ribavirin treated plants after 30 days were
exposed to the temperature of 38°C for 16 hours and 22°C for 8 hours. Results
of chemotherapy showed that complex of PNRSV and ArMV was efficiently
eradicated from plantlets that were grown on MS medium containing 10 and
30 mg/l for 40 and 20 days respectively. The virus elimination rates of 63.33
and 85.18% were obtained for ArMV and ArMV+PNRSV, respectively.
However, thermotherapy along with chemotherapy (containing 30 mg/l)
during the period of four weeks was the most effective treatment for plantlet
regeneration and virus elimination. Explant cultures with ribavirin appears to
be a simple and effective way for obtaining virus-free roses in vitro.
Effect of resistant catch crops on population decline of sugar beet cyst
nematode
A. Mohammad Deimi (1), S. BAROUTI (1)
(1) Dept. of Plant Pathology, Faculty of Agriculture and Natural Resources,
Islamic Azad University, Tehran, IRAN
Phytopathology 100:S84
The use of resistant catch crops is an important element of crop rotation in
integrated nematode management. Therefore, during the 2009, experiments
were undertaken to determine the effects of some cultivars of oil radish
(Raphanus sativus), white Mustard (Sinapis alba), Buckwheat (Fagopyrum
esculentum) and Phacelia tanacetifolia on population decline of sugar beet
cyst nematode (sben). Plants were grown after the harvest of winterwheat and
were collected 130,100 and 78 days later respectively. Ratio between final
populations (pf) to initial population (pi) of the nematode was 60% for S. alba
Cv. Maxi and R. sativus Cv’s Nemex and pegletta and the difference was
significant at the 1% level comparing to the untreated controls.
Characterization of plant cell wall-degrading enzymes produced by
Pantoea stewartii subsp. stewartii, the causal agent of Stewart’s wilt of corn
M. MOHAMMADI (1), L. Burbank (1), C. M. Roper (1)
(1) University of California, Riverside, CA, U.S.A.
Phytopathology 100:S85
Mojtaba Mohammadi, Lindsey Burbank and Caroline Roper Pantoea stewartii
subsp. stewartii (Pnss), a xylem-dwelling bacterium, is the causal agent of
Stewart’s wilt and blight of sweet corn and maize. The goal of this study is to
investigate if Pnss produces extracellular enzymes and to determine their
contribution to virulence on corn. Carboxymethyl cellulase (CMCase), -1,4endoglucanase, -1,4-xylanase and mixed-linkage 1,3-1,4 glucanase activities
were detected and measured in sonicated cells and culture supernatants of
Pnss (DC283). Maximum specific activities occurred during the mid-log and
early stationary phases, suggesting these enzymes are produced in a celldensity dependent manner. Electrophoresis of non-denaturing polyacrylamide
gels impregnated with either -D-glucan or carboxymethyl cellulose (CMC)
revealed the presence of two isoforms of CMCase and -1,4-D-endoglucanase
whose molecular weights were in the range of 55–60 kDa. Furthermore,
HPLC analysis of the reducing sugars released during the incubation of Pnss
(DC283) culture supernants with either CMC or -1,4 glucan revealed the
presence of several oligosaccharide hydrolysis products indicative of
enzymatic degradation. Additionally, work is currently under way to
characterize mutants of Pnss DC283 lacking the -1,4-endoglucanase and
xylanase genes to elucidate their role in virulence on corn seedlings.
Effect of VAM colonization in pistachio rootstock on growth, nutrition
and Phytophthora root rot
A. Mohammadi (1), Z. BANIHASHEMI (2)
(1) Iran Pistachio Research Center, Rafsenjan, IRAN; (2) Shiraz University,
Shiraz, IRAN
Phytopathology 100:S85
Phytophthora root and crown rot caused by Phytophthora spp. is one of the
major limiting factors in pistschio inflicting economic losses in Iran.
Arbuscular Vesicular Mycorrhizal (VAM) fungi are the most common type of
plant symbiosis in natural and agricultural ecosystems. The objective of the
present experiment was to study the effect of Glomus mosseae colonization on
growth, plant nutrition and root rot caused by P. drechsleri in susceptible
(Sarakhs) and tolerant (Qazvini and Atlantica) pistachio rootstocks under
greenhouse conditions. G. mosseae was inoculated to pistachio rootstocks
prior to pathogen inoculation. In the absence of P. drechsleri, mycorrhized
pistachio seedlings had higher shoot and root dry weight, height and
concentration of P, K, Ca, Mg, Fe, Cu, Zn and Mn than non-mycorrhized
plants. Inoculation of P. drechsleri in non-mycorrhized susceptible seedlings
caused in significant reduction of the above mentioned parameters compared
to none inoculated control, but low or no reduction was observed in tolerant
rootstocks. Non-mycorrhized susceptible cultivar died 40 days after
inoculation but the mycorrhized one delayed and decreased their mortality. It
is concluded that mycorrhizal colonization in pistachio seedlings increased
growth, nutrition and decreased mortality by P. drechsleri than nonmycorrhizal one especially in susceptible rootstocks.
Activity of hydrolytic enzymes and antioxidants in mycorrhized pistachio
root infected by Phytophthora drechsleri
A. Mohammadi (1), Z. BANIHASHEMI (2)
(1) Iran Pistachio Research Center, Rafsenjan, IRAN; (2) Shiraz University,
Shiraz, IRAN
Phytopathology 100:S85
The role of hydrolytic enzymes (chitinase, chitosanase, -1,3-glucanase),
antioxidants (GPX, CAT, SOD) and PAL was evaluated in Glomus mosseae
(Gm) colonized susceptible (cv. Sarakhs) and tolerant (cvs. Qazvini and
Atlantica) pistachio rootstocks in the presence of Phytophthora drechsleri
(Pd). Increasing activity of hydrolytic enzymes coincided with the increase of
Gm colonization. After establishment of mycorrhiza, the enzymatic activity
was decreased but was higher than the non-mycorrhizal controls. In
mycorrhized seedlings, PAL, GPX and CAT activity increased at the first
stages of growth and establishment of Gm and then decreased but SOD
activity was unchanged or in some cases had increasing trend. Inoculation of
Pd, resulted in higher activity of antioxidants and PAL which began earlier
and higher in tolerant than in susceptible rootstocks. In Gm+Pd treatments,
PAL and antioxidants activity was unchanged or in some cases increased
slightly and then decreased but hydrolytic enzymes activity increased in all
rootstocks. In tolerant cultivars, higher and earlier activity of the enzymes was
observed than susceptible one. Reduction of Pd population in the
mycorrhizosphere resulted in the reduction of hydrolytic enzyme activity.
These results indicated that inoculation of Gm in pistachio rootstocks prior to
Pd can activate defense related enzymes and protect pistachio seedlings
against root rot especially in susceptible rootstocks.
Effect of pH on the growth of Rhizoctonia spp. from cereal-based
cropping systems in eastern Washington State
A. MOHD JAAFFAR (1), D. M. Weller (2), T. C. Paulitz (2), L. S.
Thomashow (2)
(1) Washington State University, Pullman, WA, U.S.A.; (2) USDAAgricultural Research Service, Pullman, WA, U.S.A.
Phytopathology 100:S85
Rhizoctonia root rot caused by Rhizoctonia solani AG 8 and Rhizoctonia
oryzae are serious root diseases in dryland cereal production in Washington
State. Isolates of Rhizoctonia spp. from fields with different cropping histories
in the low- (<12 inches) and moderate- (>12 inches) precipitation zones of
eastern Washington State were identified by using six PCR primer sets
specific for AG 8, AG 2-1, AG 10, AG I (Ceratobasidium spp.) and 2 groups
of R. oryzae. Of 205 isolates, AG 8, AG I, AG 2-1, R. oryzae group 2, R.
oryzae group 3, and AG 10 comprised 28%, 19%, 17%, 15%, 13%, and 8% of
the isolates, respectively. AG 8 comprised 67% and 33% of the isolates in the
low and moderate precipitation zones. The soil pH in fields in eastern
Washington varies considerably (<pH 4 to >pH 7.5) because of decades of use
of ammonium fertilizer, which lowers soil pH. Rhizoctonia isolates were
tested for growth on 1/5-strength PDA adjusted to a range of pH values from
5.6 to 7.6. AG groups differed significantly in growth at the various pH values
and in general, growth was significantly reduced above pH 6.8. These results
suggest that the composition of Rhizoctonia isolates in a field may be
determined in part by the soil pH and precipitation.
Identifying heterokaryon incompatibility loci in Aspergillus flavus and
Aspergillus parasiticus using array-Comparative Genome Hybridization
(aCGH)
J. T. MONACELL (1), B. W. Horn (2), R. Singh (3), E. A. Stone (4), I.
Carbone (3)
(1) Department of Plant Pathology and Bioinformatics Research Center, North
Carolina State University, Raleigh, NC, U.S.A.; (2) National Peanut Research
Laboratory, USDA, ARS, Dawson, GA, U.S.A.; (3) Department of Plant
Pathology, North Carolina State University, Raleigh, NC, U.S.A.; (4)
Department of Genetics and Bioinformatics Research Center, North Carolina
State University, Raleigh, NC, U.S.A.
Phytopathology 100:S85
Heterokaryon incompatibility is the inability of two strains to undergo fusion
of vegetative fungal cells. This vegetative compatibility system is dictated by
a series of heterokaryon incompatibility (het) loci whose alleles must all be
identical for stable hyphal fusions to occur. Het loci have been identified in
several filamentous fungi, but are currently unknown in Aspergillus flavus
and A. parasiticus. These species are agriculturally important plant pathogens
that produce the potent carcinogens, aflatoxins. Fungal individuals can be
grouped into vegetative compatibility groups (VCGs) based on their ability to
undergo hyphal fusions and potentially form heterokaryons. We performed
aCGH for eleven VCGs and a total of 51 strains in Aspergillus section Flavi,
including A. flavus, A. parasiticus, A. oryzae, A. caelatus, A. tamarii and A.
nomius. We conducted an initial screening of these data for signatures of
balancing selection around single-feature polymorphism markers on
chromosomes 2, 3, 4, and 6, which associated one-to-one with VCG. Our
screening for evidence of balancing selection has revealed several putative het
loci showing distinct patterns of trans-speciation, which is typical of other loci
under balancing selection in these fungi, such as mating-type genes and the
aflatoxin gene cluster. Among the candidate het loci we identified using
aCGH was a previously annotated putative het locus on chromosome 2.
Cultivar susceptibility influences the leaf wetness duration (LWD) and
temperature relationship of Alternaria alternata citrus infection
S. N. Mondal (1), L. P. Timmer (1), M. M. DEWDNEY (1)
(1) CREC, University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S85
Alternaria brown spot (ABS), caused by A. alternata, is an important fresh
market disease of tangerines and tangerine hybrids. The Alter-Rater, a
predictive model for better timing fungicide applications, was developed with
the very susceptible Minneola but it was unknown whether the LWDs and
temperatures needed for infection would be similar for all tangerine hybrids.
Leaf wetness duration and temperature relationships were tested on 5
tangerine hybrids: Dancy, Minneola, Murcott, Nova and Sunburst. The LWDs
were 2, 4, 8, 16, 24 and 30 h at temperatures of 20, 24, 28 and 32°C. The
rating scale for number of lesions/leaf was: 0=0; 1=1-2; 2=3-5; 3=6-10; 4=1115; 5=>15 lesions/leaf and the data were taken from 15 leaves/plant. Cultivar
differences were observed with two susceptibility groups: highly susceptible,
Minneola and Dancy, and moderately susceptible Murcott, Nova and Sunburst
(P < 0.0001). As few as 2 h of LWD were needed for light infection but 16-24
h, severe infection occurred on all cultivars and temperatures. The optimal
temperature range for lesion production was 24 and 28°C for all LWDs and
the quadratic effect of temperature was highly significant (P < 0.0001). The
Vol. 100, No. 6 (Supplement), 2010
S85
interactions of temperature and cultivar as well as temperature and LWD were
highly significant (P < 0.0001) but LWD and cultivar was not. Results will be
incorporated into the Alter-Rater model so that less susceptible tangerine and
tangerine hybrids are not sprayed unnecessarily.
Decay incidence was not significantly reduced on pomegranates treated with
the mixture 30 kPa CO2 + 70 kPa CO2. On pomegranates stored at 5°C, both
gaseous shocks reduced gray mold incidence by 80 and 40% after 12 and 26
days of storage, respectively, but the treatments lacked persistence.
Evaluation of transgenic grapefruit (Citrus paradisi) for resistance to
citrus scab caused by Elsinoë fawcetti
S. N. Mondal (1), M. Dutt (1), G. A. Barthe (1), J. W. Grosser (1), M. M.
DEWDNEY (1)
(1) CREC, University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S86
Evaluation of nematicides for the management of Rotylenchulus
reniformis across management zones created using soil electrical
conductivity
S. R. MOORE (1), K. S. Lawrence (1), B. V. Ortiz (1), J. N. Shaw (1), J.
Fulton (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S86
Citrus scab, E. fawcettii (anamorph Sphaceloma fawcettii), is a common foliar
fungal disease, occurring in most humid citrus growing areas of the world.
The scab pathogen infects juvenile leaves, twigs and fruit of most citrus
species, including grapefruit, and can result in severely blemished fresh fruit.
Since no grapefruit cultivar is resistant to scab, transgenic plants offer
potential resistance to this important disease. In the present study, several
transgenic grapefruit lines containing a synthetic cecropin A-melittin chimeric
lytic peptide gene (LIMA) under control of a constitutive d35S promoter were
evaluated for resistance to E. fawcettii. Detached leaf assay results with the
Russel-15 isolate (Florida broad host range pathotype) indicated that several
lines produced significantly fewer raised pustules when compared to the
control (P < 0.05). Similar results were recorded in greenhouse assays where 6
transgenic lines produced significantly less scab compared to control (P <
0.05). In sporulation assays, repeated measures ANOVA revealed significant
variation in the effect of transgenic lines on spore production over time. All
transgenic lines produced significantly fewer conidia in all blocks. Pustules of
untransformed grapefruit leaves increased conidia production unlike most
transgenic lines evaluated. Northern blots indicated varying levels of gene
expression while PCR and Southern blots confirmed stable integration of the
gene in the plant genome.
Identification and characterization of genes involved in the type VI
secretion system in Xanthomonas axonopodis pv. manihotis
N. MONTENEGRO (1), A. Alvarez (1), S. Restrepo (1), A. J. Bernal (1)
(1) Universidad de Los Andes, Bogotá, COLOMBIA
Phytopathology 100:S86
The type VI protein secretion system (T6SS) in gram-negative bacteria has
been shown to participate in several cellular processes, including
pathogenesis. During pathogenesis of Pseudomonas aeruginosa and Vibrio
cholerae, this system secretes effector proteins that interact with target
proteins in the host cell, thus contributing to the infection process. The system
has been studied in animal pathogens and some substrates have been
identified with roles in a diverse set of processes. However, there is a paucity
of data for the importance of this system in plant pathogenic bacteria-host
interactions. In this study, we have identified the presence of the T6SS in
Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of bacterial
blight in cassava (Manihot esculenta). Bioinformatics analysis of nine
structural genes (e.g. clpB, vgrG, ompA, hcp and fha) and six candidate genes,
confirmed the existence of a full T6SS in Xam. We subsequently used sitedirected mutagenesis of some of the structural genes involved in the T6SS.
These mutants were inoculated in susceptible plants and the results suggest an
importance of this secretion system in the pathogenicity of Xam. This study
shows the importance of the novel T6SS in the virulence of Xanthomonas and
it is considerably relevant for the understanding of plant- pathogen interactions.
Brief gaseous shocks to inhibit postharvest gray mold on ‘Mollar de
Elche’ pomegranates
C. Montesinos-Herrero (1), V. Taberner (1), M. del Río (1), A. Guardado (1),
L. PALOU (1)
(1) IVIA, Postharvest Technology Center, Montcada, València, SPAIN
Phytopathology 100:S86
Spain is the first European Union producer and exporter of pomegranates
(Punica granatum L.) and ‘Mollar de Elche’ is the most important cultivar.
Studies show remarkable benefits from pomegranate consumption on human
health mainly due to the high antioxidant activity of this fruit. As new markets
based on the manufacture of derived functional food products are arising,
longer storage life of fresh pomegranates is demanded. Decay due to gray
mold, caused by Botrytis cinerea, is one of the most important factors limiting
storability of pomegranates. In Spain, no postharvest chemical treatments are
permitted and alternative antifungal methods are required. In this work,
pomegranates cv. ‘Mollar de Elche’ were artificially inoculated with B.
cinerea and exposed 24 h later to air (control), 95 kPa CO2, or 30 kPa O2 + 70
kPa CO2 at 20°C and 90% RH for 48 h, and subsequently stored at either
20°C for 12 days or 5°C and 90% RH for 2.5 months. On pomegranates
incubated at 20°C, exposure to 95 kPa CO2 reduced gray mold incidence by
97 and 63% compared to control fruit after 2 and 5 days, respectively.
However, this reduction gradually decreased along the incubation period.
S86
PHYTOPATHOLOGY
Management zones delineated by soil electrical conductivity and nematode
populations are currently being examined as an option for control of
Rotylenchulus reniformis on cotton. A trial to evaluate differences in the
effectiveness of selected nematicides within each zone was established in a 26
hectare field in south Alabama in 2009. The field was delineated into low,
medium, and high risk zones, with soils ranging from sandy loam to silt clay
and initial populations of R. reniformis averaging 134, 746, and 1,775 per 150
cm3 of soil, respectively. The nematicide treatments of Telone II (1, 3dichloropropene) 28 L/ha and Temik 15G (aldicarb) 3.9 kg/ha increased (P <
0.1) the total number of bolls per plant in the high risk zone compared to the
untreated control. All nematicides increased the average weight per boll in the
high risk zone by an average of 1.75 g, with the treatments of Avicta
Complete Pak + Vydate CL-V 1.2 L/ha and Avicta Complete Pak +Temik
15G 3.9 kg/ha + Vydate CL-V 1.2 L/ha significantly (P < 0.1) increasing
average weight per boll. Within the medium risk zone cotton treated with
nematicides produced a comparable number of bolls per plant and average
weight per boll compared to the untreated control. Nematicides in the low risk
zone also produced a total boll number comparable to the untreated control,
however average weight per boll was increased (P < 0.1) by Telone II 14 L/ha
over the untreated control.
Histopathology of Colletotrichum acutatum on citrus leaves
S. G. MORAES (1), N. A. Peres (2), N. S. Massola (3)
(1) University of Sao Paulo, Piracicaba, BRAZIL; (2) University of Florida,
Wimauma, FL, U.S.A.; (3) University of Sao Paulo / ESALQ, Piracicaba,
BRAZIL
Phytopathology 100:S86
Colletotrichum acutatum is the causal agent of two citrus diseases, postbloom
fruit drop (PFD) and Key lime anthracnose (KLA). The infection process of
C. acutatum was studied using scanning electron microscopy (SEM).
Detached leaves of sweet orange and Key lime were inoculated with
suspension of 105 conidia/mL in a previously marked area. Isolates FSHCLB-2 and KLA-CRD-CV-1, collected respectively from sweet orange
flowers and Key lime anthracnose-affected leaves, were used. Inoculated
leaves were incubated in humid chambers at 25°C during continuous wetness
periods of 24, 48, 72, 96 and 120 h. Afterward, inoculated tissues were
removed and processed for the visualization by SEM. Secondary conidia of
both isolates were observed 24 h after inoculation on sweet orange and Key
lime leaves. These conidia originated from hyphae or primary conidia.
Acervuli were observed 72 hours after inoculation (h.a.i) on sweet orange
leaves inoculated with the FSH-CLB-2 and KLA-CRD-CV-1 isolates. On Key
lime leaves, acervuli were observed 96 h.a.i when inoculated with the KLACRD-CV-1 isolate.
Infected fruit as source of inoculum and infection dynamic on olive
anthracnose caused by Colletotrichum acutatum
J. Moral (1), M. VICENTE (1), A. Trapero-Casas (1)
(1) Universidad de Cordoba, Cordoba, SPAIN
Phytopathology 100:S86
Anthracnose of olive (Olea europaea) caused by Colletotrichum species is the
most serious fruit disease of this crop. Mummified fruit are the main inoculum
source for olive anthracnose. However, effect of weather conditions on
inoculum production by mummified fruit has not been studied. Mummified
fruit were independently incubated at several temperatures (from 5 to 35°C)
for 72 h and various wetness periods (from 0 to 168 h) at 22°C. Conidial
production reached the maximum at 20°C. Conidial production increased
linearly with wetness period from 0 to 96 h, although there was an important
reduction at 168 h. When mummified fruit were subjected to successive
incubation/washing treatments, conidial production decreased exponentially
with the number of treatments. On rotted and non-mummified fruit, the
pathogen produced significantly more conidia on the susceptible cvs.
Hojiblanca (4.7 × 105 conidia/mm2) and Picudo (4.2 × 105 conidia/mm2) than
on resistant cv. Picual (2.6 × 105 conidia/mm2). Under field conditions,
mummies placed on the olive canopy produced a high number of conidia
during all year with peaks occurring in early fall. However, conidial
production was greatly reduced when mummies were placed on the soil
surface or buried. Disease progress on infected fruit over time was faster for
the susceptible cvs. Hojiblanca and Picudo than for the resistant cv. Picual.
Besides, there was a positive correlation between rainfall and disease severity
for all cultivars during the three years of study.
Alteration of cytokinin biosynthesis by Ustilago maydis: Impacts on
pathogenesis
E. N. MORRISON (1), R. Emery (1), B. J. Saville (1)
(1) Trent University, Peterborough, ON, CANADA
Phytopathology 100:S87
Late blight resistance assessing of a segregating population of diploid
potatoes (Solanum phureja)
J. G. MORALES (1), B. Franco (2), C. E. Ñústez (3), J. M. Cotes (2)
(1) Universidad Nacional de Colombia sede Medellín, Depto. Ciencias
Agronómicas, Medellin, COLOMBIA; (2) Universidad Nacional de
Colombia, Medellín, COLOMBIA; (3) Universidad Nacional de Colombia,
Bogotá D.C., COLOMBIA
Phytopathology 100:S87
Common smut of corn, caused by Ustilago maydis, is characterized by the
appearance of tumors that form on the aerial portions of the plant. Early
studies identified increased cytokinin [CK]-like activity associated with these
tumors. However, the role of U. maydis CK biosynthesis during infection has
not been thoroughly investigated. We created solopathogenic strains (SG200)
of U. maydis in which the sole tRNA-isopentenyltransferase (tRNA-IPT gene)
has been deleted. The first and rate-limiting step in cytokinin biosynthesis in
plants is catalyzed by isopentenyltransferases (IPTs). In fungi, related IPTs are
usually tRNA-isopentenyltransferases (tRNA-IPTs). We determined, by
liquid-chromatography-electrospray ionization-tandem mass spectrometry,
LC- (ESI) MS/MS, that none of the major CKs produced in wild type cultures
are detectable in the tRNA-IPT deletion mutant strains. These strains have
different disease development profiles than wild type strains. Further
investigation of the roles of CK production by U. maydis in disease
development was investigated through deletion of the tRNA-IPT gene in
compatible haploids and over expression of the tRNA-IPT gene in
solopathogens and compatible haploids. A comparison of CK production and
pathogenic development by these strains will be presented.
Potato late blight caused by the oomycete Phytophthora infestans is the most
important disease of this crop. Several chemical applications are required
every crop season to achieve effective disease control. Plant resistance is the
most effective strategy for disease control. Solanum phureja is a cultivated
diploid potato from South America and an important source of late blight
resistance. At Universidad Nacional de Colombia a breeding programme has
been established looking for resistant potato varieties. 500 clones from a
diploid S. phureja segregant population obtained from a cross between one
resistant and one susceptible genotype were tested for late blight resistance
using the Area Under The Disease Progress Curve (AUDPC) relative to the
susceptible control method. The experiment was performed during two
different time periods. 22 genotypes showed equal or higher values than the
susceptible control indicating high disease pressure during the evaluation time
period. 31 genotypes scored a value of 0 indicating immunity. Intermediate
values ranging from 0.6 to 7.3 were found for the remaining genotypes. These
results suggest that several genotypes within the S. phureja collection are
important sources for late blight resistance and may be used for potato
breeding against this important disease.
Isolation and characterization of P. chlamydospora from grapevines in
Mexico
L. G. Morales-Pedraza (1), R. HERNANDEZ-MARTINEZ (1), C.
Valenzuela-Solano (2)
(1) Microbiology Department, CICESE, Carretera Ensenada-Tijuana No. 3918
Zona Playitas, Ensenada, Baja California, MEXICO; (2) INIFAP, Campo
Experimental Costa de Ensenada, Ensenada, Baja California, MEXICO
Phytopathology 100:S87
Grapevine is the most important fruit crop in Ensenada, Baja California,
Mexico. Over the last few years an increasing number of vineyards showing
decline, vascular discoloration and dead of spurs, arms, and cordons had been
found in this region. In a survey conducted from 2007–2009, six isolates of
Phaeomoniella chlamydospora were obtained from young grapevines
showing Petri disease symptoms and ten from old vines showing esca in five
different localities. The infected cultivars were Cabernet Sauvignon, Merlot
and Mission. Trunks and cordons from these grapevines showed, in
lengthwise-section brown wood streaking, and in cross-section black spots in
a continuous ring around the central pith. The pathogen was also isolated from
the graft union of some plants. Isolates were identified based on a previous
morphological description charts and by internal transcribed spacer (ITS15.8S-ITS2) rDNA sequences. To our knowledge, this is the first report of P.
chlamydospora on grapevine in Mexico.
Spatial analysis of lethal chlorosis cucurbits caused by Zucchini lethal
chlorosis virus (ZLCV)
A. S. Moreira (1), C. R. Costa (1), J. C. Barbosa (1), A. B. FILHO (1), J. A.
Rezende (1), J. S. Giampan (2)
(1) Escola Superior de Agricultura “Luiz de Queiroz”/Universidade de São
Paulo, Piracicaba, BRAZIL; (2) Instituto Agrônomico do Paraná, Londrina,
BRAZIL
Phytopathology 100:S87
The lethal chlorosis of cucurbits, caused by Zucchini lethal chlorosis virus
(ZLCV), has been an important viral disease that can occur in high incidence
and can cause severe yield damages specially on zucchini squash crops. It is
transmitted by the thrips Frankliniella zucchini. Four experiments were
conducted between December 2006 and January 2009 in order to study the
spatial distribution of the disease. Each experiment was conducted by using
300 plants and which were evaluated every 3 days. Disease incidence was
observed by the presence of symptoms and later confirmed by serological test
PTA-ELISA. Forty one assessments were carried out over the 4 trials. The
parameters of Taylor’s power law suggested random distribution of the
disease and the values of dispersion index were significantly equal to 1.0. This
result indicates the predominance of primary infection on the field, and
probably the pathogen migrated from alternative host plants carried by
viruliferous thrips.
Distribution and prevalence of strains of Potato virus Y (PVY) in North
Western Iran as determined by RT-PCR
L. MOUSAVI (1), J. Mozafari (1), F. Rakhshandehroo (2), S. Ghadamyari
(3), N. Sokhandan (3)
(1) Department of Genetics and National Plant Gene-Bank, Seed and Plant
Improvement Institute, Karaj, IRAN; (2) Department of Plant Pathology,
Islamic Azad University, Science and Research Branch, Tehran, IRAN; (3)
Department of Plant Pathology, Tabriz University, Tabriz, IRAN
Phytopathology 100:S87
In order to survey distribution and prevalence of strains of Potato virus Y
(PVY) in North Western Iran 381 symptomatic infected samples were
collected from potato fields of this main potato growing region during the
years 2007 and 2008. Collected samples were first tested for PVY infection
using a double antibody sandwich enzyme linked immune-sorbent assay
(DAS-ELISA) technique. Seventy nine samples (20.73% of collected
samples) were tested positive for PVY infection. The highest level of infection
was observed In Gilak-Abad district of Sarab County, while the lowest
infection of the virus was observed in Oughan district in suburb of Sarab City.
RT-PCR detection of PVY strains using specific primers resulted in
amplification of DNA fragments of 725bp, 1553bp, 352bp, and 616bp specific
to PVY strains NTN, C, O, and N, respectively. The highest strain diversity in
PVY was detected in Shirehjin district of Sarab County and the lowest in
Ghaleh Jugh district of Bostan-Abad County. Both infection types of single
and multiple infections of PVY stains were observed in the region. Out of 79
PVY infected samples 77.21%, were infected with strain O, 62.02% strain C,
39.24% strain N and 8.86% with strain NTN. The highest level of multiple
infections was observed for combination of strains C+O (27.84%) and the
combination of triple strains O+N+C (15.18%). This is the first report on the
detection of the PVY strain NTN in North Western Iran.
Integrated management of Fusarium crown rot of wheat using fungicide
seed treatment, cultivar resistance, and induction of systemic acquired
resistance
E. A. MOYA (1), A. T. Dyer (1), B. J. Jacobsen (1)
(1) Montana State University, Bozeman, MT, U.S.A.
Phytopathology 100:S87
Fusarium crown rot (FCR) of wheat (Triticum aestivum L.) is a perennial
problem for wheat producer worldwide. In glasshouse trials, difenoconazole
(0.65 g/100 g seed) (Dividend-Syngenta) fungicide seed treatment reduced
FCR severity by 29.3% compared to the control on cultivar Hank. The cultivar
Volt had the highest innate activity levels of three pathogenesis-related (PR)
proteins in apoplastic fluids among five non-inoculated spring wheat cultivars
and the lowest disease severity (P < 0.05). Induction of systemic acquired
resistance (SAR) with foliar applications of Bacillus mycoides isolate BmJ
(1.5 × 108 cfu/ml) or acibenzolar-S-methyl (1.0mM) (ASM [ActigardSyngenta]) on the cultivars Hank, Knudson and Volt reduced FCR severity by
10% compared to a control (P < 0.05). BmJ application increased
concentrations of peroxidase and chitinase, while ASM increased -1, 3glucanases levels in Volt and Hank compared to water controls (P < 0.05).
Integration of the management tools: difenoconazole seed treatment, cultivar
resistance, and SAR induction, showed integration of all of them did not
reduce disease severity more than use of cultivar resistance plus fungicide
seed treatment or SAR induction in greenhouse trials. In a dryland field trial,
integration of all three management tools reduced disease severity and FCR
Vol. 100, No. 6 (Supplement), 2010
S87
populations more than individual tools (P < 0.05), while in an irrigated field
trial SAR induction with BmJ provided similar control to difenoconazole.
Seasonal ascospore release by Erysiphe necator and impact upon epidemic
severity of grape powdery mildew
M. M. MOYER (1), D. M. Gadoury (1), W. F. Wilcox (1), R. C. Seem (1)
(1) Cornell University NYSAES, Geneva, NY, U.S.A.
Phytopathology 100:S88
Differences in quantity of ascosporic inoculum and in-season weather can
cause substantial year-to-year variation in severity of epidemics of grape
powdery mildew (Erysiphe necator). Studies of established disease foci in
2008 and 2009 demonstrated that when focal intensity was below a threshold
level, severity of fruit infection was directly proportional to time of
establishment. However, when focal intensity is high, even late establishment
led to severe fruit disease. To assess variable availability of ascosporic
inoculum, cleistothecia from NY, NJ, WA, NC, GA, and VA were collected
and overwintered at collection sites or in NY in 2006–2010. In lab assays
from January to June, ascospores were released as early as February, and
between 51% and 98% of the season total ascospores were released before
grapevine budbreak, indicating possible release if conducive events (rain > 2.5
mm coincident with > 10C) occurred in the field. Locally, from 3 to 10
conducive events were recorded in 2000–2009 prior to budbreak. Subsequent
analysis showed that the date of inoculum depletion was related to spring
degree day accumulation, i.e., warmer temperatures resulted in earlier
depletion dates. Model development for estimating inoculum load and results
of volumetric spore trap studies will be discussed.
Surveys for Tomato ringspot virus in central Maryland vineyards
W. MSIKITA (1), K. A. Rice (1)
(1) Maryland Dept. of Agriculture, Annapolis, MD, U.S.A.
Phytopathology 100:S88
A study was initiated to confirm the presence of Tomato ringspot virus
(ToRSV) in central Maryland vineyards, and to test the sensitivity of a
commercially available enzyme-linked immunosorbent assay (ELISA) kit to
detect the virus. Initial surveys covered 9 commercial vineyards in four
counties (Baltimore County, Carroll, Frederick, and Montgomery). In the
field, grape plants were visually inspected for characteristic symptoms of
ringspost, leaf mottling, stunting, reduction in fruit size, abortion of the
berries, and death. Suspected plants were sampled and tested in ELISA. Based
on ELISA test analyses, a commercial vineyard in Carroll county was selected
for intensive diagnostic surveys that included quantification of Xiphinema spp.
nematodes in soil samples, and detection of the virus by ELISA and the
reverse-transcription polymerase chain reaction (RT-PCR) in nematodes,
grape and dandelion hosts. ELISA consistently was unable to detect the virus
in 76 grape samples from the 9 vineyards, but detected the virus in dandelion
and nematode samples from a Carroll county vineyard. RT-PCR was able to
detect the virus in all three types of samples. Of the 967 nematodes extracted
from a composite soil sample, 278 (28%) were Xiphinema spp., and of the 51
dandelion leaf samples, 3 (6%), tested positive to the virus. The sensitivity of
ELISA to detect ToRSV in grapes is discussed with a view to raise awareness
of this important detection tool in ToRSV disease certification.
Defense related enzymes and gene expression after resistance induction
by rhizobacteria and silicon against Ralstonia solanacearum in tomato
H. K. MULAT (1)
(1) Gottfried Wilhelm Leibniz University of Hannover, Hannover, GERMANY
Phytopathology 100:S88
Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive
diseases in tomato production. Silicon and rhizobacteria were tested in single
and simultaneous application to elicit active defense responses in tomato
against this pathogen. Individual application of silicon and rhizobacteria
significantly reduced bacterial wilt incidence by 50.7 and 26.8% in tomato
genotypes KK2 (moderately resistant) and L390 (susceptible) (silicon
amendment), and by 31.1, and 22.2%, respectively, (rhizobacteria
application). The elicitors also reduced bacterial populations in the mid-stem
of tomato but not in simultaneous application of the two elicitors. Silicon
amendment significantly increased the silicon content in the roots of both
genotypes but not in the stem, which is typical for silicon non-accumulator
plants. Non-significant increases of peroxidase and phenylalanine ammonia
lyase activity were observed in the individual treatments of silicon and
rhizobacteria upon inoculation with R. solanacearum, while the activity of
lipoxygenase was significantly decreased in the pathogen inoculated silicon
amended, but increased in the rhizobacteria treatment. In simultaneous
application of silicon-rhizobacteria, the activity of the three enzymes was
significantly reduced. To elucidate the molecular mechanisms underlying
silicon-rhizobacteria mediated induced resistance, results of transcriptome
analysis of up and down regulated genes will be presented.
S88
PHYTOPATHOLOGY
Association of ‘Candidatus Liberibacter solanacearum’ with psyllidaffected carrots in Europe
J. E. Munyaneza (1), T. W. Fisher (2), V. SENGODAGOUNDER (3), S. F.
Garczynski (1), A. Nissinen (4), A. Lemmetty (4)
(1) YARL, USDA-ARS, Wapato, WA, U.S.A.; (2) Department of
Entomology, Washington State University, USDA-ARS, Wapato, WA,
U.S.A.; (3) Department of Plant Pathology, Washington State University,
YARL, USDA-ARS, Wapato, WA, U.S.A.; (4) Agrifood Research Finland,
Plant Protection, Jokioinen, FINLAND
Phytopathology 100:S88
Carrot psyllid (Trioza apicalis) is a serious pest of carrots (Daucus carota) in
northern and central Europe. Carrots exhibiting symptoms of psyllid damage
were observed in commercial fields in southern Finland in 2008. Symptoms in
affected plants included leaf curling, yellow and purple discoloration of
leaves, stunted growth of shoots and roots, and proliferation of secondary
roots. Given recent association of liberibacter with several crops affected by
psyllids, an investigation on whether this bacterium is associated with carrots
with psyllid symptoms was conducted. PCR primer pairs OA2/OI2c and
LsoF/OI2c, specific for the 16S rRNA gene from “Candidatus Liberibacter
solanacearum”, generated amplicons of 1,168-bp and 1,173-bp, respectively,
from DNA extracted from field-collected and laboratory-reared psyllids, and
symptomatic carrots. In contrast, no PCR products were detected in DNA
extracted from insect-free plants. The DNA sequences of amplicons of the
genes encoding liberibacter 16S rRNA from psyllids and carrots were
identical. The DNA of the 16S rRNA gene sequences determined from carrots
and psyllids were over 99.9% identical to analogous sequences of “Ca. L.
solanacearum” amplified from several solanaceous crops and the potato
psyllid, vector of this bacterium. This is the first report of “Ca. L.
solanacearum” associated with a non-solanaceous species, and the first report
of this pathogen outside of North and Central America and New Zealand.
Genotyping Xylella fastidiosa strains using multiplex PCR
B. A. MYERS (1), J. A. Brady (2), F. L. Mitchell (2), H. B. Rathburn (1)
(1) Tarleton State University, Stephenville, TX, U.S.A.; (2) Texas AgriLife
Research, Stephenville, TX, U.S.A.
Phytopathology 100:S88
Xylella fastidiosa is a gram negative, xylem-limited, nutritionally fastidious
bacterium that causes leaf scorch diseases such as plum leaf scald (PLS),
oleander leaf scorch (OLS), and Pierce’s disease (PD) of grapevines. Four
different subspecies and numerous strains of the bacterium have been
recognized in North America. OLS is caused by X. fastidiosa subsp. sandyi,
PD strains belong to X. fastidiosa subsp. fastidiosa, and X. fastidiosa subsp.
multiplex causes PLS and a number of tree leaf scorch diseases. The recently
described X. fastidiosa subsp. tashke causes leaf scorch in chitalpa trees.
While each subspecies can occupy a large number of host plants, they cause
disease symptoms in a very small subset of potential hosts. Genotyping the
different bacterial subspecies and strains for epidemiological studies can be
time consuming and expensive using currently available approaches.
Additionally, current approaches lack marker density necessary for a
substantive assessment of recombination. We are developing a multiplex PCR
assay covering hundreds of genetic loci that will distinguish even closely
related X. fastidiosa isolates. The assay has proven to be robust, inexpensive,
and provides a highly informative genetic fingerprint that will facilitate an
understanding of plant/vector/pathogen relationships.
Maize chiA as a potential genetic marker for Stenocarpella maydis ear rot
resistance
T. A. NAUMANN (1)
(1) USDA/ARS/NCAUR, Peoria, IL, U.S.A.
Phytopathology 100:S88
Stenocarpella maydis (Diplodia maydis) is the most prevalent ear rot pathogen
in nearly all countries where maize is produced. The genetic basis of plant
resistance to S. maydis appears to rely on multiple genetic factors, none of
which are known. We previously reported that S. maydis secretes a protein,
Stm-cmp, that modifies maize ChitA, a chitinase that is produced abundantly
during seed development. We also demonstrated that ChitA protein from
inbred B73 is highly susceptible to Stm-cmp modification while ChitA from
inbred LH82 is resistant. These ChitA proteins are encoded by alleles of the
chiA gene that encode proteins with six polymorphisms. Here I report
cDNA cloning of both chiA genes, construction of yeast strains that produce
the ChitAs, purification of yeast-produced ChitAs, and their in vitro
modification by fungal St-cmp. In addition, I created yeast strains that
produce mutant versions of ChitA. By comparing the susceptibility to Stmcmp modification of the mutant ChitA proteins I determined that a single
amino acid, encoded by a chiA single nucleotide polymorphism (SNP) results
in resistance. This SNP may be a useful marker for breeding resistance to S.
maydis ear rot.
Effects of seed treatment on root diseases and yields of soybean
S. S. NAVI (1), X. Yang (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S89
The objective of the study was to evaluate the effects of fungicide seed
treatment on sudden death syndrome (Fusarium virguliforme), Rhizoctonia
root rot (Rhizoctonia solani), and Sclerotinia stem rot (Sclerotinia
sclerotiorum) and their effects on soybean yields. Field tests against F.
virguliforme and R. solani was conducted at Ames, and against S.
sclerotiorum at Nashua, during 2005 to 2009. Products from various
companies and an unregistered bio-fungicide from our lab were tested.
Experiments were laid out in RCBD with four replications. Percent incidence
and severity due to R. solani was recorded at 7, 14, and 21 days after
emergence and for F. virguliforme and S. sclerotiorum at flowering, pod
formation and maturity growth stages. The central four rows of each plot were
harvested and plot yields bushels/acre (adjusted to 13% grain moisture) was
recorded. Some of the products tested were effective in minimizing the
diseases and increased yields from 3 to 9 bushels per acre over untreated
controls.
A predictive model for carpogenic germination of Sclerotinia sclerotiorum
A. NEPAL (1), L. E. del Rio Mendoza (1)
(1) Dept. Plant Pathology, North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S89
A predictive model for carpogenic germination (CG) of S. sclerotiorum was
developed using soil moisture data. Two types of soils from Fargo (Fargo
Silty clay) and Kindred (Aylmer-Bantry fine Sand), ND were mixed in
proportions of 1:0, 2:1, 1:1, 1:2, and 0:1 v/v to create different textures.
Sclerotia were buried in samples from each soil texture set at constant 100%,
75%, 50% or 25% soil saturation; or to conditions fluctuating back and forth
between 100 to 0; 75 to 0; 50 to 0; and 25 to 0% saturation with 25%
saturation intervals. Samples were incubated at 14/18°C day/night for 82 days.
CG was recorded at five-day intervals. CG data were expressed as binary
values using 15 and 20% CG as thresholds. This data was split into two
portions, one was used for model development using logistic regression analysis
and the other was left for model validation. The area under cumulative
moisture curve and rate of moisture accumulation were calculated for every
time interval in all treatments and used, along with percentage of clay and silt,
as predictor variables for the model. The best model had c = 0.96. When the
model was validated using the independent data set, it produced a true
negative proportion of 73% and a true positive proportion of 100% for an
overall accuracy of 85%. Field validation of this model will be discussed.
Brown girdling root rot as a potential threat to canola production in
North Dakota
A. NEPAL (1), L. E. del Rio Mendoza (1)
(1) Dept. Plant Pathology, North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S89
Rhizoctonia solani was observed causing brown girdling root rot of canola
plants in research plots in Langdon, ND in 2008 and 2009. Since R. solani is
known to cause important yield reductions in other canola producing areas of
the world, a study was conducted to assess the potential threat this pathogen
represents to the canola industry in ND. To evaluate the protection provided
by conventional seed treatment, seeds from four commercial canola cultivars
were washed in running water to eliminate the chemicals from the seeds.
Washed and non-washed seeds were planted in replicated trials in greenhouse
soil mix infested or not with canola seeds colonized by an R. solani AG 4
isolate. Pots were maintained at 21°C and 14 hours light daily. Germination
and plant standing were quantified 14 and 20 days after planting, respectively.
The experiment was repeated once. The seed treatment did not increase
germination nor plant standing significantly (α = 0.05). Germination and plant
standing were reduced from 90 to 70% and from 87 to 57%, respectively,
when seeds were planted in R. solani-infested soils. Cultivars IX08-7121R
and Hyclass 712 had significantly less germination (mean of 64%) than
cultivars DKL30-42 and G75449 (mean of 77%). To get a better estimate of
the potential impact of this disease on the canola industry in North Dakota, a
larger study involving 59 commercial cultivars and R. solani isolates from
three AG groups (4, 2-1, and 2-2) is under way.
Effects of temperature and wetness duration on sporangia germination
and infection of cucurbit varieties by Pseudoperonospora cubensis
K. NEUFELD (1), P. Ojiambo (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S89
Cucurbit downy mildew caused by Pseudoperonospora cubensis is considered
the most damaging disease of cucurbitaceous crops worldwide. Recently, we
develop a model to quantify the combined effects of temperature (t) and leaf
wetness duration (w) on sporangia germination and infection by P. cubensis
using cantaloupe. However, the influence of host type on predictive ability of
the model has not been determined. The objective of this study was to
determine the effect of host type on the infection parameters of P. cubensis to
combined effects of t and w. Three cucurbit types (cantaloupe, cucumber and
squash) were inoculated with P. cubensis and exposed to a range of leaf
wetness durations (2–24 h) and fixed temperatures (5–30°C) in growth
chambers. Germination was assessed at the end of each wetness period and
infection was recorded at 5 and 7 days after inoculation. Data were fitted to a
Weibull function of the form f(w,t) = f(t)•(1-exp{-[B × w]D}). Host type, t, w,
and t × w significantly (P < 0.05) affected infection parameters. Optimum
range of temperature for the infection parameters was found to be between
10–25°C. Infection parameters had a minimum leaf wetness duration of 8 h
with broader optimums with increasing wetness. Host types varied in their
response to w at a given t. Host based nomograms were developed to predict
the potential risk of cucurbit downy mildew epidemics based on observed or
forecasted temperature and leaf wetness duration.
Analysis of transgenic American chestnut
A. E. NEWHOUSE (1), A. B. Zhang (1), L. Northern (1), C. A. Maynard (1),
W. A. Powell (1)
(1) SUNY-ESF, Syracuse, NY, U.S.A.
Phytopathology 100:S89
Chestnut blight (caused by Cryphonectria parasitica) is a classic example of
the devastating impact an invasive pathogen can have on a well-established
native tree species. One strategy to combat this disease is to modify the host
by adding one or more new genes to enhance disease resistance. Several lines
of transgenic American chestnut (Castanea dentata) have been produced
through Agrobacterium co-transformation of somatic embryos. Cotransformation allows constructs containing a marker gene and a putative
resistance-enhancing gene to be inserted independently within the genome of
the host. Selection for co-transformed events is both visual (i.e. GFP) and
physiological (antibiotic resistance). PCR confirms the presence of both genes
early in the regeneration process, and once individual transgenic lines are
established in tissue culture, they are multiplied and regenerated into whole
plants. Subsequent analyses (Southern hybridization, quantitative real-time
PCR, or both) have determined transgene copy number for all established
lines. Of five initial lines, three had a single copy of the putative resistanceenhancing gene, one had two copies, and one had three copies. Transgene
expression has been analyzed with a colorimetric enzyme assay (oxalate
oxidase) and with real-time PCR. Greenhouse and laboratory tests are also
underway to screen immature trees for disease resistance based on
inoculations with C. parasitica.
Causal organisms of black spot on postharvest rambutan in Mexico
D. NIETO (1), M. G. Hernandez (1), D. Teliz (1), C. Nava (1), M. T. Martinez
(2), N. Bautista (1)
(1) Colegio de Postgraduados, Texcoco, MEXICO; (2) Universidad
Autonoma Chapingo, Texcoco, MEXICO
Phytopathology 100:S89
The rambutan is a tropical fruit introduced to Mexico. It’s marketing is limited
by a short shelf life and a postharvest disease which is a dark brown spot in
the pericarp. The aim of this study was to identify the causative agent of black
spot in postharvest rambutan. Pieces of infected pericarp were disinfested for
2 min in 2% NaOCl, plated on potato dextrose agar and incubated for 7 days.
One of the fungi was white uniform and radial growth with acervuli. The
second fungi showed abundant black mycelium and pycnidia. For replication
of symptoms, 60 fruit were disinfested 2 min in 2% NaOCl. After, these were
inoculated by smitten with a drop of spore solution (1 × 103 spores/ml) and
placed in a moist chamber for 7 days. The symptoms appeared after 3 days
and on the sixth day developed abundant mycelium. Reisolated fungi were
identical to the originals. The first fungus had 5 cells, the basal and apical were
hyaline, while the intermediate two were brown. There were three appendices
in the apical and basal cells. The second fungus forms black conidia, bicellular,
both cells with a nucleus. The fungi were identified as Pestalotiopsis thea and
Lasiodiplodia theobromae based on characteristics of conidia and molecular
analysis (GenBank accession AY681477.1 and FJ478102.1). Lasiodiplodia
sp. and Pestalotiopsis sp. already been reported in other countries where it is
grown rambutan. However, this is the first report of L. theobromae and P.
thea causing black spot and fruit rot on rambutan in Mexico.
Curtoviruses in leafy greens in Arizona
C. NISCHWITZ (1)
(1) University of Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S89
Curtoviruses are transmitted by the beet leafhopper (Circulifer tenellus) and
can cause severe losses in sugar beets and vegetable crops in the western U.S.
In spring 2009, spinach in southern Arizona showed severe curly top-like
Vol. 100, No. 6 (Supplement), 2010
S89
symptoms and numerous beet leafhoppers were observed in the field. Samples
submitted to a commercial diagnostic lab were negative for curtoviruses in a
general curtovirus PCR screen. However, using primers developed in our lab,
a curtovirus, Pepper curly top virus, was detected in symptomatic spinach. As
a result of the failed detection by a commercial lab, a survey was conducted
on leafy greens including spinach and table beets, weeds and crops near leafy
green fields to determine which curtoviruses are present and if they are
established in Arizona. Fields in four locations in Arizona were selected and
samples were collected twice in each field throughout the season. DNA was
extracted from the samples and tested for curtoviruses using PCR. The PCR
products of selected samples were sequenced. One or more samples of
spinach, table beets and chard in all locations tested positive for curtoviruses.
Reported weed hosts Sisymbrium irio and Chenopodium sp. and a new weed
host Funastrum hirtellum were identified. Perennial alfalfa in the vicinity of
some spinach fields also tested positive. These findings indicate that
curtoviruses are established in the farmscape in Arizona, and curly top disease
in leafy greens is caused by various curtoviruses.
Analysis of rice CHROMOMETHYLASE 3 (OsCMT3) in RNA silencing
mediated by geminivirus
M. NISHIGUCHI (1), Y. Suizu (1), H. Chen (1), N. Yamaoka (1)
(1) Ehime University, Matsuyama, JAPAN
Phytopathology 100:S90
Chromomethylase (CMT) is one of three classes of cytosine
methylatransferase in Arabidopsis. CMT3 is preferentially involved in
cytosine methylation in CpNpG sequences. Based on the nucleotide sequence
of CMT3, we selected a rice mutant where a retrotransposon Tos17 was
inserted in a rice homolog of CMT3 (OsCMT3). In the mutant (oscmt3), RNA
silencing induction was examined by particle bombardment with a plasmid
expressing GFP (p35S-GFP) in combination with a silencing inducer, pGFP
RNAi or pWI-GFP RNAi (recombinant Wheat dwarf geminivirus), each
carrying inverted repeat (IR)-DNA of GFP sequence. When rice tissues were
bombarded with the mixture of p35S-GFP and pGFP RNAi, interestingly,
GFP expression was reduced in OsCMT3 but not in oscmt3, implying that
OsCMT3 is required for IR-DNA induced silencing. When the doublestranded RNA of in vitro transcripts from GFP DNA was used instead of
pGFP RNAi, GFP expression was reduced in both OsCMT3 and oscmt3. This
suggests that OsCMT3 functions at the level of DNA before transcription.
When we bombarded with the mixture of pWI-GFP RNAi and p35S GFP,
GFP expression was also reduced in both rice types. These results suggest that
OsCMT3 is indispensable for IR-DNA induced silencing but not for that
mediated by Wheat dwarf geminivirus.
A survey for grapevine viruses in Virginia vineyards
M. NITA (1), T. Mekuria (2), N. A. Rayapati (3)
(1) Virginia Tech, Winchester, VA, U.S.A.; (2) Washington State University,
IAREC, Prosser, WA, U.S.A.; (3) Washington State University, Prosser, WA,
U.S.A.
Phytopathology 100:S90
Commercial vineyards in the commonwealth of Virginia were surveyed for
grapevine leafroll disease (GLRD). In red-berried wine grape cultivars (Vitis
vinifera), leaves of GLRD affected vines exhibited green veins, inter-veinal
reddening and downward rolling. In white-berried cultivars, leaves of infected
vines showed mild yellowing and downward rolling. During 2009 growing
season, leaf samples were collected from about 500 grapevines planted in 45
vineyards. Petiole extracts were tested for Grapevine leafroll-associated virus
2 (GLRaV-2), GLRaV-3 and Grapevine fleck virus (GFkV) by one tubesingle step RT-PCR using primers specific to a portion of the heat-shock
protein-70 homolog (HSP70h) of GLRaV-2 and -3 and replicase gene of
GFkV. Nearly 70% of the samples tested positive for one of the three viruses,
with a significant majority testing positive for GLRaV-3. The proportion of
positive samples containing GLRaV-3 alone was higher than those with either
GLRaV-2 or GFkV. In addition, GLRaV-2 and GFkV were found as mixed
infection with GLRaV-3. Majority of the vineyards that tested positive for
GLRaV-2 or GLRaV-3 were planted in 80’s or before. The RT-PCR
fragments amplified from select number of positive samples were cloned and
their sequences compared with corresponding sequences available in
GenBank. Results indicated the presence of genetically distinct isolates of
GLRaV-2, GLRaV-3 and GFkV in Virginia vineyards.
Specific patterns of co-occurrence of grapevine viruses in Washington
vineyards
M. NITA (1), T. Mekuria (2), N. A. Rayapati (3)
(1) Virginia Tech, Winchester, VA, U.S.A.; (2) Washington State University,
IAREC, Prosser, WA, U.S.A.; (3) Washington State University, Prosser, WA,
U.S.A.
Phytopathology 100:S90
S90
PHYTOPATHOLOGY
A survey for grapevine viruses conducted in Washington vineyards during
2005 and 2009 revealed the presence of Grapevine leafroll-associated virus 1
(GLRaV-1), -2, -3, -4, -5 and -9), Grapevine rupestris stem pitting-associated
virus, Grapevine Virus A (GVA), GVB and Grapevine fanleaf virus in many
wine grape cultivars. These viruses were found occurring as single and/or
mixed infections in individual grapevines. In order to determine the scale of
association (or lack of) of viruses in mixed infections at the individual plant
level, the data sets from the survey were analyzed by the Jaccard association
analysis to measure the probability of any two viruses co-occurring in
individual grapevines. Results based on the Jaccard similarity index indicated
that some grapevine viruses such as GLRaV-2 and -4 were significantly
positively associated and others like GLRaV-1 and -2 were negatively
associated. Multivariate analysis of the data sets showed that co-occurrence of
GLRaV-3 and GVA, which are known to be transmitted by similar vector(s),
were highly correlated. Determining the nature of an association (positive,
negative, or neutral) between taxonomically disparate viruses may provide
valuable information on the epidemiology of component viruses and to
develop robust sanitation programs for preventing spread and mitigating
negative impacts of viruses.
Macroarray detection of fungal turfgrass pathogens
E. N. NJAMBERE (1), B. Clarke (1), N. Zhang (1)
(1) Rutgers The State University of New Jersey, New Brunswick, NJ, U.S.A.
Phytopathology 100:S90
Early and accurate detection and identification of fungal pathogens is critical
for turf disease management. Traditionally, diagnosticians use direct
observation or culturing of specimens to identify pathogens. DNA macroarray
is a new molecular tool, which offers a fast, culture-independent alternative
for pathogen detection. The advantage of this technique is its high throughput
compared to other detection methods. In this study, we aim to increase the
array detection sensitivity. We designed a macroarray for two turf pathogens,
Rhizoctonia solani and Pythium aphanidermatum. The array included 9 probes
specific to each species. Positive controls and internal controls were also
spotted on the array. Array sensitivity was optimized by hybridizing labeled
ITS PCR products of the two target species with three sets of probes: 1)
monomer oligonucleotide probes (20-25 nt), 2) dimers: two tandem repeats of
the monomers (40-50 nt) and 3) dimers with an intervening poly-A between
the two repeats (50-60 nt). The use of repeat sequence probes increased the
array sensitivity. However, specificity was compromised when an intervening
poly-A sequence was included. Therefore, dimers, without the poly-A,
performed best in terms of both sensitivity and specificity. These findings will
be used to develop a multiplex detection/identification system for major
fungal and oomycete pathogens of turfgrasses that will facilitate early
diagnosis and improved disease management.
Viability staining of naturally and artificially molded sorghum caryopses
using tetrazolium violet
L. W. Noll (1), D. N. Butler (1), C. R. LITTLE (1)
(1) Kansas State University, Manhattan, KS, U.S.A.
Phytopathology 100:S90
Sorghum grain mold (GM) is a yield-limiting disease characterized by damage
to embryo tissues. Caryopses were obtained from naturally weathered panicles
and panicles inoculated with Fusarium thapsinum (FT) and Curvularia lunata
(CL) at anthesis. Caryopses were assayed for tetrazolium violet (TZ) viability,
embryo damage, germination, and pathogen incidence. In naturally weathered
and artificially inoculated caryopses, Tx430 (GM-susceptible) showed low
whole embryo viability, whereas Sureno and Tx2911 (GM-resistant) showed
higher values. Tx430 had the greatest levels of non-staining scutellum,
coleoptile, plumule, and radicle tissues and highest levels of embryo damage
when inoculated with either pathogen. In naturally weathered grain, TZ
staining overestimated germination for some genotypes (especially Tx430),
whereas TZ staining underestimated germination in most artificially
inoculated material. Correlations between TZ viability and germination were
positive and significant for naturally weathered and artificially inoculated
caryopses. FT and F. proliferatum (FP) were the most commonly isolated
fungi from naturally weathered grain. Correlations between FT (yellowpigmented isolates), FP, and CL incidence from naturally weathered sorghum
caryopses and TZ viability were negative and significant. This study showed
that TZ viability differs in a genotype- and pathogen-dependent manner.
Therefore, this approach may have utility in screening sorghum germplasm
for GM resistance at the whole embryo and tissue levels.
Efficacy of ametoctradin + dimethomorph for control of Phytophthora
species infecting ornamental plants in the Eastern United States
D. J. NORMAN (1), M. M. Benson (2), M. L. Daughtrey (3)
(1) University of Florida, Apopka, FL, U.S.A.; (2) North Carolina State University, Raleigh, NC, U.S.A.; (3) Cornell University, Long Island, NY, U.S.A.
Phytopathology 100:S90
Efficacy of fungicide active ingredients varies against Phytophthora species.
In ornamental plant production, multiple species of Phytophthora may be
found within a geographic region, within a single production facility, and even
within a crop species grown in one production facility. The new fungicide
product Orvego contains two active ingredients, ametoctradin and
dimethomorph. Orvego is a foliar and root penetrant with translaminar and
locally systemic activity making it an effective control against multiple
Phytophthora species. Preliminary tests of Orvego on both annual and
perennial ornamental crops under both greenhouse and field conditions have
been conducted. Results indicate that Orvego was very effective in controlling
P. cryptogea, P. dreschleri, P. cinnamomi, P. tropicalis, and P. nicotianae
species on Gerbera daisy, Rhododendron, English ivy, and pansy. Orvego has
multiple modes of action against the pathogen and it should be an effective
rotation partner with other Oomycete control products for resistance
management.
effects of cultivation (aerification, verticutting and sand topdressing) and time
of nitrogen (N) fertilization on large patch development were evaluated on
inoculated plots in a split-plot design with four replications. The whole plot
treatment was cultivation vs no cultivation. The subplot treatment was
fertility, with either polymer-coated urea equivalent to 2 lb N per 1000 ft2
applied as one application during summer or as split applications of urea at 1
lb N each during spring and fall. Large patch severity was assessed through
patch size measurements and digital image analysis. Patch sizes were
significantly reduced in 2009 but not in 2008 in cultivated plots. Cultivation
and summer fertilization resulted in smaller patches compared with noncultivation and fertilization in spring and fall. However, recovery of zoysia
from large patch infection during summer was faster in non-cultivated plots,
irrespective of the timing of fertilization, than in cultivated plots. Cultivation
and summer fertilization could potentially reduce the fungicide volume
required for large patch management.
Selection of transformed somatic embryos by antibiotic drench technique
L. C. NORTHERN (1), C. A. Maynard (2), W. A. Powell (2)
(1) SUNY-ESF, Jamesville, NY, U.S.A.; (2) SUNY-ESF, Syracuse, NY,
U.S.A.
Phytopathology 100:S91
Evaluation of 15 new zoysiagrass lines for resistance to large patch
disease caused by Rhizoctonia solani AG 2-2 LP
K. OBASA (1), M. Kennelly (1)
(1) Kansas State University, Manhattan, KS, U.S.A.
Phytopathology 100:S91
Co-transformation of American chestnut (Castanea dentata) somatic embryos
takes approximately 16 months. To shorten the selection phase of this
procedure, an antibiotic drench was added. Co-transformations were
performed using a 1:5 ratio of Agrobacterium EHA 105 containing a binary
vector with BAR and GFP (pGFP), plus EHA 105 carrying the laccase gene of
interest and NPT2 (pESF-KB-LOE). Non-transformed embryos were treated
with the same conditions as negative controls. The combination of Finale®,
paromomycin, carbenicillin, and cefotaxime in solid selection media has been
used successfully to select for transformed embryos. A supplementary dose of
antibiotics, at the same concentration as the solid medium, was added as a
drenching solution when transferring the embryos to fresh media. We
hypothesize that this would allow the antibiotics to penetrate the cluster of
embryo tissue better than simple absorption from the surface of solid media
and that this would increase the rate of mortality of the non-transformed cells.
Transformed embryos without this extra dose of antibiotics were used as
controls. Extra doses were added each time that the embryos were transfer to
fresh media (every 2 weeks on three consecutive transfers), by covering them
with the antibiotic solution for a total of 6 hrs, and then aspirating away any
remaining liquid. Preliminary results indicate that the treatment was not
effective in reducing the time required to eliminate non-transformed cells.
Fifteen new zoysiagrass lines were evaluated for resistance to large patch
disease caused by Rhizoctonia solani AG 2-2 LP under growth chamber and
field conditions in 2009. Field plots measured 5 × 5 ft and were arranged in a
randomized complete block design (RCB) with three replicates. Plots were
maintained at a mowing height of 0.56 inches and fertilized in May with 1 lb
and July and August with 0.75 lb nitrogen per 1000 ft2 respectively using
plain urea. The center of each plot was inoculated with R. solani-infested oat
kernels in September of 2008. Large patch development in each plot was
assessed through patch size measurement and digital image analysis. Growth
chamber evaluations consisted of fifteen small plastic pots of each new line
and the standard cultivar ‘Meyer’ inoculated with 8 to 10 R. solani-infested
oat kernels and maintained at 25°C and >95% relative humidity under a 13 h
photoperiod. At 5-day intervals, three pots of each line and Meyer were
removed and rated individually for disease incidence by determining the
percentage of shoots with distinct water-soaked lesions on the leaf sheath. No
single line offered consistently better disease resistance than Meyer under
both experimental conditions. However, five lines 5313-46, 5313-71, 532118, 5313-34 and 5325-11 had recovery rates from the disease that were
comparable to Meyer.
Organic and polyethylene mulches with biofungicides for managing
diseases in organic tomato production system
L. M. NYOCHEMBENG (1), R. N. Mankolo (1), S. R. Mentreddy (1)
(1) Alabama A&M University, Normal, AL, U.S.A.
Phytopathology 100:S91
Plant disease management in organic production systems is a challenge hence
there is significant interest in developing effective strategies for mitigating
diseases and weeds. Experiments were conducted to evaluate the individual
and combined effects of spent mushroom substrate (SMS) and sudan x
sorghum hybrid (SS) organic mulches and polyethylene soil bed covers with
or without biofungicides on the severity of diseases in two tomato varieties. In
experiment 1, the organic mulch combination SMS + SS significantly
enhanced tomato fruit weight, number of fruits and weed suppression
compared to SS mulch and the control. In experiment 2, there were significant
differences between the polyethylene mulches white plastic, reflective silver
plastic and control for number of fruits/plant and powdery mildew severity.
However, the plastic mulches were better than control for fruit weight and
plant height irrespective of mulch type. Although polyethylene mulch
enhanced plant height and fruit yield, powdery mildew was more severe in the
mulch treatments. Tomato var. ‘Celebrity’ outperformed var. ‘Amelia’ in fruit
yield and showed more susceptibility to powdery mildew. Spent mushroom
substrate as biofungicide under polyethylene mulch also significantly
enhanced tomato fruit yield compared to the control. These results show that
organic and polyethylene mulches, while ineffective in reducing powdery
mildew severity, significantly enhanced tomato yield and weed suppression in
organic production system.
Effects of fertility and cultivation practices on large patch disease of
zoysiagrass, caused by Rhizoctonia solani AG 2-2 LP
K. OBASA (1), R. St. John (1), D. Bremer (1), J. Fry (1), M. Kennelly (1)
(1) Kansas State University, Manhattan, KS, U.S.A.
Phytopathology 100:S91
Zoysiagrass (Zoysia japonica and Z. matrella) is a warm-season (C4) turfgrass
that is appropriate for many uses in the central and southern United States.
Large patch, caused by Rhizoctonia solani AG 2-2 LP, is the most common
and severe disease of zoysiagrass and is managed primarily by fungicides. The
Towards understanding coronatine-dependent suppression of innate
immunity in Arabidopsis guard cells
N. OBULAREDDY (1), S. Panchal (1), M. Melotto (1)
(1) University of Texas, Arlington, TX, U.S.A.
Phytopathology 100:S91
Recent studies have shown that stomatal pores in the leaf epidermis close as a
part of the plant innate immune response against bacterial invasion of plant
tissues. Counteracting this response, the plant pathogenic bacteria Pseudomonas
syringae pv. tomato strain DC3000 has evolved the virulence factor
coronatine, an important strategy contributing to pathogenesis. The mode of
action of coronatine in plant cells has beginning to be elucidated. Two
components of the coronatine receptor complex have been identified, namely
COI1 (the F-box subunit of E3 ligase) and JAZ (a repressor of jasmonic acid
pathway) proteins, suggesting that coronatine acts in the plant by inducing the
degradation of proteins and hijacking the jasmonic acid signaling pathway.
However, an unanswered question is whether coronatine induce stomatal
opening using the same molecular mechanism. Here, we report that among all
JAZ genes, JAZ1, JAZ2, JAZ3, and JAZ9 are induced in guard cells within 30
minutes of exposure to 60 µM coronatine. In addition, coronatine-dependent
binding of COI1 and these JAZ proteins has been demonstrated using yeasttwo-hybrid system. We have developed single and multiple knock out plants
for these genes. The phenotype of these plants and the biological significance
of coronatine action in the guard cell will be further discussed.
SoilGard 12G (Gliocladium virens strain GL-21): A solution for
controlling lettuce drop (Sclerotinia minor/sclerotiorum) in conventional
and organic systems
S. C. OCKEY (1)
(1) Certis USA, Yakima, WA, U.S.A.
Phytopathology 100:S91
Lettuce drop is one of the most frequent and destructive diseases encountered
by commercial lettuce growers throughout the U.S. The causal agents
Sclerotinia minor & S. sclerotiorum produce sclerotia requiring long rotations
and annual fumigation in fields where the disease has become established.
Heavily infested fields have incurred losses of up to 70%. SoilGard® 12G
Microbial Fungicide contains spores of Gliocladium virens strain GL-21
which has proven to be an effective fungicide in laboratory and field studies.
Vol. 100, No. 6 (Supplement), 2010
S91
SoilGard® 12G was tested under field conditions in Greenfield, CA during
2009. The trial was set up as a randomized complete block design with 4
replicate plots/treatment each 2 rows x 6m. SoilGard was applied at 4.48
Kg/Ha a total of three times with a CO2 pressurized backpack sprayer
operating at 701-795 L/Ha and 276KPa. The initial application was made 1
day post planting followed by the two additional applications at approx. 4
week intervals. Disease incidence was rated throughout the trial and compiled
at harvest. SoilGard 12G and the commercial standard of Endura® Fungicide
at 11 oz/acre resulted in 7% disease incidence each which was significantly (p
= 0.05) lower than the incidence for the untreated check with 23% incidence.
Trials are planned to evaluate alternate application types with additional crops
sensitive diseases caused by Sclerotina sp.
Effect of multiple virus infections on seed transmission in cowpea
K. E. OGUNSOLA (1), C. A. Fatokun (2), C. O. Ilori (3), P. Lava Kumar (2)
(1) International Institute of Tropical Agriculture (IITA), and Crop Protection
and Environmental Biology Department, University of Ibadan, Ibadan,
NIGERIA; (2) International Institute of Tropical Agriculture (IITA), Ibadan,
NIGERIA; (3) Crop Protection and Environmental Biology Department,
University of Ibadan, Ibadan, NIGERIA
Phytopathology 100:S92
Multiple viral infections occur naturally in cowpea and these viruses may
interact synergistically causing a change in virus concentration of one or all of
the viruses. We studied the effect of multiple virus infections on virus seedtransmission in eight cowpea genotypes that differ in relative susceptibility to
viruses. Blackeye cowpea mosaic virus (BlCMV, genus Potyvirus), Southern
bean mosaic virus (SBMV, genus Sobemovirus) and Cucumber mosaic virus
(CMV, genus Cucumovirus), all of which were known to be seed-transmitted
in cowpea, were used to mechanically inoculate test plants at seedling stage
singly and in all possible combinations. Seeds were harvested at maturity and
assessed for seed-transmission by grow-out tests and enzyme-linked immunosorbent assay. Seed-transmission was found to be genotype specific and also
influenced by co-infection. In cowpea cv. IT98K-133-1-1, BlCMV was not
seed-transmitted either singly or in any combinations. Seed-transmission of
SBMV was not observed in single infections, however, seed-transmission was
detected in plants co-infected with CMV, but not BlCMV. CMV was seed
transmitted in single infections as well as mixed infection with BlCMV and
CMV. This study suggests possibility of synergistic interaction between coinfected viruses in facilitating seed-transmission of apparently non-seed
transmitted viruses, in this case SBMV, with CMV acting as a helper virus.
A tomato 14-3-3 protein (TFT7) positively regulates immunity-associated
programmed cell death mediated by diverse disease resistance proteins
C. OH (1), G. B. Martin (2)
(1) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (2)
Boyce Thompson Institute for Plant Research and Cornell University, Ithaca,
NY, U.S.A.
Phytopathology 100:S92
Immunity-associated programmed cell death (PCD) is triggered when a plant
resistance (R) protein recognizes a corresponding pathogen effector protein.
For example, the tomato R protein Pto recognizes the Pseudomonas syringae
effector AvrPto causing localized PCD that is associated with disease
resistance. Previously, we reported that both MAPKKKα (mitogen-activated
protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7)
positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana.
Moreover, we reported that TFT7, unlike MAPKKKα, is required for PCD
mediated by four other R proteins. We therefore investigated why TFT7 is
broadly required for PCD induced by R proteins in plants. We discovered that
a MAP kinase kinase (MAPKK), that acts downstream of MAPKKKα, also
interacts with TFT7 in yeast and plant cells. Gene silencing experiments
revealed that the MAPKK and TFT7 are each required for PCD induced by
the same set of R proteins. We will discuss further how TFT7 regulates this
MAPKK for induction of PCD in N. benthamiana.
In vitro assessment of Sclerotinia homoeocarpa resistance to fungicides
and plant growth regulators
C. Ok (1), J. T. POPKO (1), K. Campbell-Nelson (1), G. Jung (1)
(1) University of Massachusetts, Amherst, MA, U.S.A.
Phytopathology 100:S92
Dollar spot (caused by Sclerotinia homoeocarpa F.T. Bennett) is a turfgrass
disease primarily controlled by fungicide application on golf courses;
however, resistance has been confirmed in three of the five fungicide classes
commonly used to control dollar spot. The main objective of this study was to
evaluate S. homoeocarpa resistance to multiple fungicide classes and plant
growth regulators (PGRs) and cross-resistance among active ingredients of the
same class. Sixty-four isolates were randomly selected and assayed for in vitro
fungicide sensitivity to six demethylation inhibitor (DMI), two dicarboximide,
one anilene, one benzimidazole fungicide and three type II plant growth
S92
PHYTOPATHOLOGY
regulators. All active ingredients from the DMI class were highly correlated
(P < 0.0001) to each other as well as to the dicarboximide (iprodione) and
plant growth regulators (flurprimidol and paclobutrazol). EC50 values of all
active ingredients assayed except for boscalid were significantly higher in
isolates resistant to thiophanate-methyl than sensitive isolates. Results indicate
that multiple class and cross-resistance of fungicides and PGRs has developed
in S. homoeocarpa and that PGRs have a fungistatic effect similar to that of
demethylation inhibitor fungicides. The high correlation of in vitro
sensitivities among PGRs and DMI fungicides further suggest that PGRs may
contribute to the selection of DMI resistant isolates or facilitate decreased
sensitivity to DMI fungicides.
Assessment of SIMBLIGHT1 and SIMPHYT1 models for prediction of
Phytophthora infestans oubreak in North-Eastern U.S. from 2004 to 2009
seasons
M. OLANYA (1), C. Honeycutt (1), R. P. Larkin (1), Z. He (1)
(1) USDA-ARS, NEPSWL, Orono, ME, U.S.A.
Phytopathology 100:S92
Accurate prediction of Phytophthora infestans outbreak during a cropping
season is crucial for effective management of late blight. The SIMBLIGHT1,
SIMPHYT1, and modified SIMPHYT1 models were assessed for prediction
of late blight outbreak relative to the NOBLIGHT model based on climatic
data from field experiments. The dynamics of late blight infection pressures
and Phytophthora efficiency (pew-values) were computed by the SIMPHYT3
model to assess conduciveness of climatic conditions for disease development.
Simulation results (recommended fungicide treatment) of SIMPHYT1 model
predicted first application dates of July 11, 21, 8, 10, 7 and 7 for 2004 to 2009,
and for the modified SIMPHYT1 model (US-version) on July 11, 22, 8, 19, 7,
and 7 for the same years. Comparison of simulation results with date of
disease outbreak in untreated plots resulted in differences of 24–65 days.
Validation of the models (differences between recommended fungicide
treatment and first blight outbreak) gave better fit for models with predicted
intervals of 6–20 days from initial fungicide application to first late blight
outbreak. The SIMBLIGHT1, SIMPHYT1, and NOBLIGHT models were
accurate and flexible in forecasting the timing of first fungicide applications
for disease control. Due to the conducive conditions for late blight potential
and infection pressures, development of predictive models that can account
for external inoculum sources will greatly improve late blight management at
regional or national scales.
Insights into sexual reproduction in Aspergillus flavus from variation in
experimental crosses and natural populations
R. A. OLARTE (1), B. W. Horn (2), J. T. Monacell (3), E. A. Stone (4), I.
Carbone (1)
(1) Department of Plant Pathology, North Carolina State University, Raleigh,
NC, U.S.A.; (2) National Peanut Research Laboratory, Agricultural Research
Service, U.S. Department of Agriculture, Dawson, GA, U.S.A.; (3) Bioinformatics Research Center, North Carolina State University, Raleigh, NC, U.S.A.;
(4) Department of Genetics, North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S92
Aspergillus flavus contaminates many important crops worldwide and is the
major producer of aflatoxins, which are cancer-causing secondary metabolites.
Biological control is the most effective means of reducing inoculum levels of
detrimental aflatoxin-producing fungal pathogens in agricultural systems;
however, the long-term efficacy of such methods may face scrutiny with the
recent discovery of the sexual cycle in these fungi. We crossed strains of
opposite mating type in A. flavus to produce offspring, which were genetically
and phenotypically analyzed to quantify gene flow and determine the
heritability of aflatoxin (AF) and cyclopiazonic acid (CPA). We found that a
single generation of sexual reproduction between a nonaflatoxigenic parent
containing a single mutation in the aflatoxin cluster and an aflatoxigenic
parent can restore aflatoxin production. The recombinant F1 progeny regained
aflatoxigenicity through a crossover event within the aflatoxin gene cluster.
Other F1 progeny in crosses between either a partial aflatoxin cluster strain or
a strain missing the entire cluster and an aflatoxigenic parent regained toxicity
via independent assortment of chromosomes. We also found that genetic
exchange and recombination are associated with increased heritability of AF
and CPA in progeny. These results suggest that a single round of sexual
reproduction in A. flavus can generate contemporary patterns of recombination
and toxin diversity.
Weather patterns and the distribution of Asian soybean rust in the
United States
R. O. OLATINWO (1), R. C. Kemerait (2), J. O. Paz (3), G. Hoogenboom (1)
(1) University of Georgia, Griffin, GA, U.S.A.; (2) University of Georgia,
Tifton, GA, U.S.A.; (3) Mississippi State University, Mississippi State, MS,
U.S.A.
Phytopathology 100:S92
Asian soybean rust (ASR) caused by the fungus Phakopsora pachyrhizi, was
reported for the first time in the United States in late 2004. The disease is
mostly restricted to the south, although it has shown a gradual northward
movement during the last few years. The fungus prefers moderate
temperatures and wet environments. The objective of this study was to
determine the effects of precipitation and temperature on the distribution of
ASR incidence in the U.S. Confirmed cases of ASR incidence on kudzu and
soybean from 20 states was evaluated using data from the Integrated Pest
Management Pest Information Platform for Extension and Education (IPM
PIPE), and weather data from the National Climate Data Center (NOAANCDC). The initial results showed a significant upward trend in the
percentage of counties with ASR infection from 2005 to 2009 in most states
including Alabama, Georgia, Mississippi, and Louisiana. When the average
statewide monthly temperature in June was ≤24.2°C only 6% of the overall
counties had ASR infection, while approximately 36% of counties had ASR
infection when the June temperature was higher. When the average statewide
June temperature was >24.2°C and the average statewide September
precipitation was ≥155.4 mm, the percentage of counties with ASR
increased to 69%. Above normal statewide precipitation increased the
percentage of counties with ASR infection. Accurate information about
favorable conditions for ASR infection could help growers make informed
management decisions.
Difenoconazole baseline sensitivity distribution of Colletotrichum coccodes
isolates from potatoes
G. OLAYA (1), A. Cochran (2), G. Neil (3)
(1) Syngenta Crop Protection, Vero Beach, FL, U.S.A.; (2) Syngenta Crop
Protection, Greensboro, NC, U.S.A.; (3) North Dakota State University,
Fargo, ND, U.S.A.
Phytopathology 100:S93
The sensitivity to the demethylation inhibitor fungicide difenoconazole of 65
monoconidial isolates of Colletotrichum coccodes was determined under invitro conditions. C. coccodes solates were collected from potatoes infected
with black dot disease that had not been previously exposed to
difenoconazole. The geographical origin of the isolates is broad and included
several potato growing regions in different States. Sensitivity of each isolate
was determined by comparing the colony radial growth on ½ strength potato
dextrose agar plates either amended or not with difenoconazole. The
sensitivity distributions (ED50 values) of C. coccodes isolates ranged from
0.034 to 0.167 with a mean value of 0.065 mg/L. Difenoconazole baseline
information will help in future difenoconazole sensitivity monitoring studies
looking for the early detection of changes in sensitivity of C. coccodes to
difenoconazole. Difenoconazole is being developed as a postharvest fungicide
for stored potatoes to control diseases caused C. coccodes, Fusarium spp. and
Helminthosporium solani.
Fludioxonil sensitivity monitoring of Penicillium expansum isolates
collected from apples in Washington State
G. OLAYA (1), A. Mulcahy (1), A. Cochran (2)
(1) Syngenta Crop Protection, Vero Beach, FL, U.S.A.; (2) Syngenta Crop
Protection, Greensboro, NC, U.S.A.
Phytopathology 100:S93
The sensitivity to the fungicide fludioxonil (commercial name Scholar®) of
103 monoconidial Penicillium expansum isolates collected from packing
houses where the fungicide have been used was determined under in-vitro
conditions. The objective of the fludioxonil monitoring was to determine if
changes in sensitivity to fludioxonil were occurring in P. expansum
isolates. P. expansum isolates were collected in 2009 from 5 different apple
varieties from commercial packing houses in Washington State. The
distribution of fludioxonil sensitivities of 103 P. expansum isolates collected
in 2009 was similar to the fludioxonil baseline sensitivity distribution that was
established in 2004 before the commercial introduction of Scholar®.
Sensitivities of isolates collected in 2009 ranged from 0.001 to 0.326
with a mean ED50 value of 0.033 mg/L, whereas the sensitivities of baseline isolates tested in 2004 ranged from 0.006 to 0.171 with a mean of
0.028 mg/L. In conclusion, no important changes in the sensitivity to
fludioxonil were detected in P. expansum isolates collected in 2009 in
Washington State.
Development of a high throughput and fast system for testing transgenic
resistance constructs derived from Grapevine fanleaf virus
J. E. OLIVER (1), M. Fuchs (1)
(1) Cornell University, Geneva, NY, U.S.A.
Phytopathology 100:S93
Grapevine fanleaf virus (GFLV) from the genus Nepovirus, family
Secoviridae is transmitted from grapevine to grapevine by the ectoparasitic
nematode Xiphinema index and causes fanleaf degeneration disease. This
disease is widespread throughout the world nearly everywhere grapes are
grown and the nematode vector is found. Management of GFLV has largely
relied on control of its nematode vector, essentially through soil disinfection
and the use of resistant rootstocks, although resistance to X. index does not
prevent GFLV translocation from rootstocks into scions or transmission in
vineyards. Resistance to GFLV has not been identified in wild or cultivated
grapes; nonetheless, transgenic grapevine rootstocks expressing the full-length
viral coat protein gene can confer resistance to GFLV. Based on recent
knowledge concerning the genetic variability of GFLV, it is unlikely that
transgenic grapevine rootstocks expressing the full-length coat protein gene
will show durable resistance. Therefore, new GFLV constructs potentially
capable of providing resistance have been developed. However, due to the
extended time and expense involved in the development and testing of
transgenic grapes for resistance to GFLV, efforts to develop a high throughput
approach for testing candidate constructs in Nicotiana benthamiana, a
systemic host, have been pursued. Results from the development and
evaluation of GFLV-derived genetic constructs for engineered resistance will
be discussed.
Races of Puccinia graminis f. sp. tritici with virulence on Sr13 and Sr9e in
durum screening nursery in Ethiopia
P. OLIVERA FIRPO (1), Y. Jin (2), A. Badebo (3), D. Singh (4)
(1) University of Minnesota, St. Paul, MN, U.S.A.; (2) USDA-ARS Cereal
Disease Laboratory and University of Minnesota, St. Paul, MN, U.S.A.; (3)
Ethiopian Institute of Agricultural Research, Debre Zeit, ETHIOPIA; (4)
International Maize and Wheat Improvement Center (CIMMYT), Nairobi,
KENYA
Phytopathology 100:S93
Durum wheat (Triticum turgidum ssp. durum) of North America has a higher
frequency of resistance to race TTKSK (or Ug99) of Puccinia graminis f. sp.
tritici than common wheat based on evaluations conducted in Njoro, Kenya.
We postulated TTKSK resistance in durum is likely due to Sr13, a common
gene in North American cultivars. However, when resistant selections were
evaluated in Debre Zeit, Ethiopia, many became susceptible to stem rust,
suggesting that local races may possess a virulence combination that
overcomes the TTKSK resistance. The objective of this study was to identify
and characterize races of P. graminis f. sp. tritici present in the Debre Zeit
screening nursery in 2009. Single-pustule isolates were derived from collected
samples and race-typed based on the North American stem rust differentials.
The isolates were further characterized on a set of universal resistant lines.
Three races of P. graminis f. sp. tritici were identified: JRCQC, TRTTF and
TTKSK. Both JRCQC and TRTTF possess virulence combination on Sr13
and Sr9e, which may explain why the TTKSK-resistant durum in Kenya
became susceptible in Debre Zeit. The virulence combination on Sr9e and
Sr13 is of big concern because these genes constitute the main components of
stem rust resistance in North American durum cultivars. In addition to
virulence on Sr9e and Sr13, race TRTTF appears to be virulent to stem rust
resistance conferred by the 1AL.1RS translocation in winter wheat in the
United States.
Resistance to race TTKSK of Puccinia graminis f. sp. tritici in tetraploid
wheat
P. OLIVERA FIRPO (1), Y. Jin (2), S. Xu (3), D. Klindworth (3)
(1) University of Minnesota, St. Paul, MN, U.S.A.; (2) USDA-ARS Cereal
Disease Laboratory and University of Minnesota, St. Paul, MN, U.S.A.; (3)
USDA-ARS, Northern Crop Science Laboratory, Fargo, ND, U.S.A.
Phytopathology 100:S93
A group of races of Puccinia graminis f. sp. tritici in the TTKS (or Ug99)
lineage possess broad virulence to wheat cultivars worldwide, and only a few
genes in the adapted cultivars have resistance to these races. In attempts to
identify new stem rust resistance genes effective against race TTKSK, we
evaluated cultivated tetraploid wheat (T. turgidum ssp. dicoccum, ssp.
carthlicum, ssp. polonicum, ssp. turanicum, and ssp. turgidum) for resistance
to TTKSK and other races with broad virulence. A high frequency of
TTKSK resistance at the seedling stage was observed, as 207 (21% of
1002 accessions) exhibited low infection types. Low infection types ranging
from 2= to 2+ to race TTKSK were predominant. Studies to determine the
genetic basis of TTKSK resistance at the seedling stage revealed that
resistance in tetraploid wheat is conferred mostly by single genes. Fifty
accessions were evaluated for resistance to TTKSK and Ethiopian races in the
field screening nursery at the Ethiopian Institute for Agricultural Research
(Debre Zeit, Ethiopia). Twenty-three accessions exhibited resistant to
moderately resistant responses to stem rust. Four accessions susceptible to
TTKSK at the seeding stage were resistant at the adult stage. These accessions
may possess adult plant resistance. Since all these tetraploid species share the
same genome as durum wheat and are in cultivated form, resistance genes
could be easily transferred to durum wheat by conventional breeding
approaches.
Vol. 100, No. 6 (Supplement), 2010
S93
Phylogenetic history and genetic diversity of Phytophthora cryptogea and
P. drechsleri isolates from floriculture crops in North Carolina
greenhouses
H. A. OLSON (1), I. Carbone (1), M. Benson (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S94
Since P. cryptogea was described in 1919 and P. drechsleri 12 years later, one
of the most challenging taxonomic situations in the genus is discerning these
species. Using isolates collected from NC floriculture crops, the evolutionary
history and genetic diversity of P. cryptogea and P. drechsleri were explored.
Initially, 66 isolates representing 13 location-host groups were sequenced at
multiple loci. Sequences of all isolates within a group were identical at all
loci, so a subset of isolates were selected, cloned to resolve heterozygous
sites, and subjected to analysis using SNAP Workbench. The internal
transcribed spacer region and cytochrome oxidase II genealogies were
congruent and indicated that P. cryptogea and P. drechsleri are wellsupported sister species diverged from a common ancestor with no evidence
of gene flow. At both loci, P. cryptogea is more genetically diverse than P.
drechsleri. In contrast, evidence of recombination between P. cryptogea and
P. drechsleri isolates was found in the beta-tubulin (btub) locus, suggesting
gene flow between species. Coalescent analysis based on a non-recombining
partition in btub showed an initial (older) split between P. cryptogea and P.
drechsleri with a later (recent) event separating the remaining P. cryptogea
haplotypes from P. drechsleri. This may indicate recent gene flow between
species or that P. cryptogea is polyphyletic and some lineages of P. cryptogea
share a recent common ancestor with P. drechsleri.
Characterization of the MADS-box family of transcription factors in
Fusarium verticillioides
C. ORTIZ (1), W. Shim (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S94
Transcription factors (TFs) are protein complexes that activate or repress
transcription of genes by specifically binding to target recognition sites.
MADS-box TFs are a superfamily of regulators that bind to the regulatory
motif CArG-box of functionally diverse target genes. This family is well
studied in plants, but little is known in fungi. MADS-box TFs have been
characterized in select ascomycetes and have been shown to play a role in
pathogenicity, cell and fruiting body development, and mating. Our research
aim is to elucidate the function of two MADS-box TFs in Fusarium
verticillioides, a filamentous fungus with worldwide distribution and direct
association with ear and stalk rots of corn. The fungus also produces the
mycotoxin fumonisin B1 (FB1) which has been linked to human and animal
illnesses. To study these TFs, we generated MADS1 and MADS2 gene knockout mutants via homologous recombination. On V8 agar, MADS1 mutant
produced a purple pigment while MADS2 did not differ from the wild type.
Additionally, when grown on corn, MADS1 and MADS2 mutants produced
significantly less (<10%) FB1 compared to the wild type progenitor. We are
in the process of generating knock-out mutants of these MADS genes in the
complementary mating type as well as a double mutant. Sexual and asexual
development assays, transcriptome analyses, and pathogenicity tests with
these mutants will help us further elucidate the function of MADS-box TFs in
F. verticillioides.
Oomycete research at undergraduate institutions: An update on
SPACES, an internet resource for and by the oomycete community
M. D. OSPINA-GIRALDO (1), C. Bentley (1)
(1) Lafayette College, Easton, PA, U.S.A.
Phytopathology 100:S94
Comprehensive information on oomycete research can be found in several
web portals, including the Oomycete Molecular Genetics Research
Collaboration Network website, which provides an excellent source of up-todate information on events, meetings, fellowships, and important news for the
community. In addition, the databases maintained by the Broad Institute, the
Joint Genome Institute, and the Virginia Bioinformatics Institute contain and
share a wealth of resources focusing on recently sequenced genomes of
several oomycetes. The increasing availability of the Internet has made these
portals easily accessible to most members of the community. A
complementary resource would be one that facilitates member interaction and
becomes a forum that makes the exchange of ideas possible. The Oomycete
Undergraduate Molecular Genetics Network (OUMGN) has been developed
as a means to fill this need; although originally geared towards members of
the oomycete research community affiliated with undergraduate institutions, it
also welcomes individuals from major universities and other organizations.
The OUMGN SPACES site is a place where OMGN members and nonmembers can post questions, comments, announcements, links of interest, etc.
Details on its characteristics, advantages, and use will be presented.
S94
PHYTOPATHOLOGY
Gene transcription patterns in Phytophthora infestans cultures grown in
vitro and in planta
M. D. OSPINA-GIRALDO (1), E. Laird (1), C. Mingora (1)
(1) Lafayette College, Easton, PA, U.S.A.
Phytopathology 100:S94
A total of 49 putative genes homologous to members of 8 families belonging
to the Carbohydrate esterase (CE) gene superfamily have been identified in
the genome of the oomycete Phytophthora infestans. It has been suggested
that CE enzymes, such as the ones classified within the CE Family 5 (the
“cutinase” family) may play a role in the infection process by targeting and
degrading the cell wall. Because no EST evidence supporting the expression
of some of these genes was available, we analyzed the expression of a
subgroup of CE-coding genes using reverse-transcription PCR (RT-PCR) and
found that most genes are expressed in mycelium of P. infestans grown in
vitro. To determine the level of expression of each of these genes, a
quantitative PCR (qPCR) analysis was conducted. In addition, because of the
potential importance of cutinase for P. infestans pathogenicity, a qPCR study
was performed using plant tissue samples obtained at different stages of the
infection process. Results of these investigations will be presented and
discussed.
Foliar chlorophyll content of ponderosa pine on black stain root
disease sites after prescribed burning and subsoiling treatment
combinations
W. J. OTROSINA (1), P. C. Spaine (1), S. S. Sung (2), W. Woodruff (3), J. T.
Kliejunas (4)
(1) USDA Forest Service, Athens, GA, U.S.A.; (2) USDA Forest Service,
Pineville, LA, U.S.A.; (3) USDA Forest Service, Susanville, CA, U.S.A.; (4)
USDA Forest Service, Kent, WA, U.S.A.
Phytopathology 100:S94
A long term study involving underburning, subsoiling and subsoiling X
underburning treatments along with untreated control plots were initiated in
2000 in the Lassen National Forest, California. The study site has active
blackstain root disease. A tree within each of three randomly selected grid
points among nine located within treatment plots was selected for foliar
sampling. Each treatment was replicated four times in a randomized complete
block design. Two branches per tree were obtained at mid crown from
opposite sides of sampled trees by shooting with a 12 ga shotgun. Harvested
needles were placed immediately in an ice chest and later analyzed for
chlorophyll a and b content. Six years of needles were commonly retained,
thus a short history of chlorophyll status is obtained for this species. Overall,
needles from severely symptomatic trees (based on ground observations of
gross needle color and appearance) had about 40% of fresh weight and needle
length of non symptomatic or slightly symptomatic trees. Total chlorophyll
content also declined with decline in needle fresh weight. Prescribed burn
treatments also affected needle retention, with the burn treatment having the
lowest average needle retention (25% for 2004) compared to the control (63%
for 2004), indicating a long term treatment effect.
Fairy ring disease of cranberry: Dissecting the life cycle and development
of control strategies
P. V. OUDEMANS (1), J. Polashock (2), J. Vaiciunas (1)
(1) Rutgers University, Chatsworth, NJ, U.S.A.; (2) USDA ARS, Chatsworth,
NJ, U.S.A.
Phytopathology 100:S94
Fairy ring is a serious disease affecting cultivated cranberries in New Jersey
and Massachusetts. The disease is expressed as expanding patches of dead
vines leading to long term yield loss. Recently, we observed dark
infection pads associated with dying vines and subsequently isolated the
pathogen and confirmed pathogenicity. Sequence analysis showed the causal
agent to be a species of Helicobasidium (teleomorph) and the anamorph
isolated from cranberry was identified as Thanatophytum sp. A second
anamorphic phase of this pathogen, Tuberculina, a rust mycoparasite,
may function as the recombinant stage of the pathogen life cycle. A
collection of isolates from New Jersey and Massachusetts revealed a high
level of diversity as determined by vegetative compatibility (VC) and RAPD
analyses. Isolates collected from individual rings were identical whereas most
isolates collected from different rings were distinct. This suggests that an
active sexual phase is responsible for dissemination. The rust mycoparasite stage may be an important component of the fairy ring life cycle. In
2009, we found the Tuberculina stage on a rust infecting greenbrier
(Smilax sp.), a common weed in cranberry beds. These isolates were identical
to the cranberry pathogen based on ITS sequence. A complex life cycle has
emerged and includes at least three distinct stages on distinct host species.
Control of the briar rust may limit spread of the disease into cultivated
cranberry beds.
Challenges and constraints impacting development of new and novel
plant disease management solutions
D. G. OUIMETTE (1)
(1) Dow AgroSciences LLC, Indianapolis, IN, U.S.A.
Phytopathology 100:S95
Significant market driving forces and constraints with current plant disease
management technology requires the continued development of new solutions
(such as fungicides) to ensure the viability of agricultural crop production.
Such driving forces include: 1) biological (fungicide resistance); 2)
regulatory; and 3) public/food chain perceptions of food safety in crops
treated with fungicides The development and registration of new fungicides
require extensive field characterization to fully understand the strengths and
limitations of the new technology, and this process can take up to ten years
from initial discovery to final product launch. Due to the complex nature of
the product characterization process, various constraints must be effectively
managed. These constraints include the following: suitable trial location and
environmental manipulation to ensure sufficiently high disease development;
reducing the impact of unwanted pests (in particular insects and non-target
diseases); government regulations requiring permits for chemical and
biological shipment; and finally technical expertise among field scientists to
implement successful trials. Global characterization strategies require
leveraging both the southern and northern hemisphere for trial locations to
take advantage of unique marketplace and environmental conditions and for
the ability to conduct research trials throughout the year. The effect of these
constraints on efficient product characterization will be discussed.
Microarray analysis of tomato gene expression reveals complex effects on
hormone signaling associated with viroid infection
R. OWENS (1), K. Tech (1), J. Shao (1), N. Mock (1), T. Sano (2)
(1) USDA ARS, Beltsville, MD, U.S.A.; (2) Hirosaki University, Hirosaki,
JAPAN
Phytopathology 100:S95
Viroids are small non-coding RNAs that use the plant transcriptional
machinery to replicate their genomes. Microarray analysis of tolerant
(Moneymaker) and sensitive (Rutgers) tomato cultivars infected with the type
strain of Potato spindle tuber viroid (PSTVd) revealed significant changes in
the expression levels of 8.8% or 24.6% of the >10,000 genes included on the
Affymetrix array. Three weeks post inoculation, as epinasty and stunting
began to intensify in systemically infected leaf tissue, evidence of a general
stress response was detected in infected Rutgers plants. Synthesis of
ribosomal proteins (primarily cytoplasmic) and ubiquitin-associated protein
turnover increased, and expression of many chloroplast-associated genes was
repressed. In addition to the salicylic acid-mediated response pathway
activated by both RNA and DNA viruses, an unusually large number of genes
associated with abscisic acid and brassinosterioid signaling were also affected
by PSTVd infection. Both gibberellin and brassinosteroid signaling appear to
be involved in PSTVd-induced stunting; in each case, these effects may be
due to changes in hormone synthesis/degradation as well as signaling per se.
The possible role of PSTVd-related small RNAs as a mediator of posttranscriptional gene silencing is under investigation using a deep sequencing
strategy.
Development of a Sweet potato leaf curl virus infectious clone for
agroinfection
V. PAHALAWATTA (1)
(1) Alcorn State University, Lorman, MS, U.S.A.
Phytopathology 100:S95
Sweet potato (Ipomoea batatas) is an important root crop and source of
industrial raw material worldwide. Virus diseases cause significant losses in
sweet potato production both in terms of quality and yield. Sweet potato leaf
curl virus (SPLCV) is among the six most important sweet potato virus
diseases prevalent in the U.S. SPLCV infection in recent years resulted in 25–
30 percent yield losses to the cultivar ‘Beauregard’ that accounts for
approximately 80 percent of the U.S. production. SPLCV is a whiteflytransmitted monopartite single-stranded DNA begomovirus. An alternative
method is being tested to infect sweet potato plants with SPLCV in the
absence of the insect vector. Full length genomic DNA of SPLCV was cloned
and sequenced. The restriction digested DNA fragment containing the viral
replication origin was ligated into the corresponding restriction sites of the
binary vector pBI121 resulting in pBI121-SPLCV-ori. Full length SPLCV
genomic DNA was then cloned into pBI121-SPLCV-ori to obtain the tandem
repeat dimer construct pBI121-SPLCV. The SPLCV infectious clone will be
introduced into sweet potato plants using an Agrobacterium tumefaciens/Ti
plasmid-mediated delivery system. Development of an effective inoculation
method independent of the whitefly vector will greatly facilitate highthroughput screening of resistant germplasm, identification of resistant genes
as well as understanding the molecular basis of SPLCV-host interactions.
Pathogenicity of Fusarium oxysporum f. sp. radicis cucumerinum and
control strategies of cucumber root and stalk rot in hydroponic systems
D. PALMERO (1), M. De Cara (2), M. Santos (2), C. Iglesias (1), J. Tello (2)
(1) Universidad Politecnica de Madrid, Madrid, SPAIN; (2) Universidad de
Almería, Almeria, SPAIN
Phytopathology 100:S95
Pathogenicity of 6 isolates of Fusarium oxysporum f. sp. radiciscucumerinum
was studied on four cucurbitaceae species, one pumpkin hybrid (Cucurbita
maxima X C. moschata), and on eight species belonging to different botanical
families. All six F. oxysporum isolates showed pathogenicity on three
cucurbitaceae, but didn’t on squash and pumpkin hybrid, and didn’t on each of
eight other species different to cucurbitaceae. Severity of the disease was
slightly higher at 17°C than 25°C. Control strategies against cucumber root
and stalk rot were also tested. Disinfection of the substrate (perlite) and use of
cucumber plants (cv Borja) grafted on hybrid pumpkin were evaluated to
control the disease. For chemical disinfection of the substrate three different
products were used: metam potassium, metam sodium and chloropicrin + 1.3
dichloropropene, these treatments were tested both with and without
solarization. Results showed that chemical disinfectants and solarization,
failed to successfully control the disease during the two usual crop periods in
the area (autumn and spring). The protection for some treatments did not
reach the 10 months that included the two cultural cycles. On the other side
eight commercial rootstocks were evaluated. All the grafted varieties
expressed a complete resistance and a significant increase in production was
observed. Keywords: Melon, cucumber syndrome, Cucurbita pepo x C.
moschata, Cucumis sativus, Fusarium disease.
Pathogenicity and fusaric acid production by Fusarium proliferatum
isolated from garlic in Spain
D. PALMERO LLAMAS (1), M. de Cara (2), W. Nosir (3), C. Iglesias (1),
M. García (1), S. Woodward (3), J. Tello (2)
(1) Technical University of Madrid, Madrid, SPAIN; (2) Universidad de
Almería, Almeria, SPAIN; (3) University of Aberdeen, Aberdeen,
SCOTLAND
Phytopathology 100:S95
Fusarium proliferatum has been reported on garlic in the north west U.S.A.,
Spain and Serbia, causing as water-soaked tan lesions on cloves. Moreover, F.
proliferatum is known to produce a range of toxins, including fumonisin B1,
moniliformin, beauvericin, fusaproliferin and fusaric acid, which are
implicated in pathogenesis. In this study six randomly selected F. proliferatum
isolates from garlic were tested for pathogenicity and screened for fusaric acid
production. Healthy seedlings of onion (Allium cepa), leek (A. porrum) and
chives (A. schoenoprasum) and garlic clones (A. sativum) were inoculated.
Onion seedlings and garlic clones were soaked in the conidial suspensions of
each F. proliferatum isolate for 24 h and then planted in flats containing soil
previously inoculated with the same isolate of F. proliferatum. Plants were
maintained in a temperature and light-controlled greenhouse (12 h/12 h
light/dark; 25/21°C). The root and bulb/clove rot disease symptoms were
graded into five classes following the method of Stankovick et al. (2007). A
disease severity index (DSI) was calculated as the mean of three plants of
each species and four test replicates. Symptoms on onion and garlic plants
were observed three weeks after inoculation. The overall effects of isolate,
host and variety were analyzed. Effects were significant for all the studied
isolates. The correlations between isolate pathogenicity and production of FA
are also discussed.
Isolation of Aspergillus section Nigri strains and incidence of ochratoxin
A in California raisins
J. D. PALUMBO (1), T. L. O’Keeffe (1), N. E. Mahoney (1), S. Vasquez (2)
(1) USDA ARS WRRC, Albany, CA, U.S.A.; (2) University of California
Coop Ext, Fresno, CA, U.S.A.
Phytopathology 100:S95
Species of Aspergillus section Nigri, particularly A. niger and A. carbonarius,
have been implicated as sources of ochratoxin A (OTA) contamination in
wine and table grapes, as well as raisins and other dried fruits. OTA
contamination of these commodities is not uncommon in Mediterranean and
South American regions, but has not been reported in California vineyards. To
investigate the occurrence of OTA-producing Aspergillus section Nigri
species in California, four raisin vineyards were sampled during the 2009
harvest. Thirty seven of the 40 raisin samples contained measurable OTA
contamination. From these raisin samples, a total of 400 strains of Aspergillus
were isolated and analyzed for production of OTA on culture media. Of these,
13 isolates, from six raisin samples, produced OTA. These isolates were
identified as A. carbonarius (12 isolates) and A. niger (1 isolate), based on
morphological characteristics and multilocus sequence analysis. A.
carbonarius was only recently reported as a causal agent of sour rot on table
grapes in California. This is the first report of OTA production by A.
carbonarius or A. niger isolated from California raisins.
Vol. 100, No. 6 (Supplement), 2010
S95
Dynamics and single nucleotide polymorphisms of rice blast resistance
alleles at the Pik locus
Q. PAN (1)
(1) South China Agricultural University, Guangzhou, Guangdong, PRC
PEOPLES REP OF CHINA
Phytopathology 100:S96
The Pik locus located on rice chromosome 11, which consists of Pik, Pik-s,
Pik-p, Pik-m, and Pik-h alleles, is an important resistance gene resource for
rice blast breeding programs in China. Dynamics of resistance of these alleles
was characterized with more than 10 populations of Magnaporthe oryzae
collected from various regions in China. Each allele was an independently and
dominantly acting gene at the Pik locus, and conditions differential reactions
against many isolates. For molecular characterization of the alleles, the
genetic and physical maps of these alleles, respectively, were constructed
using genomic position-ready markers. Then, the alleles were isolated,
respectively, through an approach called map-based cloning, in silico. The
function of each allele was dissected using both forward and reverse genetic
approaches. The evolutionary relationships among alleles were determined by
the allele-specific single nucleotide polymorphisms (SNPs) using a large
range of rice germplasms. The detailed results will be presented in the
meeting.
Genotypic and pathotypic diversity of the Xanthomonas oryzae pv.
oryzicola in southern China
R.-W. Pan (1), W.-C. Zou (1), D.-G. XU (1), R.-Q. Pan (1), C.-Y. Ji (1)
(1) Department of Plant Pathology, South China Agricultural University,
Guangzhou, PRC PEOPLES REP OF CHINA
Phytopathology 100:S96
Rice bacterial leaf streak (BLS), caused by the pathogen Xanthomonas oryzae
pv. oryzicola (Xoc), is currently one of the most important diseases in
southern China, and the utilization of resistance is the major measure to
control the disease. Total 179 strains of the pathogen were collected from four
provinces in southern China, and the pathotype and the population genetic of
the pathogen were studied. PCR-based DNA fingerprint and virulence
analysis were used to evaluate the genetic diversity and population structure.
Three specific primers ERIC, BOX, and J3, each produced 150, 137, and 126
haplotypes, respectively. Combined the data from three primers, at a similarity
level of 0.73, 11 clusters were grouped. Cluster D, represented 31.3% of the
strains, was the predominant group in southern China. The value of the
genetic diversity (0.826) indicated the highly genetic diversity of the pathogen
population in southern China. These strains were classified into ten groups on
the basis of the phenotypic reaction on six differential rice cultivars (IRBB4,
IRBB5, IRBB14, IRBB21, IR24, and Jingang30). Strains obtained from four
provinces or different planting season shared various DNA fingerprints.
Pathotype I was the predominant group in southern China including 31.28%
of the strains. No correlation was observed between DNA fingerprints and
pathotypes of the pathogen.
Identification of soybean genes that contributes to Rpp2-mediated defense
against Asian soybean rust using VIGS
A. K. PANDEY (1), C. Yang (1), C. Zhang (1), K. F. Pedley (2), M. Graham
(3), J. H. Hill (1), S. A. Whitham (1)
(1) Plant Pathology, Iowa State University, Ames, IA, U.S.A.; (2) Foreign
Disease-Weed Science Research Unit, United States Department of
Agriculture-Agricultural Research Service, Ft. Detrick, MD, U.S.A.; (3) Corn
Insects and Crop Genetics Research Unit, United States Department of
Agriculture-Agricultural Research Service, Ames, IA, U.S.A.
Phytopathology 100:S96
Asian soybean rust (ASR) is an aggressive foliar disease caused by the
obligate biotrophic fungus Phakopsora pachyrhizi. On susceptible cultivars of
soybean, the disease symptoms are characterized by tan lesions and abundant
uredinia. None of the commercially grown elite cultivars of soybean are
resistant to all the isolates of P. pachyrhizi. Germplasm screening efforts have
identified five different genes that provide varying levels of resistance to
specific isolates of P. pachyrhizi (Rpp1 - Rpp5). Resistance in Rpp2 plants
(PI230970) is characterized by the formation of reddish-brown (RB) lesions,
limited fungal growth, and little or no sporulation. In the present work, we
utilized virus-induced gene silencing (VIGS) to identify genes that contribute
to Rpp2-mediated resistance. We screened approximately 140 genes and
identified eleven that compromise resistance when silenced. Gene silencing
was confirmed by semi-quantitative RT-PCR and fungal growth was
measured by quantifying P. pachyrhizi α-tubulin transcript in the silenced leaf
tissue. A clear correlation was observed between the increase in fungal growth
and the silencing of the eleven soybean genes identified, suggesting their
involvement in Rpp2-mediated disease resistance Our results provide
fundamental information on the signaling networks that contribute to
resistance towards ASR, and will assist further efforts to engineer durable
resistance.
S96
PHYTOPATHOLOGY
Identification of Xanthomonas sp. using resonance Raman spectroscopy
M. L. Paret (1), S. K. Sharma (2), A. K. Misra (2), T. Acosta (2), A. S. de
Silva (1), T. Vowell (1), A. M. ALVAREZ (1)
(1) Department of Plant and Environmental Protection Sciences, University of
Hawaii at Manoa, Honolulu, HI, U.S.A.; (2) Hawaii Institute of Geophysics
and Planetology, Honolulu, HI, U.S.A.
Phytopathology 100:S96
We have used resonance Raman spectroscopy with 514.5 nm laser excitation
for characterizing xanthomonadin in numerous plant pathogenic
xanthomonads including Xanthomonas campestris pvs. campestris,
armoraciae, abberans, citrumelo, X. citri, X. vesicatoria, X. translucens, X.
axonopodis pvs. dieffenbachiae and manihotis. Resonance Raman
spectroscopy has been extensively utilized with visible laser excitation in the
characterization of carotenoids, which have structurally unique conjugated
polyene chain that gives these pigments absorption in the blue-green region of
the spectrum. A molecule similar to carotenoids is xanthomonadin, the
brominated aryl-polyene pigment in the bacterial genus Xanthomonas.
Xanthomonadin is a unique marker for the genus, but the identification
technique currently used is thin-layer chromatography, which requires a long
processing time, and also can have poor resolution. The Raman bands
representing the vibrations (v1, v2, v3) of the polyene chain of xanthomonadin
is 1004 (v3), 1136 (v2), 1529 (v1), 2267 (2v2), and 2656 (v1+ v2). These
separated out from the Raman fingerprints of other bacterial genera, and
results were obtained within 5 minutes.
AiiA-mediated quorum-quenching does not affect virulence or toxoflavin
experession in Burkholderia glumae SL2376
J. PARK (1), S. Han (1), Y. Lee (2), B. B. McSpadden Gardner (3), Y. Kim (1)
(1) Gwangju, SOUTH KOREA; (2) Suwon, SOUTH KOREA; (3) Wooster,
OH, U.S.A.
Phytopathology 100:S96
Burkholderia glumae causes bacterial rice grain rot and produces a broad-host
range phytotoxin, called toxoflavin, which is a key pathogenicity factor. The
quorum sensing signaling pathway and its connection with pathogenicity of B.
glumae have yet to be clearly elucidated. In an effort to investigate the effects
of quorum-quenching on the pathogenicity of B. glumae, the N-acylhomoserine lactonase (aiiA) gene from Bacillus sp. 240B1 was expressed in
B. glumae under the control of a constitutive promoter. Production of acyl
homoserine lactones in the aiiA-transformants was significantly reduced, but
no evidence of loss of virulence was observed in terms of causing rice
seedling rot and rice grain rot. The aiiA-expressing strains displayed wild-type
levels of transcription from the genes in the toxoflavin biosynthetic operon
and toxin production. The aiiA-expressed strains swarmed and formed flagella
similar to the wild-type strain. However, the aiiA-expressing B. glumae strain
reduced the severity of soft-rot when co-inoculated with the soft-rot pathogen,
Pectobacterium carotovorum SCCI, consistent with its lactonase activity. Our
results show that the aiiA-mediated quorum-quenching does not affect
virulence or toxoflavin production in B. glumae SL2376.
Protein profile differences between soybean accessions resistant and
susceptible to soybean rust (Phakopsora pachyrhizi)
S. PARK (1), Z. Chen (1), M. C. Ganiger (1), A. A. Fortunato (1)
(1) Louisiana State University Agricultural Center, Baton Rouge, LA, U.S.A.
Phytopathology 100:S96
Asian soybean rust, caused by fungus Phakopsora pachyrhizi, was first
discovered in the continental U.S. in late 2004 and has the potential to cause
severe yield losses due to that all U.S. commercial soybean varieties are
susceptible. In this study, thirteen accessions were evaluated with rust spores
collected in Louisiana. Two accessions (PI417089A and PI567104B) showed
consistent immune response in both detached leaf assay and greenhouse
inoculation. Fungal biomass as determined using qRT-PCR increased
significantly 2 days after infection in susceptible lines whereas no or little
increase was detected in resistant lines. Protein profiles of these two resistant
and two susceptible lines (PI548631 and 93M60) were compared to
understand compatible and incompatible host-pathogen interactions at the
molecular level using proteomics. Differentially expressed proteins were
observed in both resistant and susceptible lines with and without infection.
Nine and 16 proteins were identified as induced spots at 1 day after infection
in both resistant accessions after comparing to PI548631 and 93M60
susceptible line, respectively. Sixteen spots were sequenced and they belong
to plant defense, signaling, and photosynthesis. The transcript levels of
differentially expressed proteins were also determined using qRT-PCR.
Distinct roles of two 9-lipoxygenase paralogs in the regulation of aflatoxin
accumulation in maize seed
Y. PARK (1), M. Kolomiets (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S96
Colonization of kernels by Aspergillus flavus is one of the major limiting
factors for maize production worldwide because they contaminate seed with
highly carcinogenic aflatoxins. Host and pathogen-derived oxygenated lipids
are implicated as signals in the regulation of biosynthesis of aflatoxins. In this
study, we tested whether disruption of the host lipoxygenases, ZmLOX4 and
ZmLOX5, have an effect on aflatoxin accumulation of seed. The two genes are
highly related to each other sharing >94% nucleotide and amino acid sequence
identity. Field-based testing for the mutants and near-isogenic wild types
showed that lox5 mutants accumulated significantly lower levels of aflatoxin,
whereas lox4 mutants did not show any significant difference. Northern blot
analyses demonstrated differential induction of the two genes in response to
infection of silks and kernels with A. flavus. Taken together, these data
indicate that despite extremely high sequence similarity between the two
paralogs, only ZmLOX5 is required for normal production of aflatoxins under
the filed conditions. Potential mechanisms of ZmLOX5 involvement in the
regulation of aflatoxin production are proposed.
Genetic stability of Magnaporthe oryzae isolates on the host and artificial
media
S. PARK (1), M. Chi (1), H. Kim (1), S. Kang (2), Y. Lee (1)
(1) Seoul National University, Seoul, KOREA; (2) Pennsylvania State
University, State College, PA, U.S.A.
Phytopathology 100:S97
Although genetic instability in the rice blast fungus is a highly controversial
subject, only a few strains have been used in genetic studies for more than two
decades. Here we report genetic stability of Magnaporthe oryzae isolates on
the host and artificial media. A total of 176 strains were obtained by culturing
single spores on artificial medium during successive round of asexual
reproduction and by infecting rice plants up to the 10th generation from
isolate 70-15. Another 20 strains were recovered from both ends of germ
tubes at basal and apical cells from 10 conidia which are generally three-cells.
Additionally, 60 strains were recovered by serial transfer on rice plants and
the medium from isolate KJ201. All strains exhibited no apparent difference
in several phenotypes, including mycelial growth, conidial morphologies,
conidial germination, appressorium formation, and pathogenicity, and in DNA
fingerprints using MGR586, MAGGY, Line, and MG-SINE as probes. Our
data strongly suggest that phenotype and genotype of M. oryzae isolates are
maintained stably, at least, up to the 10th successive generations on the
cultural medium and the host plant.
Phylogenetic and functional characterization of putative forkhead
transcription factors in the rice blast fungus
J. PARK (1), J. Jeon (2), S. Park (1), S. Kong (1), J. Park (1), Y. Lee (1)
(1) Department of Agricultural Biotechnology, Center for Fungal Genetic
Resources, and Center for Fungal Pathogenesis, Seoul National University,
Seoul, SOUTH KOREA; (2) Department of Agricultural Biotechnology,
Seoul National University, Seoul, SOUTH KOREA
Phytopathology 100:S97
Forkhead-box protein, named from the two spiked-head mutants of
Drosophila, is a transcription factor (TF) playing essential roles in a wide
range of biological processes such as cell cycle progression, growth
regulation, and developmental differentiation in eukaryotic organisms.
Although a few forkhead TFs were characterized in yeasts, very little
knowledge is available for filamentous fungi. To examine the phylogenetic
relationships, a total of 62 putative forkhead TFs, including 4 of Magnaporthe
oryzae, from 16 fungal species were identified and analyzed. This analysis
showed that 2 of M. oryzae forkhead TFs (named as MoFKH1 and MoHCM1)
are yeast-related and others (MoFOX1 and MoFOX2) are filamentous fungispecific. Deletion mutants of MoFOX1, a filamentous fungi-specific forkhead,
were indistinguishable to the wild-type in their phenotypes. No deletion
mutant of MoFOX2 was obtained notwithstanding several attempts of
transformation and extensive screening of transformants, indicating the
possibility of an essential gene. However, deletion mutants of both MoFKH1
and MoHCM1 exhibited pleiotrophic phenotype defects, such as conidial
germination, appressoria formation, mycelial growth, and pathogenicity.
Furthermore, defects in septa formation were only observed in deletion mutant
of MoFKH1, but not in MoHCM1. These results suggested that yeast-related
forkhead TFs in M. oryzae showed functional links to the corresponding TFs
of Saccharomyces cerevisiae.
FEDB: The integrated platform for expressed sequence tags in fungal
kingdom
J. PARK (1), S. Kim (2), W. Song (3), S. Kim (3), S. Jang (3), K. Cheong (1),
D. Kim (1), J. Choi (1), K. Ahn (3), B. Park (4), D. Choi (5), S. Kang (4), Y.
Lee (1)
(1) Fungal Bioinformatics Laboratory, Center for Fungal Genetic Resources,
Center for Fungal Pathogenesis, and Department of Agricultural
Biotechnology, Seoul National University, Seoul, KOREA; (2) Fungal
Bioinformatics Laboratory, Department of Plant Science, and Department of
Agricultural Biotechnology, Seoul National University, Seoul, KOREA; (3)
Fungal Bioinformatics Laboratory, Seoul National University, Seoul,
KOREA; (4) Department of Plant Pathology, The Pennsylvania State
University, University Park, PA, U.S.A.; (5) Deaprtment of Plant Science,
Seoul National University, Seoul, KOREA
Phytopathology 100:S97
Regulation of gene expressions plays an essential role in modulating diverse
biological processes. One conventional way to survey the expression level is
expressed sequence tags (ESTs) which provide messenger RNA sequences
under certain conditions. With the combination of new genome sequencing
technologies and many fungal genome projects, large amount of fungal ESTs
have been generated; however, there is no proper data warehouse for these
ESTs. Fungal Expression Database (FEDB; http://fedb.snu.ac.kr/)
standardized fungal ESTs with Comparative Fungal Genomics Platform
(CFGP; http://cfgp.snu.ac.kr/), allowing free-data exchange with various
bioinformatics databases. Currently FEDB archives 3.1 million ESTs from
101 fungal species. To generate unigenes, three-step pipeline was constructed: i) crossmatch was integrated for removing vector sequences,
ii) polyA/T filter for eliminating poly A/T sequences, and iii) CAP3 and Phrap
for generating unigenes. To provide user-interactive web interfaces,
i) Taxonomy Browser to browse ESTs along with fungal taxonomy, ii)
SNU Genome Browser (SNUGB; http://genomebrowser.snu.ac.kr/) to present
ESTs matched to proper fungal genomes, and iii) Favorite, the personalized
virtual space containing various EST analysis tools, were integrated. With the
tools implemented in FEDB and the free-data exchange model, FEDB will
provide the environment for diverse in-depth analyses of fungal
transcriptomics.
Differentiation of Xylella fastidiosa
environmentally-mediated genes
J. K. PARKER (1), L. De La Fuente (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S97
strains
via
analysis
of
Attempts to elucidate the genetic relationships between strains of Xylella
fastidiosa (XF) have shown that genotypes tend to cluster into groups based
on the host plant species from which they were isolated. Comparative genetic
analyses have been conducted for isolates from host plants of economic
importance including grapes, citrus, and almonds. However, differentiation of
strains from within host types (e.g. between grape isolates) and
characterization of strains from newly identified hosts (e.g. blueberries) have
been limited and/or inconclusive. Here, sequence analysis of environmentallymediated genes (genes thought to be influenced by environmental factors) was
applied to identify strain relationships. Multi-locus sequence analysis (MLSA)
was used for genes related to processes important for establishing XF
infections such as surface attachment, biofilm formation, virulence, and
nutrient transport and utilization. These types of genes may be more relevant
to host-based genetic variability. Genes of interest were identified from
previous studies and PCR primers were designed from the available whole
genome sequences of XF. Target genes were PCR-amplified and sequenced
from XF strains isolated from a variety of host plants from several geographic
regions in the U.S. Phylogenetic analyses of the resulting sequence
information show host-based and geographic origin-based genetic
relationships which provide new insights into the epidemiology of populations
of XF.
Diversity of the tobacco black shank pathogen, Phytophthora nicotianae,
in Virginia
V. PARKUNAN (1), C. Johnson (2), C. Hong (1)
(1) VPI & State University, Virginia Beach, VA, U.S.A.; (2) VPI & State
University, Blackstone, VA, U.S.A.
Phytopathology 100:S97
Black shank, caused by Phytophthora nicotianae, is a significant problem in
tobacco-growing regions throughout the world, including Virginia. A total of
217 isolates of P. nicotianae were collected from stem pith samples from fluecured, burley, and dark fire-cured tobacco sampled from fields in 12 Virginia
counties in 2006–09. Isolate race identities were determined using host
differential assays. Seventy-six percent of the isolates were race 1, 21% race
0, and 3% race 3. The mating type was determined based on a conventional
pairing assay using P. meadii and/or P. nicotianae testers. In contrast to an
earlier North Carolina study, 94 percent of the isolates were of the A2 mating
type; only 6 percent of the isolates belonged to the A1 mating type. A single
mating type in most of Virginia’s tobacco fields may indicate a lower
possibility for sexual recombination, possibly creating a biological bottleneck
to adaptation by the pathogen population. A detailed genetic diversity study is
underway employing simple sequence repeats and random amplified
polymorphic DNA markers.
Vol. 100, No. 6 (Supplement), 2010
S97
New races of the rust pathogen in the United States affect Ur-3 a broadly
used rust resistance gene in common bean
M. A. PASTOR-CORRALES (1)
(1) USDA ARS, Beltsville, MD, U.S.A.
Phytopathology 100:S98
Common bean rust is caused by Uromyces appendiculatus, a hyper variable
pathogen that is notorious for its capacity to recurrently produce new virulent
strains. Rust symptoms were found on dry bean varieties with Ur-3, a gene
that had previously conferred resistance to bean rust and had been used
extensively in the development of dry bean cultivars. Two new strains of this
pathogen were found in Michigan and North Dakota in 2007 and 2008,
respectively. These two states are the largest producers of dry beans in the
U.S. Our virulence studies revealed two similar but not identical races; one
from Michigan (22-2) and the other from North Dakota (20-3). Both races
infected the differential cultivars Aurora (Ur-3) and Golden Gate Wax (Ur-6).
Neither race infected Early Gallatin (Ur-4), Mexico 309 (Ur-5), Pompadour
Checa 50 (U-9, Ur-12), and PI 181996 (Ur-11). These races differed in their
virulence; only 22-2 infected Redlands Pioneer (Ur-13) and only 20-3 infected
Great Northern 1140 (Ur-7). Other sources of resistance, such as CNC and PI
260418, with uncharacterized rust resistance genes, were also resistant to both
new races. Approximately 40 U.S. common bean cultivars were also
inoculated with the two new races. The results revealed new dry bean
cultivars, some with two or more rust resistance genes possessed a broad rust
resistance to the new races from Michigan and North Dakota and to different
races from other parts of the world.
Differential reactions of sources of Rpp-resistance to an Rpp-virulent
isolate of Puccinia polysora
J. K. PATAKY (1), W. E. Dolezal (2), J. L. Brewbaker (3)
(1) University of Illinois, Dept. Crop Sciences, Urbana, IL, U.S.A.; (2)
Pioneer Hi-Bred International, Inc., Johnston, IA, U.S.A.; (3) University of
Hawaii-Manoa, Dept. Tropical Plant and Soil Sciences, Manoa, HI, U.S.A.
Phytopathology 100:S98
Southern rust, caused by Puccinia polysora, occurs on maize grown in
subtropical or tropical regions and in temperate climates of the continental
United States. It is distinct from common and tropical rusts, caused by P.
sorghi and Physopella zeae. A single dominant gene Rpp9, described from PI
186208 (Boesman yellow flint), is used in North America as a source of a
chlorotic fleck, resistant reaction against P. polysora; however, this gene is
ineffective in many parts of the world due to virulent isolates. The Rpp9 gene
is located on chromosome 10S about 1.6 cM from the Rp1 region of genes
conveying resistance to P. sorghi. Several additional sources of Rppresistance map to the same region of 10S as Rpp9 or they are allelic with Rpp9
based on resistance of testcross progeny. In 2008, isolates of P. polysora
virulent on corn lines with the Rpp9 gene, including PI 186208, were collected
in Georgia. Several sources of resistance thought to carry the Rpp9 gene were
resistant when inoculated with a wild-type isolate of P. polysora from Illinois,
susceptible when subjected to wild type isolates at Waimanalo, Hawaii, but
had differential reactions when inoculated with Rpp-virulent isolates from
Georgia. The differential reactions of these lines suggests that genes in
addition to Rpp9 are involved in the southern rust resistance of some of these
Rpp-resistant lines and/or that the Rpp9 gene is a complex region of multiple
resistance genes similar to the Rp1 region for common rust resistance.
Characterization of Type IV pilus in the bacterial biocontrol agent
Lysobacter enzymogenes strain C3
N. PATEL (1), B. Hillman (1), D. Kobayashi (1)
(1) Rutgers The State University of New Jersey, New Brunswick, NJ, U.S.A.
Phytopathology 100:S98
Gliding motility and pathogenesis of lower eukaryotes are distinct features of
the Gram negative bacterium Lysobacter enzymogenes. While both are
predicted to play important roles in microbial antagonism by the biological
control agent L. enzymogenes strain C3, the molecular mechanisms that
contribute to these traits are poorly understood. Type IV pili (T4P) are known
to contribute to a variety of roles in bacteria, including gliding motility, host
adherence and pathogenesis. Genes encoding for the biogenesis of a type IV
pilus (T4P) have been identified in three unlinked pil loci within the genome
sequence L. enzymogenes strain C3. To evaluate the role of T4P in various
functions in L. enzymogenes C3, a deletion mutation within the major pilin
subunit pilA gene was constructed. Colonies of the resultant mutant strain
appeared as dry circular colonies with lobate margins during growth on 10%
tryptic soy agar, in contrast to fluid circular colonies with entire margins
produced by the wildtype strain. The mutant strain also lacked the spreading
phenotype of gliding motility on solid growth media. During interactions with
fungal hosts, the pilA mutant strain was capable of infecting fungal cells
intracellularly in similar manner to the wildtype strain, but it remains unclear
whether virulence of the mutant is reduced.
S98
PHYTOPATHOLOGY
Probability modeling of pecan scab using weather variables as inputs
A. F. PAYNE (1), D. L. Smith (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S98
Pecan scab, caused by the fungus Cladosporium caryigenum (syn.
Fusicladium effusum), affects foliage and fruit of pecan trees resulting in
reduced quality and yield. Improving the accuracy of models that predict scab
infection would be useful for timing fungicide sprays. Visual ratings of fruit
scab severity were collected in 1994, 1995, 1996, and 2009 growing seasons.
Daily weather data were compiled from the Oklahoma Mesonet. Moving
averages were calculated with respect to days between disease ratings for
selected weather variables. Rainfall (≥2.5 mm) and disease severity (≥25%)
thresholds were converted to dichotomous variables where 1 was above and 0
below the threshold for each variable. Weather variables were used as
independent variables and disease severity (DS) as the dependent variable
using generalized estimating equations. Goodness of fit was determined using
the quasi-log-likelihood under the independence model information criteria
adjusted for number of model parameters (QICu). Minimizing QICu
demonstrates better model fit. Weather variables examined were temperature
(T), relative humidity (RH), dew point (DP), solar radiation (SR), and total
rainfall (R). Model 1 included T, RH, SR, and R to describe DS (QICu =
167.99). Model 2 included DP, SR, and R (QICu = 177.14). The modest
increase in QICu of Model 2 demonstrates that DP can be used as accurately
as T and RH when modeling DS. These data suggest that DP may be an
important predictor of pecan scab.
One Step Construction of Agrobacterium Recombination-ready plasmids
(OSCAR), an efficient and robust tool for ATMT gene deletion
construction in fungi
Z. Paz (1), S. E. GOLD (1), D. L. Andrews (1), S. J. Klosterman (2), M. D.
García-Pedrajas (3), L. Baeza-Montañez (3)
(1) University of Georgia, Athens, GA, U.S.A.; (2) USDA-ARS, Salinas, CA,
U.S.A.; (3) Laboratorio de Micologia Estacion Experimental La Mayora,
CSIC, Algarrobo-Costa, Malaga, SPAIN
Phytopathology 100:S98
We describe OSCAR, a novel method for the rapid generation of gene
deletion constructs for Agrobacterium tumefaciens-mediated transformation
(ATMT) in fungi. We designed i) a marker vector with a hygromycin B
resistance gene with the Aspergillus nidulans trpC promoter, flanked by the
attP1r and attP4 recombination sites and ii) a modified binary vector
containing the ccdB gene flanked by attP2r and attP3 recombination sites. 5′
and 3′ gene flanks with attB2r/attB1r and attB4/attB3 recombination sites,
respectively are generated by PCR. A single BP clonase reaction containing
both the above vectors and PCR flanks results in a deletion plasmid containing
the resistance cassette surrounded by the upstream and downstream gene
flanks, all contained between the T-DNA borders. The genome sequence of
Verticillium dahliae (http://www.broad.mit.edu/) allows functional analysis
using OSCAR. Deletion constructs for six genes were obtained at high
frequency. One OSCAR construct (VDAG_02161) was tested in fungal
ATMT and resulted in a complete gene deletion. Additionally, the method has
been validated in several collaborating laboratories and is being used in an
undergraduate course at the UGA. In summary, OSCAR combines PCR and
Gateway technology, replacing the more time consuming and expensive
Invitrogen MultiSite Gateway® Technology, to rapidly and robustly generate
precise deletion constructs for ATMT based homologous gene replacement.
Correlation between antibody-binding properties and seasonal disease
severity indices of Dickeya sp. that cause bacterial heart rot of pineapple
G. D. Peckham (1), A. de Silva (1), J. M. Berestecky (2), A. M. ALVAREZ
(1)
(1) University of Hawaii, Honolulu, HI, U.S.A.; (2) Kapiolani Community
College, Honolulu, HI, U.S.A.
Phytopathology 100:S98
Bacterial heart rot of pineapple in Hawaii is caused by strains initially
identified as Erwinia chrysanthemi, now reclassified as Dickeya sp. The
species designation of pineapple strains awaits full genetic characterization.
Based on BOX-PCR the pineapple strains fell into five genetic groups (A-E).
Differences in seasonal disease severity were observed between BOX-PCR A
and C types, C types being the most severe. Monoclonal antibodies (MAbs)
were generated to facilitate rapid detection and identification of these virulent
strains from diseased plant material. One antibody (Pine-1, clone 2D11G1;
IgG1 with kappa light chain) recognized all of the strains isolated from
Hawaiian pineapple (A-E). Another antibody (Pine-2, clone 2A7F2; IgG3 with
kappa light chain) recognized only a subset of these strains (BOX-PCR types
BCD). In the initial outbreak both A and C type strains were frequently
isolated. However, in subsequent years only C types have been rediscovered.
Since the antibodies distinguish between A and C types, they have value in
on-going studies to evaluate the spread and establishment of the most virulent
bacterial populations associated with the initial disease outbreak.
Evaluation of runner bean germplasm for virus resistance
E. Peña-Reyes (1), G. Bascur (1), P. Mendoza (1), P. Sepúlveda (1), I.
ROSALES VILLAVICENCIO (1)
(1) Instituto de Investigaciones Agropecuarias, Santiago, CHILE
Phytopathology 100:S99
Common bean (Phaseolus vulgaris L.) is one of the most important grain
legumes and the leading source of low cost quality proteins. For several years
INIA has developed a bean-breading program whose main objectives are to
breed cultivars with special plant habits such as high yields, adaptability to
mechanical harvesting and resistance to viral diseases, which are a limiting
factor for bean production in Chile. Considering that the most effective way to
control viral diseases is the use of resistant material, the main objective of this
work was to search for sources of resistance against the most important viral
agents affecting this crop in Chile: Bean common mosaic virus (BCMV),
Bean yellow mosaic virus (BYMV), Alfalfa mosaic virus (AMV) and
Cucumber mosaic virus (CMV). Sixteen accessions of runner bean (Phaseolus
coccineus L.) were mechanically and naturally infected with each one of the
four viruses under study. The plants were regularly evaluated for viral
infection by using diagnostic techniques specifically developed for the
identification of the pathogens, such as RT-PCR and immunotissue blot. The
results indicated that only two accessions were immune to all viruses studied,
and 10 of them show resistance to the new and emerging viral complex
affecting bean production, which is caused by CMV and AMV. Additionally,
we are at this time confirming the first identification of Bean common mosaic
necrosis virus (BCMNV) in bean fields located at INIA Research Station.
Management of clubroot (Plasmodiophora brassicae) with microbial and
synthetic fungicides
G. Peng (1), S. Hwang (2), M. McDonald (3), R. Lahlali (1), B. D. GOSSEN
(1), R. H. Hynes (1), S. M. Boyetchko (1)
(1) Agriculture & Agri-Food Canada, Saskatoon, SK, CANADA; (2) Alberta
Agriculture and Rural Development, Edmonton, AB, CANADA; (3)
University of Guelph, Guelph, ON, CANADA
Phytopathology 100:S99
Clubroot, caused by Plasmodiophora brassicae, is an emerging threat to
canola (Brassica napus) production in western Canada and a serious problem
on Brassica vegetable crops worldwide. In inoculated trials on canola under
controlled conditions, the biofungicides Serenade® (Bacillus subtilis) and
Prestop® (Gliocladium catenulatum), and the synthetic fungicides fluazinam
and cyazofamid reduced clubroot severity substantially when applied as a soil
drench at 1.4 Kg, 10 L, 2.9 L, and 0.54 L/ha, respectively. Suspensions of the
biofungicides made with pure bacterial or fungal cultures were as efficacious
as the formulated products. Cell-free product filtrates were slightly less
effective. These fungicides were also evaluated as in-furrow treatment at the
same rates as above in three field trials on canola in Alberta and Ontario, and
one napa cabbage (B. rapa subsp. Chinensis var. utilis) trial in Ontario.
Resistant (R) and susceptible (S) cultivars were assessed in each trial. Plants
(20-25 per rep) were assessed for clubroot severity using a 0–3 scale at bloom
(canola) or 8 wks after seeding (napa cabbage). Severe spring drought
conditions in Alberta delayed germination and reduced seedling
establishment. None of the products reduced clubroot on canola in the Alberta
trials. For the napa cabbage trial, a rain event occurred 2 d after seeding. The
fungicides reduced clubroot severity on the S cultivar by 50–85%. R cultivars
reduced severity by 87–93% on canola and 99% on napa cabbage when
compared to S cultivars.
Resistance of triploid watermelon cultivars to Fusarium wilt caused by
Fusarium oxysporum f. sp. niveum
K. PENNERMAN (1), K. Everts (2)
(1) University of Maryland, College Park, MD, U.S.A.; (2) University of
Maryland and University of Delaware, College Park, MD, U.S.A.
Phytopathology 100:S99
Yield loss in watermelon due to Fusarium wilt (Fusarium oxysporum f. sp.
niveum) was managed for many years through the use of cultivars that were
resistant to F. o. niveum race 1. However, the prevalence and severity of
Fusarium wilt in watermelon has been increasing in the eastern U.S. in the
past decade. This increase has coincided with an increase in production of
triploid watermelon cultivars, most of which lack resistance to race 1. A trial
was conducted in 2009 in a field at the University of Delaware’s Research and
Education Center, Georgetown to evaluate thirteen triploid watermelon
cultivars that had previously been identified as possessing some level of
resistance to one or more races (races 0 and 1) of F. o. niveum and a
susceptible check cultivar. Watermelons had been continuously planted in the
field for eleven years resulting in a moderate level of F. o. niveum. ‘Matrix’,
‘Sweet Delight’, ‘ACX4674T FR’, ‘ACX5117TSS FR’, ‘ACR6277TSS FR’,
‘ACR6177TSS FR’, and ‘ACX5727 FR’ had significantly lower wilt
incidence than ‘Ruby’ and ‘Indiana’ on 21 July. However, many cultivars did
not perform as well as expected. Isolates of F. o. niveum were collected and
evaluated to determine if F. o. niveum race 2 was present in the field. The
isolates of F. o. niveum along with reference isolates of races 0, 1, and 2 were
compared on four differential watermelon genotypes using the root-dip
inoculation method. The presence of F. o. niveum race 2 was confirmed.
Update on olive pathologies in Argentina
B. A. PEREZ (1), M. L. Otero (2), E. Oriolani (3), M. Roca (4), N. Brancher
(5), A. C. Matias (6)
(1) IMYZA-INTA, Hurlingham, Buenos Aires, ARGENTINA; (2) INTAIFFIVE, Cordoba, ARGENTINA; (3) INTA-EEA, Mendoza, ARGENTINA;
(4) SENASA, La Rioja, ARGENTINA; (5) Ministerio de la Produccion,
Catamarca, ARGENTINA; (6) INTA EEA, Catamarca, ARGENTINA
Phytopathology 100:S99
Olive (Olea europaea) is grown in Catamarca, La Rioja, Mendoza, San Juan,
south Buenos Aires, northwest Cordoba, and eastern Rio Negro provinces.
The aim was to determine the current health status in the traditional and new
olive growing area throughout 2009. Disease surveys included several
locations of the cited provinces. Plant samples were analyzed by routine
laboratory techniques. Olive knot disease was in both young and old plants in
Mendoza, San Juan, and south Buenos Aires in orchards with hail damage.
Root rot diseases were recorded in field plants in Buenos Aires (Aparicio,
Dorrego), Catamarca (Central Valley), La Rioja (Chilecito), Mendoza (Atuel,
Lavalle, Lujan de Cuyo, Maipu), Rio Negro (Conesa, Viedma.), and San Juan
(Pocito). Sclerotium basal rot was detected in nursery plants in Catamarca, La
Rioja, and San Juan. Plants with Verticillium wilt were present in Chilecito,
Lavalle, La Rioja Capital, Lujan de Cuyo, and Maipu. Phomopsis twig blight
was noted in field plants (Central Valley). Foliar and fruit diseases were more
evident in older orchards and less in younger plants throughout the surveyed
olive zone. Partially dry plants were more evident in La Rioja but were also
observed in Mendoza, and San Juan. Crown gall is restricted to north and
northwest Cordoba. Symptoms such as bleeding in branches and trunks,
vitiligo, lumps and lattices appeared in young and old plants in La Rioja
(Aimogasta, La Rioja Capital) and Catamarca (Central Valley, Tinogasta).
Differente leaf distortions (sickle, bifida, bell and cup shaped leaves) were
widespread in the olive zone.
Blueberry pathologies in nursery plants
B. A. Perez (1), E. R. WRIGHT (2), M. Divo de Sesar (2)
(1) IMYZA-INTA, Hurlingham, Buenos Aires, ARGENTINA; (2) Facultad
de Agronomía, Universidad de Buenos Aires, Ciudad de Buenos Aires,
ARGENTINA
Phytopathology 100:S99
Blueberry plants (Vaccinium corymbosum) are obtained by vegetative
propagation under different conditions. The purpose of this study was to
identify the fungi present in plants raised in nurseries under a black shading
net in Buenos Aires. Leaf samples cvs. Sharp Blue and Misty with dark and
red spots were collected in summer months (January to March) in 2008 and
2010 and processed in laboratory using routine techniques. A stereo
microscope was used to observe rust pustules, to prepare slides for
microscopic observations and transfer fungal spores that developed on leaves
incubated in moist chambers to 2% WA. Fungal purification was made by
subculturing the point of hyphae in WA and then cultured in GYE and MYE
agar media. The fungi Colletotrichum gloeosporioides and Pestalotiopsis
guepini were identified. Rust pustules and urediospores of Pucciniastrum
vaccinii were recorded in the underside of the leaves in correspondence with
dark spot on the upper side of affected leaves of cv. Sharp Blue. This cultivar
had a susceptible reaction to rust and was also the most susceptible under field
conditions in San Pedro area (Buenos Aires).
Alterations of capsid protein amino acid positions internal to virions of
Cucumber mosaic virus disrupt nonpersistent virus transmission by
aphids
K. PERRY (1), C. Bricault (1)
(1) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S99
In the atomic model of Cucumber mosaic virus (CMV), six amino acid
residues form stabilizing salt bridges between subunits of the asymmetric unit.
To evaluate the effects of these positions on virion stability and aphid vector
transmission, six charged amino acid residues were individually mutated to
alanine. Five of the six engineered viruses, mutants D100A, K127A, D176A,
D179A, and K182A, were viable and able to systemically infect Nicotiana
tabacum and to locally infect Chenopodium quinoa; mutant K101A could only
be recovered with difficulty. In order to assess the physical stability of
mutants, virions were purified from plants and tested in a urea disruption
assay. All five mutant viruses were stable during purification in the presence
Vol. 100, No. 6 (Supplement), 2010
S99
of 1.5 M sodium and chloroform and exhibited wild type levels of virion
stability in the presence of urea. Aphid vector transmissibility of three of the
five mutants, D100A, K127A, and D176A, was nearly eliminated. After a
series of mechanical passages, additional second site mutations were selected
in four of the five mutant viruses, including in one compensatory mutation
that restored wild type levels of aphid transmission. It is hypothesized that
non-surface associated amino acids involved in acid-base pairing affect the
dynamic properties of virions that are in turn required for aphid vector
transmission.
A tospovirus new to North America: Virus detection and discovery
through the use of a macroarray for viruses of solanaceous crops
K. PERRY (1), X. Lu (1)
(1) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S100
Winter planted tomato plants with virus-like symptoms of leaf distortion,
mottle-mosaic, and necrotic stem streaking were observed in south Florida in
January 2010. Extraction of total plant RNAs and analysis using a macroarray
for the detection of viruses of solanaceous crops revealed hybridization to 70mer oligonucleotide probes designed for the detection of tospoviruses. Probes
with hybridization signals contained sequences from the genomes of Tomato
chlorotic spot virus (TCSV), Impatiens necrotic spot virus, and Tomato
spotted wilt virus, with the majority from TCSV. The macroarray did not
contain probes for the related Groundnut ringspot virus (GRSV), as this virus
had not been reported from solanaceous plants. Sequences from the S RNA
showed 91% identity with GRSV but only 78% identity with TCSV.
Sequencing of an M RNA derived, cDNA fragment suggests a closer
relationship with TCSV. A more complete analysis of the viral genome and
investigation of its vector relationships in progress will provide a better
understanding of the nature of the virus.
Host proteins implicated in the aphid transmission of cereal yellow dwarf
virus-RPV
K. PETER (1), M. Cilia (2), F. Gildow (1), T. Thannhauser (2), S. Gray (3)
(1) Penn State University, University Park, PA, U.S.A.; (2) USDA-ARS,
Robert W. Holley Center for Agriculture and Health, Ithaca, NY, U.S.A.; (3)
USDA-ARS, Robert W. Holley Center for Agriculture and Health and Cornell
University, Department of Plant Pathology and Plant-Microbe Biology,
Ithaca, NY, U.S.A.
Phytopathology 100:S100
carrots may still enter storages, where lateral spread of the pathogen from
diseased to healthy carrots can occur rapidly. To manage the post-harvest
spread of the pathogen, applications of low rates of fludioxonil as a dip
treatment as carrots enter storage was shown to virtually eliminate lateral
spread of the pathogen in storage crates. Therefore, a comprehensive
management strategy for SRC will include careful decisions on cultivar
choice, seeding rate and fertility inputs as well as the integration of canopy
trimming and post-harvest fludioxonil application.
The relationship between genetic diversity of Ralstonia solanancearum
and mechanical transmission in tobacco
P. D. PETERSON (1), B. A. Fortnum (1), A. L. Mila (2)
(1) Clemson University, Florence, SC, U.S.A.; (2) North Carolina State
University, Raleigh, NC, U.S.A.
Phytopathology 100:S100
Mechanical flower and leaf removal in tobacco have a major role in the spread
of bacterial wilt in the southeastern U.S.A. Ralstonia solanacearum typically
infects tobacco plants through direct penetration of roots, but infection can
also occur through topping wounds, leaf scars and stem abrasions. The
objectives of this study were to evaluate the aggressiveness of diverse R.
solanacearum isolates when applied to foliar tobacco plant parts during flower
removal and to determine if an Avr-induced resistance response found with
certain isolates in root tissue also occurs in stem tissue. The experiment was
conducted at the Pee Dee Research & Education Center using the tobacco
variety K346 and 23 isolates of R. solanacearum selected for differences in
genetic diversity and aggressiveness. Plots consisted of a single row of 10
plants and the experimental design was a RCB with 4 replications. Inoculation
simulated mechanical flower removal – a steel cutter blade was misted with a
1 × 106 cells/ml suspension of each isolate. A water inoculated treatment was
used as the control. Plants were assessed weekly starting 21 days after
inoculation. Stem necrosis was recorded at the final disease assessment date.
Results showed significant differences in the amount of disease caused by the
selected isolates of R. solanacearum when inoculated to foliar plant parts. The
resistance mechanism functioning against tomato strains in root infections
also appears to function in tobacco stem tissue.
Resistance to DMI fungicides in Venturia inaequalis from Pennsylvania
E. E. PFEUFER (1), J. W. Travis (1), H. K. Ngugi (1)
(1) Penn State University, Biglerville, PA, U.S.A.
Phytopathology 100:S100
The circulative, persistent transmission of cereal yellow dwarf virus-RPV
(CYDV-RPV; Luteoviridae) is orchestrated by interactions between virus and
aphid proteins. Here we present evidence that plant proteins are also required
for successful virus transmission. Treatment of CYDV–RPV-infected oat
tissue homogenate during virus purification with commonly used additives
EDTA and sodium sulfite rendered the purified virus non-transmissible by
aphids. Virus purified in the absence of additives was aphid transmissible.
TEM analysis demonstrated virion structure was similar, and the
elecrophoretic migration of the capsid proteins in 1-D SDS PAGE was
identical. However, the number and molecular weights of additional proteins
consistently co-purifying with virus were different between preparations.
Using mass spectrometry, proteins involved in respiration, carbon metabolism
and carbohydrate breakdown, and histones were identified that co-purified
with both transmissible and non-transmissible virus, indicating these proteins
are closely associated with virions during infection, but are not sufficient for
aphid transmission. Two key enzymes in fructose metabolism co-purified with
only transmissible virus. These results demonstrate that luteovirus
transmission is influenced, in part, by host proteins binding to, or closely
associating with virions. Additives can affect protein structure and proteinprotein interactions and therefore may alter critical interactions necessary for
aphid transmission.
Apple scab, caused by Venturia inaequalis, is the most economically
important disease of apple in the eastern United States. Over the past 25 years,
apple growers have relied on sterol demethylation inhibiting fungicides
(DMIs) for scab control, but reduced efficacy has recently been noted. The
aim of this study was to evaluate the sensitivity of V. inaequalis isolates from
Pennsylvania to DMI fungicides. In 2009, leaves and immature fruit with scab
symptoms were collected from 14 commercial orchards. Growers provided
management history of the sampled plots. A total of 296 single-spore cultures
were isolated from the tissues and maintained individually. Each isolate was
tested for sensitivity to DMIs on 1/4-strength PDA plates amended with a
range of concentrations of myclobutanil, fenbuconazole, or difenoconazole.
Relative growth (RG) values were calculated and isolates with RG >75% on
plates amended with 0.5 mg/ml were scored as resistant to the particular
fungicide. About 14% of the isolates were cross-resistant to all three
fungicides. Age of trees, size of orchard, number of DMI sprays in 2009, and
lack of dormant copper sprays were positively correlated (0.0001 < P < 0.05)
with the incidence of resistant isolates. Use of dormant copper sprays reduced
the odds of an isolate being resistant to myclobutanil by about half (odds ratio
= 0.446; 95% confidence interval = 0.239 to 0.832; P = 0.011). Management
practices that reduce the risk of resistance to DMI fungicides in V. inaequalis
were identified.
A comprehensive management strategy for Sclerotinia rot of carrots
R. D. PETERS (1), K. R. Sanderson (1)
(1) Agriculture & Agri-Food Canada, Charlottetown, PE, CANADA
Phytopathology 100:S100
Control of Cylindrocladium black rot of peanut with Propulse, a 1:1
mixture of prothioconazole and fluopyram
P. M. PHIPPS (1), D. P. Tylenko (1), G. H. Musson (2)
(1) VPI&SU, Suffolk, VA, U.S.A.; (2) Bayer CropScience, Research Triangle
Park, NC, U.S.A.
Phytopathology 100:S100
Sclerotinia rot of carrots (SRC), caused by Sclerotinia sclerotiorum, is a
devastating disease that causes yield losses in the field and in storage.
Research in Prince Edward Island (PEI), Canada on carrots grown in mineral
soils has demonstrated that factors that increase canopy density, including
higher seeding rates and nitrogen inputs, can significantly increase the
severity of SRC. As well, cultivars with a propensity to grow lush canopy
architectures are also more susceptible to disease. In an attempt to ameliorate
these effects, a prototype carrot foliage trimmer (CFT) was designed and
manufactured in PEI in 2006. Several years of field evaluation has indicated
that trimming at row closure can significantly (P = 0.05) reduce the incidence
of SRC on foliage and in carrot roots by about 75%, without compromising
carrot yield or quality. Even with these disease reductions, some diseased
S100
PHYTOPATHOLOGY
Two field trials were conducted in 2009 to assess control of Cylindrocladium
black rot (CBR) of peanut with foliar sprays of Provost 433SC
(prothioconazole 0.084 + tebuconazole 0.169 kg a.i./ha or prothioconazole
0.113 + tebuconazole 0.226 kg a.i./ha), and seed-furrow treatments with
Proline 480SC (prothioconazole 0.2 kg a.i./ha) or Propulse 400SC
(prothioconazole 0.215 + fluopyram 0.215 kg a.i./ha). All plots received three
foliar sprays of Provost at the low or high rate and one final spray of
chlorothalonil 1.263 kg a.i./ha. Treatments were mixed in water for delivery in
140 liter/ha in foliar sprays and 48 liter/ha in-furrow. The standard for CBR
control was metam sodium 32 kg a.i./ha applied under each row 2-wk prior to
planting. Plots were four, 10.7-m rows spaced 0.9-m apart and treatments
were randomized in four complete blocks. Low and high rates of Provost
provided excellent control of foliar diseases but showed no differences in
CBR incidence. Proline in-furrow significantly reduced CBR incidence by 43
and 33% while the Vapam standard reduced incidence by 52 and 55% in trial
1 and 2, respectively. Propulse in-furrow reduced CBR incidence by 83 and
57% in both trials. Yields were increased significantly by Propulse and by
metam sodium in trial 1. Yields in trial 2 were not different; however,
Propulse and metam sodium made the highest yields. Propulse is the first infurrow fungicide to show promise as a replacement for metam sodium in
control of CBR.
First report of Fusarium oxysporum f. sp. lycopersici race 3 on tomato in
Iran
S. PIRAHESH (1), H. Zamanizadeh (1), S. Rezaee (1), B. Morid (2)
(1) Islamic Azad University-Science and Research Branch, Tehran, IRAN; (2)
Islamic Azad University-Takestan Branch, Takestan, IRAN
Phytopathology 100:S101
Fusarium wilt of tomato, caused by Fusarium oxysporum f. sp. lycopersici, is
a devastating disease in major tomato-growing regions worldwide and has
been reported in at least 32 countries. Race 1 described in 1886 and race 2 was
first reported in 1945. Race 3 was observed in Australia in 1978 and was
subsequently reported in several U.S. states. Thirty five isolates of Fusarium
oxysporum were morphologically identified from wilted tomato plants in
greenhouses in Sistan and Baluchestan Province. The pathogenic types (forma
speciales and races) of Fusarium oxysporum was identified with polymerase
chain reaction (PCR) technique. the partial nucleotides sequence of
polygalactoronase genes amplified with specific primer sets (uni, sp13 and sp
23). Results indicated that eleven isolates could be classified as Fusarium
oxysporum f. sp. lycopersici, race 3. This technique could be helpful in
epidemiological and etiological studies to monitor the behavior of the
pathogen in disease plants. This is the first report of presence of Fusarium
oxysporum f. sp. lycopersici race 3 on tomato in Iran.
In vitro evaluation of native Trichoderma isolates against fungal trunk
disease pathogens in Ensenada, Mexico
J. A. Plata-Caudillo (1), C. Valenzuela-Solano (2), R. HERNANDEZMARTINEZ (1)
(1) Microbiology Department, CICESE, Carretera Ensenada-Tijuana No.
3918, Zona Playitas, Ensenada, Baja California, MEXICO; (2) INIFAP,
Campo Experimental Costa de Ensenada, Ensenada, Baja California,
MEXICO
Phytopathology 100:S101
In Mexico, Ensenada, Baja California produces around 95% of the wine.
Trunk diseases, caused mainly by fungi are among the main factors limiting
vineyard longevity and productivity in the area. Due to the importance of
these pathogens, finding control strategies is a priority. Here, we isolated
Trichoderma spp. from field-grown grapevines, and evaluated their potential
to control L. theobromae (Lt), D. seriata (Ds), N. vitifusiforme (Nv), D.
corticola (Dc), N. australe (Na), Cylindrocarpon sp. (Cy), P. aleophium (Pa),
and P. chlamydospora (Pc), under laboratory conditions. Six native isolates
were compared with a commercial strain of T. harzianum (T-22). Percent of
inhibition of the pathogens growth by T-22 vary from 43% to 50%, while
inhibition by the isolates CCMT01-1 and SACH26-1, were significant higher
for Na (75% and 96%), Ds (53% and 82%), Nv (66% and 79%), Dc (55% and
95%) and Lt (56% and 57%) respectively. CCMT01-1 and SACH26-1 were
also capable of inhibit the growth of Pa and Pc by producing volatile and
nonvolatile compounds. Based on their morphological characteristics and the
analysis of their internal transcribed spacer region (ITS-5.8S-ITS2),
CCMT01-1 and SACH26-1 were identified as members of T. gamsii. These
results open the possibility to use CCMT01-1 and SACH26-1 as biological
control agents of trunk diseases fungi affecting grapevines in the Ensenada
Region.
Association of multiple virus infections with apple disease in western
Colorado
R. POKHAREL (1), R. Mock (2), R. Li (2), G. Kinard (2), H. Larsen (1)
(1) Colorado State University, Grand Junction, CO, U.S.A.; (2) Beltsville,
MD, U.S.A.
Phytopathology 100:S101
Gala and Golden Delicious apple trees with small leaves and small, deformed
fruit were observed in an apple orchard in western Colorado. The symptoms
on Gala were more severe than on Golden Delicious. To understand the
possible association of viruses and viroids with the disease, three leaf and 20
fruit samples were collected from these trees, and asymptomatic Red
Delicious trees, in October 2009 and tested for Apple chlorotic leafs pot virus
(ACLSV), Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV),
Apple stem pitting virus (ASPV) and Cherry rasp leaf virus (CRLV) by RT-
PCR, and Apple dimple fruit viroid, Apple fruit crinkle viroid, Apple scar skin
viroid and Pear blister canker viroid by dot-blot hybridization. All five viruses
were detected in fruit samples with different incidences, but no viruses were
found in leaf samples. ACLSV was detected in all samples, and ASGV was
detected in all Red Delicious and Golden Delicious trees but only in 3 of 8
Gala trees. CRLV was detected in all Golden Delicious samples, 7 of 8 Gala
samples, and was not found in Red Delicious samples. ASPV was detected in
3 Golden Delicious and 1 Gala samples. ApMV was detected in only one Gala
sample. Viroids were not detected in any samples. Asymptomatic Red
Delicious trees were infected with only two viruses, while symptomatic Gala
and Golden Delicious trees were infected with up to four viruses, suggesting a
possible synergistic role of these viruses in symptom expression.
Taxonomic status of Acanthorhynchus vaccinii: reassessment of the
identity of the cranberry pathogen Physalospora vaccinii
J. POLASHOCK (1), P. Oudemans (2), J. Crouch (3)
(1) USDA ARS, Chatsworth, NJ, U.S.A.; (2) Rutgers University, Chatsworth,
NJ, U.S.A.; (3) Cereal Disease Laboratory, USDA-ARS, St. Paul, MN, U.S.A.
Phytopathology 100:S101
Fruit rot of cranberry is caused by complex of phytopathogenic fungi.
Physalospora vaccinii (Shear) Arx & E. Müll. is one of the most common and
widespread causal species of fruit rot in both native and cultivated habitats.
This species is easily identified from cranberry hosts based on characteristic
perithecia, ascospores, and large appressoria. We have identified two distinct
morphological types of P. vaccinii in North America that are easily
differentiated by colony color (gray vs. white) and ascospore size and shape.
Using molecular phylogenetics based on a six-gene dataset, it was clear that
these distinct morphological types are evolutionarily distinct taxa that should
be named as separate species. Surprisingly, the molecular data also showed
that these fungi are unrelated to other members of the genus Physalospora
(family Xylariomycetidae). Instead, both modern isolates and the type
specimens of P. vaccinii fall within the family Sordariomycetidae. Based on
these data, we propose to resurrect the genus name Acanthorhynchus
originally applied to this group by Shear in 1922, to include the two species
pathogenic to cranberry, A. vaccinii and A. alba.
Application of intragenic technology for development of disease-resistant
potato
G. P. PONCIANO (1), C. M. Rommens (2), D. R. Rockhold (1), K. F. McCue
(1), M. C. Whalen (3), W. R. Belknap (1)
(1) USDA ARS, Albany, CA, U.S.A.; (2) J. R. Simplot Company, Simplot
Plant Sciences, Boise, ID, U.S.A.; (3) Pacific West Area USDA-ARS,
Albany, CA, U.S.A.
Phytopathology 100:S101
Intragenics (also known as ‘cisgenesis’) is a plant transformation technology
that consists of employing only genes, regulatory, and transfer DNA
sequences from the plant genus to be transformed. Current status of our use of
the technology to produce disease-resistant potatoes will be presented. The
late blight disease caused by Phytophthora infestans continues to be potato’s
most serious disease worldwide. Using an intragenic transformation vector
developed by J.R. Simplot Company, and the recently cloned RB resistance
gene effective against a wide spectrum of P. infestans races, we aim to
develop a late blight-resistant potato. We are also developing a strategy for
intragenic potato plants resistant to viruses. We took a computational
approach to identify virus homologous sequences present in the potato
genome and in potato EST sequences. The identified genomic sequences are
being evaluated to determine their potential to serve as si/miRNA templates to
induce plant viral disease resistance via RNAi.
Application of a simple extraction method for the detection of viruses in
plants and insect vectors
S. POOJARI (1), G. Karthikeyan (2), O. J. Alabi (3), T. A. Damayanti (4), K.
Manoranjitham (5), N. Balakrishnan (6), R. A. Naidu (3)
(1) Washington State University, Prosser, WA, U.S.A.; (2) Department of
Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, INDIA;
(3) Washington State University, Irrigated Agriculture Research and Exten
Ctr, Prosser, WA, U.S.A.; (4) Faculty of Agriculture, Bogor Agricultural
University, Bogor, INDONESIA; (5) Department of Fruit Crops, Tamil Nadu
Agricultural University, Horticultural College and Research Institute,
Coimbatore, INDIA; (6) Dept. of Plant Molecular Biology and Biotechnology,
Tamil Nadu Agricultural University, Coimbatore, INDIA
Phytopathology 100:S101
Accurate diagnosis of plant viruses is critical for developing management
strategies to mitigate their negative impacts. A wide range of methods have
been used for the extraction of viral nucleic acid prior to detection by reverse
transcription (RT)-PCR or PCR, depending on whether the test sample is
infected with RNA- or DNA-containing virus, respectively. In this study, we
used a relatively simple buffer for extraction of fresh leaf and fruit tissues and
Vol. 100, No. 6 (Supplement), 2010
S101
for elution of nucleic acids from samples spotted on FTA® cards or
nitrocellulose membranes. The extracts were used directly in one step-single
tube RT-PCR or PCR for amplification of virus-specific DNA bands with
species-specific primers. The amplicons were cloned and sequenced, and the
derived sequences compared with corresponding sequences in the databases to
confirm the presence of virus(es) in a given sample. The sample extraction
buffer was also tested in combination with RT-PCR for the detection of
tospoviruses in vector thrips and ampeloviruses in grape mealybugs. The
practical utility of this protocol for the detection of RNA- and DNAcontaining viruses will be discussed.
Timing and methodology of application of Azoxystrobin to control
Rhizoctonia solani in sugarbeet
S. Pooran DeSouza (1), M. D. Bolton (2), M. F. KHAN (1)
(1) NDSU Plant Pathology, Fargo, ND, U.S.A.; (2) USDA - ARS, Fargo, ND,
U.S.A.
Phytopathology 100:S102
Rhizoctonia solani AG 2-2 is the causal agent of Rhizoctonia root and crown
rot of sugar beet (Beta vulgaris) in North Dakota and Minnesota. This disease
is a major limiting factor to sugar beet production. Management strategies
currently include using partially resistant cultivars and fungicides.
Azoxystrobin is the most widely used fungicide for disease control. Our
objective was to determine the best time for application of azoxystrobin and to
compare the efficacy of azoxystrobin in foliar and soil drench applications in
greenhouse trials. Azoxystrobin was applied as a hypocotyl drench at 0, 3, 10,
14, and 21 days post-inoculation, at 0, 7, 14, and 28 days pre-inoculation, and
applied to either foliage or soil of inoculated sugar beet hypocotyls at the fourleaf stage. Based on disease severity means, azoxystrobin applied at preinoculation was not significantly different from non-inoculated check. Post
inoculation applications at 0 and 3 days had low root disease severity and at
21 and 28 days disease severity were similar to inoculated checks. In efficacy
trials, foliar application of azoxystrobin was significantly different from soil
drench application and had the highest disease severity that was similar to
inoculated checks. Azoxystrobin application as a soil drench at the base of the
hypocotyls and prior to infection was effective in controlling disease.
Field efficacy of propiconazole on diverse Sclerotinia homoeocarpa
population structures
J. T. POPKO (1), C. Ok (2), M. T. McGrath (3), G. Jung (2)
(1) Department of Plant, Soil and Insect Science, University of MassachusettsAmherst, Hadley, MA, U.S.A.; (2) Department of Plant, Soil and Insect
Science, University of Massachusetts-Amherst, Amherst, MA, U.S.A.; (3)
Department of Plant Pathology and Plant-Microbe Biology, Cornell
University, Riverhead, NY, U.S.A.
Phytopathology 100:S102
Dollar spot (Sclerotinia homoeocarpa) is a major turfgrass disease requiring
fungicide application to maintain acceptable conditions for golf.
Demethylation inhibitors (DMI) are the most frequently used fungicide class
with confirmed resistance in S. homoeocarpa. Due to the quantitative nature
of DMI resistance, a clear correlation between in vitro sensitivity values and
reduced field efficacy has yet to be established for S. homoeocarpa. The study
objective was to determine field efficacy of propiconazole rates on S.
homoeocarpa populations with differing in vitro sensitivities. Prior to
fungicide application, DMI sensitivity of populations on fairways of four golf
courses and the UMass turf center was characterized by determining relative
mycelium growth percentage (RMG%) on 0.1 g a.i./ml propiconazoleamended medium. The UMass site consisted of a sensitive population (0–20%
RMG) and the golf course sites consisted of two insensitive populations (50–
80% RMG) and two with discrete bimodal populations (0–40% and 60–90%
RMG). S. homoeocarpa was completely controlled with all propiconazole
rates at the UMass site for all but one rating date. Reduced efficacy was
observed at all four golf course sites on multiple rating dates, and 90% of
isolates sampled 7 and 14 days after DMI application had RMG values >60%.
Results indicate isolates with >60% RMG are likely responsible for reduced
DMI field efficacy, despite quantitative variation of in vitro sensitivity values.
Effects of soybean cyst nematode on growth of kidney and navy bean
S. H. POROMARTO (1), B. D. Nelson (1)
(1) Dept. Plant Pathology, North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S102
Phaseolus vulgaris is a host of soybean cyst nematode (SCN; Heterodera
glycines), but the effects of SCN on growth of plants are poorly studied. The
effects of SCN (HG type 0) on cultivars Montcalm (kidney bean) and
Mayflower (navy bean) were evaluated in the field in 2008 and 2009. Arveson
loam soil was pasteurized and re-infested with 0, 1,000, 2,500 and 5,000
eggs/100 cc soil for Montcalm in 2008. In 2009 for Montcalm and Mayflower,
treatments were similar but without the 1,000 eggs/100 cc. Soil was placed in
10 L plastic pots that were buried in the field with the bottoms removed. In
S102
PHYTOPATHOLOGY
2008 there were no significant effects on growth of Montcalm in two field
experiments, but in 2009, in a single experiment, pod weight (PW), seed
number (SN), seed weight (SW), and total dry weight (TDW) of the aboveground-plants were significantly (P < 0.05) less at 2,500 and 5,000 eggs/100
cc soil compared with the control. There were also significant (P < 0.05)
reductions of plant height (PH), PW, SN, SW, and TDW of Mayflower in
2009 in one field, but not a second field, when infested with 2,500 or 5,000
eggs/100 cc soil compared to the control. In both cultivars there were no
significant differences among the two SCN treatments. SCN has now been
shown to cause a yield loss in three dry bean classes and is a potential threat to
the dry bean industry in the Dakota-Minnesota region.
Lack of adaption toward greater reproduction of soybean cyst nematode
on dry bean
S. H. POROMARTO (1), B. D. Nelson (1)
(1) Dept. Plant Pathology, North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S102
Phaseolus vulgaris is a host of soybean cyst nematode (SCN; Heterodera
glycines), a pathogen now in the major bean production area of North Dakotanorthern Minnesota. The nematode reproduces less on most bean classes
compared to soybean, but can still reduce plant growth. An important question
is the following: will SCN adapt to dry beans and over time increase in ability
to reproduce on roots? To answer this question the following experiments
were conducted. The cultivars Premiere and Cirrus (navy), Buster and Othello
(pinto), and Eclipse and Jaguar (black) were grown in ‘Conetainers’ in sand in
plastic pots immersed in a water bath at 27 degrees C in the greenhouse.
Seedlings were inoculated with 2000 eggs per plant of SCN HG 0 and cysts
were harvested and counted after 40 days. The eggs were immediately
extracted from those cysts and seedlings were inoculated again and grown for
40 days using the same methods. Soybean Lee 74 was used as a control. A
female index (number of cysts produced on the test plant divided by the
number of cysts produced on Lee 74) was calculated for each bean cultivar
after each 40 days. This procedure was repeated until 8 generations of eggs
were completed. There was no significant (P ≤ 0.05) change overtime in the
female index on the six bean cultivars. Therefore, no evidence of an
adaptation toward higher reproduction on dry bean cultivars was detected
during two 11 month periods of continual reproduction of HG 0 on roots.
Aggressiveness and management of metalaxyl-resistant Pythium ultimum
L. D. PORTER (1)
(1) USDA ARS, Prosser, WA, U.S.A.
Phytopathology 100:S102
Pythium ultimum is a major seed rotting pathogen of green pea in the Pacific
Northwest, U.S.A. Metalaxyl (m), a systemic fungicide, is commonly used to
manage seed rot due to Pythium spp. Recently, m-resistant isolates of P.
ultimum were isolated from soil in ID, OR and WA. A m-resistant and msensitive isolate were recovered from each of five fields: a single field in ID,
and two fields in WA and OR, respectively. Paired isolates were assessed for
growth at 7.2 and 10°C on corn meal agar. These temperatures represent
common soil temperatures when seeding green pea. Growth of m-resistant
isolates was either signficantly greater or greater than the paired sensitive
isolate at both temperatures in replicated tests, except for a single isolate in
one of two trials at both temperatures. Faster growth rates for m-resistant
isolates than for m-sensitive may indicate that resistant isolates are more
aggressive than sensitive isolates in colonizing peas at these temperatures.
Also, potential fungicide seed treatments (phosphorous acid, potassium
silicate, metalaxyl, metalaxyl-M, fosetyl aluminum and cyazofamid) to
manage seed rot by m-resistant isolates were assessed in infested soil at 10°C.
Cyazofamid was the only fungicide that improved germination above 50%
when soil was inoculated with a m-resistant isolate. Germination of seed
treated with m or m-M was reduced by 58% when soil was infested with a mresistant compared to a m-sensitive isolate of P. ultimum.
Biological control of seven woody invasive plant species with the fungal
pathogen Chondrosterium purpureum
A. R. POST (1), C. A. Judge (2), D. Little (3), J. C. Neal (3), M. Benson (4)
(1) Virginia Tech, Blacksburg, VA, U.S.A.; (2) BASF Corporation, Dewitt,
MI, U.S.A.; (3) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S102
The plant pathogen Chondrostereum purpureum is a wood rotting fungi
currently registered in the United States and Canada as Chontrol™. It is
intended for use in forests and on rights-of-ways as a biocontrol herbicide for
invasive woody species. The objective of this study was to evaluate the
effectiveness of cut-stump Chontrol applications against seven invasive
woody species including empress tree, Chinese wisteria, Chinese privet,
oriental bittersweet, red maple, tulip poplar and sweet gum. Trials were set up
in a randomized complete block design with four to eight replications and four
treatments plus a nontreated check. Treatments included cut-stump and wet-
blade applications of Chontrol in paste and liquid forms, as well as the
industry standard against woody species, triclopyr. Each replication included
plants of uniform stem diameter. All trials were repeated. Regrowth was
measured 12 months after treatment for all species. No differences were
observed between the stems treated with Chontrol and the nontreated check.
Triclopyr controlled all species 100% with no regrowth observed. Though
Condrostereum purpureum is documented to control some invasive woody
species, current formulations and application methods failed to provide
control of common woody invasive species in North Carolina. It is possible
this is due to application timing or improper environmental conditions for
infection.
wildtype alone inoculation. The relative accumulation of defense-related
enzymes (POX, PAL, and -1,3 glucanase), salicylic acid (SA) and
pathogenesis-related proteins (PR-proteins) from plants treated with mutant
and wildtype was greater upregulation compared to single inoculation. Result
suggests categories of overrepresented genes expressing coordinately in
response to rpfF mutant-wildtype interaction.
Virulence factors in xanthomonads pathogenic on pepper increase in
planta growth of Xanthomonas perforans
N. POTNIS (1), G. V. Minsavage (1), D. J. Norman (2), J. B. Jones (1)
(1) Department of Plant Pathology, University of Florida, Gainesville, FL,
U.S.A.; (2) Mid-Florida Research & Education Center, University of Florida,
Apopka, FL, U.S.A.
Phytopathology 100:S103
Poultry litter (PL), a major byproduct produced in large quantities on
corporate poultry farms for which new uses are needed, was evaluated for
potential use as a biocontrol material against sclerotia of Sclerotium rolfsii in
soil. Survival of sclerotia was evaluated following their incubation within
porous membrane filters in plates containing a sandy loam soil with and
without PL amendments at 4 and 8% (dry weight equivalents). Sclerotia
survived and germinated at relatively high percentages (70–100%) in Control
soil after 2 wk, while survival and/or germination were consistently reduced in
PL-amended soils. Physical destruction of sclerotia was usually greatest with
4% PL, whereas loss of viability of recovered sclerotia was usually greatest
with 8% PL. Pre-incubation of soil with PL for 2 wk prior to addition of
sclerotia gave less reduction in survival than occurred without pre-incubation.
Incubation of sclerotia in soil for 4 wk rather than 2 wk reduced their survival
in both Control and PL-amended soils. Results indicate that PL is efficacious
for biocontrol of sclerotia of S. rolfsii in soil, and they suggest that different
mechanisms of biocontrol may be involved at the 4% and 8% PL
concentrations in soil.
Comparative genomics has been widely used for identifying host
specificity/virulence factors. Four Xanthomonas species pathogenic on tomato
were sequenced. These include X. euvesicatoria (Xcv85-10) (already
sequenced and published), and three draft sequences of X. vesicatoria
(Xv1111), X. perforans (Xp91-118), X. gardneri (Xg101). Xcv8510, Xv1111
and Xg101 are also pathogenic on pepper, whereas, Xp91-118 is not.
Comparison of genomes of pepper pathogens Xcv8510, Xv1111, Xg101 to X.
perforans draft genome revealed a number of genes shared by pepper
pathogens, which could be candidate virulence factors on pepper. We cloned
three candidate genes and conjugated individually and in combination in
ME24 (91-118ΔavrXv3), which no longer elicits an HR in pepper; however, in
planta growth of ME24 is more similar to that of an avirulent strain than a
virulent pepper strain. ME24 transconjugants carrying these genes showed
increased in planta growth and also comparatively increased number of
lesions on pepper cv. ECW when compared to ME24. The contribution of
these genes towards pepper specificity is discussed.
Epidemiological studies on Blackberry yellow vein associated virus
B. POUDEL (1), W. M. Wintermantel (2), S. Sabanadzovic (3), I. E.
Tzanetakis (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) USDA-ARS,
Salinas, CA, U.S.A.; (3) Mississipi State University, Starkville, MS, U.S.A.
Phytopathology 100:S103
Blackberry production in the Southeast is threatened by blackberry yellow
vein disease (BYVD), a disorder caused by virus complexes. Blackberry
yellow vein associated virus (BYVaV), a recently identified crinivirus, is the
most prevalent virus in the BYVD complexes. BYVaV is asymptomatic in
single infection and acts synergistically during co-infecton with other viruses
to cause disease. The objective of this study was to acquire information on the
epidemiology of BYVaV including identification of initial sources of
infection, vector(s) and alternate hosts. Several isolates of the virus infecting
cultivated and wild blackberries were collected from different states with high
BYVD incidence. The variability was determined after sequencing and
analysis of four genomic regions of the virus; these regions being the most
genetically diverse among viruses in the family Closteroviridae. Alternate
host identification was performed by testing plant species present in
blackberry fields with high BYVaV incidence. Whiteflies, known vectors of
criniviruses, have been tested to identify species that can transmit BYVaV.
The results of this study clarify factors contributing to the epidemiology of
BYVaV, the primary component of the emerging BYVD, by identifying the
geographical origin of the BYVaV, its potential vectors and alternate hosts.
The rpfF mutation of Xanthomonas axonopodis pv. glycines reduces
bacterial pustule disease and activates defense in soybean
S. PRATHUANGWONG (1), W. Chuaboon (1), T. Chatnaparat (1), J.
Thowthampitak (1)
(1) Kasetsart University, Chatuchack, Bangkok, THAILAND
Phytopathology 100:S103
RpfF is a protein similar to enoyl-CoA hydratase that synthesizes a diffusible
signal factor (DSF) demonstrating as a virulence regulator in various plant
pathogenic bacteria including Xanthomonas axonopodis pv. glycines (Xag), a
cause of soybean bacterial pustule. Preliminary experiments reported that rpfF
mutant of Xag failed to produce DSF, extracellular polysaccharide,
siderophore, and many extracellular enzymes. In this study, we investigated
the expression of rpfF on disease severity and defense response at the
biochemical and transcriptional levels. Virulence assay was conducted by
exploring the function of rpfF on soybean that symptoms and severity caused
by the mutant were delayed and decreased respectively. Coinoculation of
mutant and wildtype resulted in decreased pustule symptoms compared with
Evaluation of poultry litter for biocontrol of sclerotia of Sclerotium rolfsii
in soil
R. PRATT (1)
(1) USDA ARS, Mississippi State, MS, U.S.A.
Phytopathology 100:S103
Evaluation of a new method for collection and detection of plant
pathogens within their vector
J. A. PRICE (1), A. Simmons (1), J. Bass (1), C. M. Rush (1)
(1) Texas AgriLife Research, Amarillo, TX, U.S.A.
Phytopathology 100:S103
Many of the important plant pathogens that affect agriculture are dependent
on some type of vector for movement and distribution. Studies in
epidemiology and vector/pathogen relations often involve detection of
pathogens, not only within the host plant, but also within a single vector.
Wheat streak mosaic virus (WSMV), which causes wheat streak mosaic, and
Candidatus Liberibacter solanacearum (CLs), causal agent of Zebra chip, are
two important plant pathogens that rely on vectors for their movement.
However, analysis of a single vector can be challenging due to their small
size. Therefore, a new method of pathogen testing is being investigated for
detection of WSMV and CLs within a single wheat curl mite or psyllid,
respectively. FTA membranes can be used to bind nucleic acids from both the
vector and pathogen for long term storage, or go directly into analysis by realtime PCR without the use of traditional RNA/DNA extraction. This method
has been used successfully in detection of WSMV and CLs within a single
mite and psyllid, respectively. Tests are currently being conducted for
detection of CLs within a single psyllid nymph collected from leaves
displaying symptoms of Zebra chip. This method has potential as a powerful
tool for analysis and quantification of plant pathogens within a single vector.
Relationships between in vivo and in vitro aflatoxin production: Reliable
prediction of fungal ability to contaminate maize with aflatoxins
C. PROBST (1), P. J. Cotty (2)
(1) University of Arizona, Tucson, AZ, U.S.A.; (2) University of Arizona,
USDA-ARS, Tucson, AZ, U.S.A.
Phytopathology 100:S103
Aflatoxins are highly carcinogenic mycotoxins frequently produced by
Aspergillus flavus. Contamination of maize with aflatoxins imposes both
economic and health burdens in many regions. Identification of the most
important etiologic agents of contamination is complicated by mixed
infections and varying aflatoxin-producing potential of fungal species and
strains. In order to know the potential importance of an isolate to cause a
contamination event, the ability of the isolate to produce aflatoxins on the
living host must be determined. Aflatoxin production in vitro (synthetic and
natural media) was contrasted with in vivo (living corn kernels) production in
order to determine ability of in vitro techniques to predict the relative
importance of causal agents of maize contamination. Several media and
fermentation techniques were compared. There was no correlation between
aflatoxin production on living corn and production in any liquid fermentation
medium using any of the fermentation techniques. Isolates that produced
aflatoxins on living corn frequently failed to produce detectable (limit of
detection = 1 ppb) aflatoxin concentrations in synthetic media. Aflatoxin
production on autoclaved corn kernels was highly correlated (r2 = 0.98) with
production on living corn kernels. The results have important implications for
Vol. 100, No. 6 (Supplement), 2010
S103
researchers seeking to either identify causal agents of contamination events or
to characterize atoxigenic isolates for biological control.
Subcellular localization of the replicase proteins encoded by a member of
the family Betaflexiviridae
S. W. Prosser (1), C. Li (1), B. MENG (1)
(1) Dept. Molecular and Cellular Biology, University of Guelph, Guelph, ON,
CANADA
Phytopathology 100:S104
Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of
the genus Foveavirus, family Betaflexiviridae. The GRSPaV genome contains
five ORFs, with ORF1 encoding a replicase polyprotein typical of members of
the Alphavirus superfamily. It is known that replication of RNA viruses
occurs in distinctive structures called “replication complexes” that can only be
formed via association with a cell membrane. It was shown recently that
translocation of the replication complexes of Tobacco mosaic virus was
achieved by the actomyosin network. As a first step in unraveling the nature
of GRSPaV replication complex and its interaction with cellular structures, we
studied the subcellular localization of the full-length and truncated versions of
its replicase polyprotein via fluorescence protein tagging and microscopic
observation. We discovered that the replicase polyprotein forms distinctive
bodies resembling the replication complexes in tobacco cells. We further
revealed that a subdomain of 76 amino acid residues in the methyl-transferase
(MTR) is responsible for formation of these bodies. Interestingly, these
punctate bodies appear to align with the microfilaments. We are testing the
possibility that these bodies may traffick on the microfilaments using the
model plant Nicotiana benthamiana co-expressing autofluorescent protein
fusions to MTR and actin marker. This is the first report on the subcellular
localization of replicase proteins encoded by a member of the
Betaflexiviridae.
Validation and haplotyping of Fusarium head blight resistance genes in a
diverse spring wheat germplasm
K. D. PURI (1), S. Chao (2), H. Bockelman (3), M. Mergoum (4), S. Zhong
(1)
(1) Department of Plant Pathology, North Dakota State University, Fargo,
ND, U.S.A.; (2) USDA-ARS Cereal Crops Research Unit, Northern Crop
Science Laboratory, Fargo, ND, U.S.A.; (3) USDA-ARS National Small
Grains Collection, Aberdeen, ID, U.S.A.; (4) Department of Plant Sciences,
North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S104
Fusarium head blight (FHB), primarily caused by Fusarium graminearum
Schw., is the most destructive disease of wheat and barley in North America.
Use of host resistance is one of the most efficient and economic strategies for
managing the disease. However, the FHB resistance sources used in the
breeding programs are very limited. In this study, we haplotyped 83 PI
accessions obtained from the National Small Grains Collections with various
levels of FHB resistance at 17 molecular marker loci associated with the
known FHB resistance QTLs on the chromosomes 2B (Triticum carthlicum
‘Blackbird’), 3A (T. aestivium ‘Frontana’ and T. dicoccoides ‘Israel A’), 3B
(T. aestivium ‘Sumai 3’ and ‘Wanshuibai’), 5A (‘Sumai 3’ and ‘Frontana’),
6B (‘Blackbird’ and ‘Wangshuibai’), and 7A (T. dicoccoides). Fifty three of
the PI lines showed different haplotypes at the marker loci on 3B and 5A of
Sumai 3 origin, suggesting that they may carry different FHB resistance genes
from Sumai 3, the most frequently used FHB resistance source in spring
wheat breeding programs. Marker data further showed that 22 of these lines
also lacked the markers associated with FHB resistance in other known
sources investigated. All the wheat lines have been evaluated for FHB
resistance under greenhouse and field conditions and the results will be
presented. The novel FHB resistance genes carried by the spring wheat lines
could be utilized to develop new wheat varieties with enhanced FHB
resistance.
Molecular studies of genotype, organ and physiological stage dependent
immunity against karnal bunt (Tilletia indica) of wheat (Triticum
aestivum)
S. PURWAR (1), S. Sundaram (2)
(1) Allahabad University, Allahabad, INDIA; (2) Center of Biotechnology,
Nehru Science Center, Allahabad, INDIA
Phytopathology 100:S104
Tilletia indica. an economically important disease -Karnal bunt (KB) of wheat
which is incited by a semi-biotrophic pathogen. It has been elucidated that
various enzymes of phenylpropanoid pathway not only enhance structural
defense and induce systemic acquired resistance but also inhibit PCD involved
in pathogenesis. Besides it, there is also involvement of cytokinins which are
interestingly involved in both suppression of PCD and induction of systemic
acquired resistance. The observation that resistant wheat varieties have high
level of ABA, which is a precursor of JA has clearly demonstrated that the JA
S104
PHYTOPATHOLOGY
dependent pathways play important role in triggering induced resistance
against the KB pathogen. The activity of protease was higher in initial stages
of resistant genotype and gradually declined in later stages after anthesis.
However, in contrast to protease activity, reverse trend has been observed for
cystatin levels in all the stages of resistant wheat spikes showing negative
correlation to each others. Therefore, stoichiometric balances of protease and
protease inhibitor play key role in controlling the resistance against KB
probably by intervention of PCD.
Pseudomonas savastanoi found in association with stem galls on
mandevilla
M. L. PUTNAM (1), M. Curtis (1), M. Serdani (1), A. J. Palmateer (2)
(1) Oregon State University, Corvallis, OR, U.S.A.; (2) University of Florida,
Homestead, FL, U.S.A.
Phytopathology 100:S104
Mandevilla splendens is a woody twining vine in the family Apocynaceae.
Rooted cuttings with stem galls were submitted to the Oregon State University
Plant Clinic and the Extension Plant Diagnostic Clinic in Homestead, FL for
diagnosis. The concern was crown gall, but symptoms were not typical of that
disease. Galls were around 0.5 in diameter, occurred above soil level near the
stem base, often at mid-stem. Vertical cracks with oozing bacteria were
abundant. Isolations for Agrobacterium tumefaciens were negative, as were
PCR assays using Agrobacterium specific primers A, C’ and E’. Isolations
produced an abundance of fluorescent bacteria which were identified by
substrate utilization (Biolog, Haywood, CA) to the genus Pseudomonas. PCR
using DNA extracted from gall tissue and the IAALF/IAALR primer pair,
which target a region of the iaaL gene (synthesis of 3-indoleacetyl-ε-L-lysine)
unique to Pseudomonas savastanoi pv. savastanoi, yielded a ~454 bp product.
This PCR product was sequenced and showed 100% identity (395/395
nucleotides) to the P. savastanoi pv. savastanoi strain NCPPB-3335 iaaL
gene. To our knowledge, this is a new host family for P. savastanoi in the
United States and the first time P. savastanoi has been associated with galls
on mandevilla. The conspicuous nature of symptoms and the economic
importance of this plant in the ornamental industry suggest that this disease
may become significant.
Factors contributing to abscisic acid-mediated predisposition to disease
caused by Phytophthora capsici
M. F. PYE (1), T. V. Roubtsova (1), M. V. DiLeo (2), J. D. MacDonald (1),
R. M. Bostock (1)
(1) University of California, Davis, CA, U.S.A.; (2) Ithica, U.S.A.
Phytopathology 100:S104
Plants respond to changes in the environment with complex signaling
networks controlled in part by phytohormones that display positive and
negative downstream crosstalk. Disease response pathways are influenced by
systemic increases in abscisic acid (ABA), such as occurs following brief
dehydration stresses. Experiments with ABA-modified tomato plants indicate
that ABA plays a critical, and perhaps dominant, role in predisposition to
Phytophthora capsici. To further assess ABA’s contribution relative to other
factors in root stress-induced predisposition, this study examines how other
phytohormones influence disease severity following an episode of salt stress,
and if plants expressing anti-apoptotic genes are altered in their predisposition
phenotype. Ethylene (ET), jasmonic acid (JA), and salicylic acid (SA) were
studied using tomato perception and deficient mutants. Several anti-apoptotic
transgenes with different modes of action were examined for their potential to
affect predisposition. ET, which can exacerbate disease symptoms in plantmicrobe interactions, did not contribute to predisposition in our assay. JA- and
SA-deficient mutants displayed a more severe disease phenotype than their
respective wildtype backgrounds in both control and salt stressed treatments.
Increased levels of ABA following salt stress might perturb SA and JA
signaling to enhance predisposition of mutants already compromised in these
defense signaling networks.
Genome sequence and comparative genomics of Pseudomonas savastanoi
pv. glycinea
M. QI (1), D. Wang (1), C. A. Bradley (1), Y. Zhao (1)
(1) University of Illinois, Urbana, IL, U.S.A.
Phytopathology 100:S104
Bacterial blight, caused by Pseudomonas savastanoi pv. glycinea (Psg), is a
common bacterial disease of soybean and occurs in most soybean growing
areas. We have previously identified an ancestral soybean line which confers
resistant to both Psg race 4 and a recently isolated strain B076. In an effort to
identify effectors responsible for inducing resistance in the ancestral line, the
genomes of both Psg strains were sequenced using 454 pyrosequencing. The
genomes of both Psg strains consist of a single circular chromosome of more
than 5.8Mb. In addition, Psg race 4 has two large (110 and 55 kb) and three
small (all less than 10 kb) plasmids. For strain B076, it contains six large
plasmids with a size of 130, 85, 80, 67, 50, and 40 kb, respectively.
Comparative genomic analyses between two Psg strains and with other
sequenced pseudomonads revealed that the genomes of Psg strains are more
closely related to that of P. savastanoi pv. phaseolicola 1448A than to P.
syringae pv. tomato DC3000 or pv. syringae B728a. Though conserved,
genome rearrangements and recombination events occur commonly within the
two Psg genomes. Type three secretion gene clusters of Psg strains are near
identical with that of strain 1448A. Coronatine biosynthetic cluster is present
in strain B076, but not in race 4. Furthermore, candidate effectors have been
identified from the two Psg genomes and determined whether they are
responsible for inducing hypersensitive response in the ancestral line.
Identification of molecular markers associated with resistance to TSWV
through genetic mapping
H. QIN (1), Y. Li (2), Y. Guo (3), G. He (4), C. Chen (5), A. Culbreath (6), S.
J. Knapp (3), D. R. Cook (7), C. C. Holbrook (8), M. Wang (9)
(1) USDA-ARS, Crop Protection and Management Research Unit, Tifton,
GA, U.S.A.; (2) Department of Plant Pathology, University of Georgia,
Athens, GA, U.S.A.; (3) Center for Applied Genetic Technologies, the
University of Georgia, Athens, GA, U.S.A.; (4) Center for Plant
Biotechnology, Tuskegee University, Tuskegee, AL, U.S.A.; (5) USDA-ARS,
National Peanut Research Laboratory, Dawson, GA, U.S.A.; (6) Department
of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (7)
Department of Plant Pathology, University of California-Davis, Davis, CA,
U.S.A.; (8) USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton,
GA, U.S.A.; (9) USDA-ARS, Plant Genetic Resources Conservation Unit,
Griffin, GA, U.S.A.
Phytopathology 100:S105
(1) University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S105
The demethylation inhibiting (DMI) fungicide tebuconazole is widely used in
Georgia to control early leaf spot of peanut, caused by Cercospora
arachidicola. In recent years, reports from Georgia and neighboring states
indicated that tebuconazole was less effective than it used to be, although
control failures have not been widespread. The objective of this study was to
develop a rapid assay to detect tebuconazole resistance in C. arachidicola. In
this assay, conidia were transferred directly from lesions to tebuconazoleamended medium and sensitivity was based on diameters of 3-day-old
colonies. Isolates were collected in 2008 and 2009 from peanut fields with or
without a history of DMI use. EC50 values were determined using the new
assay and compared to EC50 values based on the standard mycelial growth
assay in microtiter plates. For the new assay, EC50 values ranged from 0.39 to
6.17 µg/ml for 21 isolates in 2008 and from 0.36 to 9.73 µg/ml for 78 isolates
in 2009. For the standard assay, EC50 values ranged from 0.017 to 4.65 µg/ml
for 29 isolates in 2008 and from 0.025 to 5.56 µg/ml for 58 isolates in 2009.
EC50 values were consistently higher for the new assay compared to the
microtiter plate assay. For combined data from both years, there was a
significant positive correlation between EC50 values from the two assays. The
main advantage of the new assay is that it can be completed in 3 days,
compared to 2–3 months for the standard microtiter plate assay.
Mechanisms of DMI resistance in field isolates of Cercospora arachidicola
J. Qiu (1), A. K. Culbreath (1), K. L. STEVENSON (1)
(1) University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S105
Peanut is vulnerable to a range of diseases, such as tomato spotted wilt virus
(TSWV), and early and late leaf spots. The objective of this study is to
construct a genetic linkage map to facilitate quantitative trait locus (QTL)
analysis and gene tagging for use in a marker-assisted breeding. Tifrunner has
been released as a resistant cultivar to TSWV and leaf spots. New breeding
line NC94022 has been identified with the highest resistance to TSWV. Two
genetic mapping populations have been developed, a total of 248 F2:7s for
Tifrunner x GT-C20 (T) and 352 F2:7s for SunOleic 97R x NC94022 (S). A
total of 4574 simple sequence repeat (SSR) markers have been collected and
screened among the parents of the populations for polymorphisms. Of the total
SSR primer pairs, 269 and 173 primer pairs (markers) were polymorphic in
these populations, respectively, and used in genotyping these RIL populations.
Genotypes of the S population has been completed and the linkage map has
been constructed which has 20 linkage groups (LG) with 186 mapped loci
(173 SSRs and 13 with two loci). In 2009, we conducted field evaluation of
F2:5s for disease resistance to TSWV with two replications, and one QTL for
TSWV resistance have been identified with 2009 phenotype data. The
phenotypic variation explained by this QTL was 40%. The seeds have been
increased, multiple field phenotypes will be conducted in 2010, and the QTLs
will be reevaluated. This map will be compared with the T population.
Mechanisms of resistance to the demethylation inhibiting (DMI) fungicides in
the early leaf spot pathogen, Cercospora arachidicola, were investigated.
Based on mechanisms of DMI resistance reported in other fungi, mutations of
the CYP51 gene, which encodes the target 14 alpha-demethylase necessary for
sterol biosynthesis, over-expression of CYP51, and active efflux of fungicide
mediated by ATP-binding cassette (ABC) transporters were evaluated in
relatively DMI-resistant and DMI-sensitive isolates of C. arachidicola
collected from peanut fields. Sequencing of the CYP51 gene revealed
alterations at codons 453 or 461 in 4 of the 10 DMI-resistant isolates. This is
the first report of mutations in the CYP51 gene associated with DMI resistance
in C. arachidicola. However, based on a RT-PCR assay, CYP51 expression in
DMI-resistant isolates of C. arachidicola was not different from that in DMIsensitive isolates. Except for one resistant isolate that became more sensitive
to tebuconazole when promazine was added, there was no apparent increase in
tebuconazole sensitivity in the presence of ABC transporter inhibitors
flavonone or promazine. DMI resistance was not associated with overexpression of CYP51 or activity of ABC transporters in the isolates tested.
However, mutations in the CYP51 gene were associated with DMI resistance
in some C. arachidicola isolates, which can be used to develop PCR-based
assays for detection of DMI resistance in populations of this pathogen.
Nematicidal activity of Gymnoascus reessii za-130 and the properties of its
nematicidal substance
J. QIU (1), J. Liu (1), T. Liu (1)
(1) Institute of Plant and Environment Protection, BAAFS, Beijing, PRC
PEOPLES REP OF CHINA
Phytopathology 100:S105
The role of Turnip crinkle virus capsid protein in viral systemic movement
in Arabidopsis
F. QU (1), M. Cao (1), X. Ye (2), K. Willie (3), J. Lin (1), A. Simon (4), M.
Redinbaugh (3), T. Morris (2)
(1) OARDC Plant Pathology, Ohio State University, Wooster, OH, U.S.A.;
(2) School of Biological Sciences, University of Nebraska, Lincoln, NE,
U.S.A.; (3) USDA ARS, Wooster, OH, U.S.A.; (4) University of Maryland,
College Park, MD, U.S.A.
Phytopathology 100:S105
Strain za-130 of Gymnoascus reessii was isolated from soil in Beijing suburbs
and it had ability to kill the root knot nematode (Meloidogyne incognita)
obviously. The nematicidal activity of the broth filtrate from za-130 against
Meloidogyne incognita was determined with the mortality of M. incognita J2,
the inhibition rates of the hatching amount of egg masses and individual eggs
by the method of immersion. The results showed the corrected mortality of J2
was over 80% and would be reached to 100% when treated 24 hours. The
hatching of individual eggs and egg masses were suppressed by broth of za130 which had been condensed 5 times, with inhibition efficacies over 85%
and 95% respectively, and the treated result of the masses one more efficacy
than individual one is the first report. No more differences for the ability of
killing compared with pesticide of Avermectin and Fosthiazate. The
nematicidal substance from za-130 displayed a higher stability to heat when
the temperature under 60°C after 2 hours and still have higher ability to
against nematode with the inhibiting rates of 80% when the temperature over
75°C up-to 100°C. The tolerant ranges of pH from 1 to 8 have no affect for
the ability of the broth and will lost the ability on pH 10. The broth could
against ultraviolet radiation when the irradiation under 4 hours and will lost all
actives after 8 hours. Treated with sunlight showed have no affect for the
active of broth.
A new rapid assay for detecting tebuconazole resistance in Cercospora
arachidicola
J. Qiu (1), A. K. Culbreath (1), K. L. STEVENSON (1)
The capsid protein (CP) of Turnip crinkle virus (TCV) is a multi-functional
protein needed for virus assembly and suppression of RNA silencing-based
antiviral defense. In this report, we have examined genetic requirements for
the different functions of TCV CP, and evaluated the inter-dependence of
these functions. A series of TCV mutants containing alterations in the CP
coding region were generated. These alterations range from single amino acid
substitutions, domain truncations, to knockouts of CP translation. The latter
category also contained two constructs in which the CP coding region was
replaced by either the cDNA of a silencing suppressor of a different virus, or
that of green fluorescence protein. These mutants were used to infect
Arabidopsis plants with diminished antiviral silencing capability. There was a
strong correlation between the ability of mutants to reach systemic leaves and
the silencing suppressor activity of mutant CP. Virus particles were not
essential for entry of the viral genome into vascular bundles in the inoculated
leaves in the absence of antiviral silencing, but were necessary for egress of
viral genome from the vasculature of systemic leaves. Our experiments
demonstrate that TCV CP allows the viral genome to access the systemic
movement channel through silencing suppression, and then ensures its smooth
egress with virus particle assembly. These results illustrate that efficient longdistance movement of TCV requires both functions of CP.
Vol. 100, No. 6 (Supplement), 2010
S105
Sequence analysis of Raspberry latent virus suggests a new genus of dicot
infecting reoviruses
D. QUITO (1), R. R. Martin (2)
(1) Oregon State University, Corvallis, OR, U.S.A.; (2) Horticultural Crops
Research Unit USDA-ARS, Corvallis, OR, U.S.A.
Phytopathology 100:S106
Currently there are three assigned genera of plant reoviruses: Phytoreovirus,
Fijivirus and Oryzavirus. With only two exceptions, all plant reoviruses infect
monocotyledonous plants. The recent characterization of Raspberry latent
virus (RpLV) isolated from red raspberry plants in northern Washington has
revealed new dicotyledonous hosts for plant reoviruses. Phylogenetic analyses
demonstrate that RpLV is related to reoviruses belonging to the genera
Oryzavirus, Cypovirus, Dinovernavirus and Fijivirus. Analysis of the
polymerase sequence showed 36% aa identity to Rice ragged stunt virus
(RRSV, an oryzavirus). As a general rule, reoviruses with an aa sequence
identity greater than 30% in the RdRp sequence are considered members of
the same genus (two exceptions have been reported). The analysis of the 5′
and 3′ terminal regions, however, indicate that RpLV and RRSV have
different conserved sequence motifs, which would suggest they are species
from distinct genera. Furthermore, the first 3 nucleotides (AGU) at the 5′
terminus of RpLV are conserved among members of the genera Cypovirus,
Dinovernavirus, and Fijivirus, supporting the relatedness among these
reoviruses. The lack of conservation between the terminal sequences of RpLV
and RRSV, the very low aa identity in sequences from segments S8 and S9,
and the distinct hosts for these viruses justify the creation of a new genus for
the classification of RpLV.
Screening of exotic and commercial sorghum accessions against a new
virulent race (P6) of Peronosclerospora sorghi causing downy mildew
disease
G. L. Radwan (1), R. Perumal (1), T. Isakeit (1), L. K. Prom (2), C. R.
LITTLE (3), C. W. Magill (1)
(1) Texas A&M University, College Station, TX, U.S.A.; (2) USDA-ARS,
Southern Plains Agricultural Research Center, College Station, TX, U.S.A.;
(3) Kansas State University, Manhattan, KS, U.S.A.
Phytopathology 100:S106
A recent outbreak of sorghum downy mildew in Texas has led to the
discovery of both metalaxyl resistance and a new pathotype (P6) in the causal
organism Peronosclerospora sorghi. To identify new sources of resistance to
P6, a total of 312 (245 mini-core lines representing diverse germplasm from
ICRISAT and 67 elite commercial accessions from Kansas) were tested in a
greenhouse inoculation study. Forty-eight mini-core and 20 Kansas accessions
had < 10% infection and were selected as resistant for further confirmation.
Out of the 48 mini-core accessions, 20 (IS1212, 4060, 4613, 4631, 5094,
12804, 12883, 12965, 22294(PMK108), 24453, 24462, 24463, 26617,
29314(PL512), 29358(PHN2), 29392(PHM36), 29606, 30092(AMM938),
30443, and IS30562) were photoperiod insensitive and showed 0% infection.
Eleven of the commercial accessions (DKS28-05, DKS37-07, DKS44-20,
DSS B64, MG4748, MG4665, MG4765, Pioneer 85Y40, Dyna-Gro 742C,
NK5418, and TRX83774) showed 0% infection. All 245 mini-core
germplasm accessions are also being screened in separate tests under
greenhouse inoculation for the identification of anthracnose, head smut and
ergot resistance sources. DNA fingerprinting has been completed with 60
SSRs (representing all ten sorghum linkage groups) using a high throughput
ABI Prism 3100 DNA sequencing system to quantify genetic diversity among
resistant accessions.
Survey of small RNA during the suppression of soybean defense
responses by Pseudomonas syringae pv. glycinae avrB
O. RADWAN (1), B. Calla (1), L. Vodkin (1), M. Hudson (1), S. J. Clough
(2)
(1) University of Illinois, Dept. Crop Science, Urbana, IL, U.S.A.; (2)
University of Illinois, Dept. Crop Science and U.S. Department of
Agriculture, Urbana, IL, U.S.A.
Phytopathology 100:S106
Bacterial effector proteins, secreted through type III secretion systems, have
been shown to trigger defense responses when recognized by resistant plants,
and to suppress defense responses in susceptible host plants. Here we
examined the hypothesis that Pseudomonas syringae pv. glycinae (Psg)
carrying the avirulence gene avrB suppresses soybean defense responses from
a soybean host that lacks the corresponding R gene (RPG1-b). Gene
expression profiling using soybean oligo microarrays indicated that while
defense genes, transcription factors, genes involved in the phenoproponoid
pathway and signal transduction components were induced in the
incompatible reaction, they were suppressed in susceptible plants inoculated
with Psg (avrB) compared to a Psg (avrB-) control. To address the possible
role of small RNA during the suppression of defense responses in susceptible
soybean, we examined the differential expression of small RNA (miRNAs and
S106
PHYTOPATHOLOGY
siRNAs) between resistant and susceptible plants inoculated by Psg carrying
avrB. Among 39887 unique reads with high quality, 35594 reads were 19 to
24 bp in length. These sequences were blasted against soybean and Psg
genome sequence databases to determine plant and bacterial origins and to
narrow the lists of interest. This information is being used to identify possible
targets of the siRNA and miRNAs. These analyses will provide insight into
the possible role of small RNA during soybean-Psg interactions.
Inhibition of Magnaporthe oryzae using cells and cell-free extracts of
several strains of Bacillus
A. RAHMAN (1), W. Uddin (1)
(1) Penn State University, State College, PA, U.S.A.
Phytopathology 100:S106
The suppressive ability of several isolates of Bacillus subtilis, B. licheniformis
and B. amyloliquefaciens to Magnaporthe oryaze, the cause of gray leaf spot
of perennial ryegrass, was evaluated through assessment of direct antagonism.
Cells, culture supernatants and methanolic supernatant extracts of each isolate
were tested in vitro for mycelial growth and conidial germination inhibition.
Solid phase columns were used to extract hydrophobic/lipophilic compounds
from the cell-free culture supernatants using different concentrations of
methanol. Cells, culture supernatants, and methanolic extracts of all isolates of
B. subtilis and B. amyloliquefaciens significantly inhibited mycelial growth.
However, the inhibition zone of mycelial growth using cell-free culture
supernatants and methanolic extracts were 20–35% smaller than that produced
by live bacterial cells. The conidial germination of M. oryzae was reduced 70–
80% by culture supernatants of B. subtilis and B. amyloliquefaciens isolates.
B. licheniformis cells, culture supernatants and extracts had no significant
effect on fungal growth or spore germination. The possible roles of specific
components of methanolic extracts in induced systemic resistance in the
perennial ryegrass-gray leaf spot pathosystem will be presented.
Effect of strawberry nursery infestation with Colletotrichum
gloeosporioides on fruiting field Anthracnose crown rot severity
M. RAHMAN (1), M. E. Carnes (1), F. J. Louws (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S106
Anthracnose crown rot caused significant losses to strawberry (Fragaria x
ananassa) nursery and fruit growers in the southeast U.S. in recent years.
There is very little epidemiological information available to develop
management strategies for this disease. Initial inoculum levels and spread in
the nursery, impact of nursery inoculum on fruiting field disease severity and
management options were studied for consecutive two years. Isolates from
non-cultivated wild hosts such as Muscadine grape (Vitis rotundifolia) and
Virginia Creeper (Parthenocissus quinquefolia) were pathogenic on
strawberry and identified as potential sources of initial inoculum to the
nursery. Assessment after 60 days of inoculating 5%, 10% or 25% mother
plants in the nursery indicated inoculum load significantly (P ≤ 0.001)
impacted latent infection severity on adjacent daughter plants. Spatial
dispersal data showed better fit using the empirical power law model
compared to an exponential model based on R2 and residual values. Steepness
of the dispersal gradient suggested rouging of plants around infection foci
could be used for removal/separation of infested plants from the healthy ones.
Bare root plants from the 10% and 25% nursery inoculation levels caused
significant (P ≤ 0.005) crown rot incidence in grow-out studies in the fruiting
field and reduced yield compared to noninoculated control or 5% inoculation
level. Dipping infested plants in selected fungicides before fall planting
significantly decreased crown rot.
Yield loss incited by orange rust (Puccinia kuehnii) on a highly
susceptible sugarcane cultivar in Florida
R. N. RAID (1), J. C. Comstock (2), N. Glynn (2)
(1) University of Florida, Belle Glade, FL, U.S.A.; (2) ARS-USDA Sugarcane
Field Station, Canal Point, FL, U.S.A.
Phytopathology 100:S106
Sugarcane orange rust, incited by Puccinia kuehnii, was initially reported in
the Western Hemisphere in 2007, when it was first observed in Florida. Since
that time, it has affected several commercial cultivars, notably CP80-1743,
CP72-2086, and CL85-1040. Experiments were conducted to quantify the
amount of yield losses on the latter, the most susceptible of these cultivars.
Varying levels of orange rust were established at the Everglades Research and
Education Center in Belle Glade, FL by applying the fungicide pyraclostrobin
at 7, 14, 21, and 28 day intervals. Experimental units measured five rows by
15 m and were arranged in a randomized complete block design with eight
replications. Non-sprayed plots served as controls. Rust severities were visibly
estimated on the top-visible-dewlap leaf minus four using established
assessment keys. The 2008 orange rust epidemic began in May and persisted
through the remainder of the growing season. Disease severities ranged from
near zero in the 7-day treatments to over 35% in the controls. Millable stalk
populations were reduced by 12% and stalk biomass by 32% when controls
were compared with the 7-day treatments. Together, these accounted for
reductions of 43% in tons of cane per hectare. Sucrose concentrations were
significantly reduced by nearly 18% on this cultivar. Cumulative losses in
terms of sugar per unit area of cane measured 53%, demonstrating the
destructive nature of this disease under favorable conditions.
Evaluation of fungicides for management of downy mildew on sweet basil
R. N. RAID (1), E. McAvoy (2), D. D. Sui (3)
(1) University of Florida, Belle Glade, FL, U.S.A.; (2) University of Florida,
Labelle, FL, U.S.A.; (3) University of Florida, West Palm Beach, FL, U.S.A.
Phytopathology 100:S107
Basil downy mildew, incited by the fungal pathogen Peronospora belbahrii, is
a newly important disease of this popular herb in the U.S. First reported in
Florida in 2007, the disease has since been reported in 17 states and Canada.
While host-plant resistance to downy mildew has been observed among
various basil species, most sweet basil varieties (Ocimum basilicum) are very
susceptible. Multiple experiments were conducted at the Everglades Research
and Education Center during 2009 to evaluate fungicides for downy mildew
control. In one replicated field trial, 19 fungicides were evaluated under
extreme disease pressures. The experiment consisted of a randomized
complete block design and chemicals were applied foliarly at weekly
intervals. Disease was visually assessed on a scale of 0 to 10, with 0
representing no disease and 10 representing the level of disease in the
untreated check. Of the compounds tested, Revus (0.7), Pristine (1.0), Ridomil
Bravo (1.0), Reason (1.0), Forum (1.0), BAS 651 (1.0), Quadris (1.7), and
Ranman (1.7) provided for the highest levels of control, in that order. These
were followed by Presidio (2.7), Gavel (4.0) and K-Phite (4.3), which
provided intermediate levels of control. Of the remaining treatments, although
Actigard (6.7), Bravo Weather Stik (6.7), Kocide 3000 (5.3), Previcur (7.7),
Regalia (7.0), Serenade (7.0), and Tanos (6.0) provided for reductions in
disease severity, the level of control was not commercially acceptable.
Self- and cross-interaction studies among Pelargonium flower break and
Pelargonium line pattern viruses coat proteins and their domains
G. RAIKHY (1), S. M. Leisner (1)
(1) The University of Toledo, Toledo, OH, U.S.A.
Phytopathology 100:S107
Pelargonium spp., native to South Africa, are widely cultivated in Europe and
North America as ornamentals. Vegetative propagation of Pelargonium
subjects them to viral infections, which have detrimental effects on quality
and quantity. Pelargonium flower break (PFBV) and Pelargonium line pattern
(PLPV) are two most widespread viruses infecting Pelargonium spp. They
both belong to the family Tombusviridae and have single-stranded positivesense genomic RNAs of ~3900 nucleotides. The multifunctional, 37 kDa, coat
protein (CP) is encoded by 3′ proximal ORF. CP of both viruses contains 3
different domains namely, R- (interacting with RNA), S- (virion shell) and P(projecting). In this study, self- and cross-interactions of complete CPs of
these two viruses along with identification of domain(s) involved is reported.
Self- and cross- interaction between two CPs was observed by yeast twohybrid analyses and confirmed by MBP pull-down assays. For PFBV, all the
three domains showed self-association as well as interaction among
themselves, and with full length CP. For PLPV, the interactions among
different domains are under investigation though preliminary results show
similar pattern to that of PFBV. When cross-interaction studies of the CP
domain were performed, the S domains were identified to interact.
Characterization of Huanglongbing associated ‘Candidatus Liberibacter
asiaticus’ from citrus relatives
C. RAMADUGU (1), K. L. Manjunath (2), S. E. Halbert (3), R. H. Brlansky
(4), M. Roose (1), R. F. Lee (2)
(1) University of California, Riverside, Riverside, CA, U.S.A.; (2) USDA
ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside,
CA, U.S.A.; (3) Florida Division of Plant Industry, Gainesville, FL, U.S.A.;
(4) University of Florida Citrus Research and Education Center, Lake Alfred,
FL, U.S.A.
Phytopathology 100:S107
Citrus greening or Huanglongbing (HLB) is a devastating disease reported
predominantly from Citrus species. Effective mitigation of HLB requires
information on all possible means of distribution of the disease including
spread by alternate hosts. Citrus relatives collected from HLB-infected regions
of South Florida were analyzed for the presence of ‘Candidatus Liberibacter
asiaticus’ (LAS). qPCR of the 16s RNA region indicated the presence of LAS
from several plant samples. Molecular confirmation of the presence of LAS
was carried out by PCR amplification, cloning and sequencing of several other
genomic regions of the bacterium. The taxonomic identity of the host plant
materials was confirmed by comparing the sequence of a nuclear gene, malate
dehydrogenase, with the sequence of known accessions from the Citrus
Variety Collection, Riverside, CA. This is the first report of detection of the
bacterium associated with HLB from naturally infected Atalantia ceylanica
and Severinia buxifolia from the United States. The sequence information
shows that A. ceylanica and S. buxifolia harbor a bacterium identical to LAS
associated with HLB. The study helps in generating information about citrus
relatives that can serve as alternate hosts for LAS. The nature of Liberibacters
from other PCR positive citrus relatives was analyzed, and the importance of
other hosts in management of HLB will be discussed.
Over-expression of the calmodulin gene SCaM-4 in soybean enhances
resistance to Phytophthora sojae
S. RAO (1), M. El-Habbak (1), J. S. Haudenshield (2), D. Zheng (2), G. L.
Hartman (2), S. S. Korban (2), S. A. Ghabrial (1)
(1) University of Kentucky, Lexington, KY, U.S.A.; (2) University of Illinois,
Urbana, IL, U.S.A.
Phytopathology 100:S107
Soybean is the major oilseed crop in the world with an annual value of 14
billion dollars in the United States. Plant pathogens inflict heavy losses on
soybean yield. One of the most important pathogens is the oomycete,
Phytophthora sojae, that causes Phytophthora root and stem rot. A set of
resistance genes (Rps) was found to confer resistance to the pathogen.
However, due to selection pressure, virulent races continue to evolve.
Calmodulin, a Ca2–binding protein, is implicated in plant defense responses in
different plant species. The cellular level of SCaM-4 is known to increase
rapidly in response to pathogen infection. Over-expression of SCaM-4 in
transgenic tobacco has been correlated with enhanced broad resistance to
pathogens. We used the bean pod mottle virus-based vector for overexpression of SCaM-4 in soybean, which was verified by RT-PCR. The
SCaM-4 over-expressing plants exhibited a distinct phenotype compared with
control plants. Mock, vector control, and SCaM4 over-expressing Harosoy
plants were inoculated with P. sojae race 3 using stem wound inoculation.
While mock and vector control plants showed expanding necrotic lesions at
the infection sites and many plants died within 10 to 15 days post-inoculation,
all SCaM-4 over-expressing plants remained vigorous, and showed only
superficial small lesions at infection sites. Experiments are underway to
silence the SCaM-4 gene in P. sojae- resistant soybean cultivars to determine
whether such treatment would jeopardize the resistance response.
Bacterial toxins as natural nematicides: A high-throughput screen using
C. elegans
A. A. RASCON (1), A. Garcia (1), S. Hanson (1)
(1) New Mexico State University, Las Cruces, NM, U.S.A.
Phytopathology 100:S107
Plant parasitic nematodes cause devastating damage to a wide variety of crops
throughout the United States and worldwide resulting in billions of dollars in
crop losses annually. Various effective chemical methods of nematode control
have been or are currently being phased out due to environmental and human
health concerns. The loss of these nematicides is likely to lead to a resurgence
in agricultural production losses caused by nematodes. Bacterial toxins such
as Bt genes have been widely and successfully used as natural insecticides in
agriculture. Therefore, the hypothesis is that bacterial toxins may be useful
controls for nematodes. The data from a simple high-throughput toxicity assay
using Caenorhabditis elegans to screen for naturally occurring soil bacteria
that are toxic to C. elegans is reported here. To date, over 40 strains of
bacteria have been identified that inhibit C. elegans growth and reproduction,
and increase the mortality rate. Data on the identity of the nematicical bacteria
and properties of the toxic molecules are included as well as discussion on the
potential use of these toxins as nematode control agents.
A qPCR assay for detection and quantification of Verticillium dahliae in
spinach seed
G. RAUSCHER (1), B. Mou (1), R. J. Hayes (1), S. T. Koike (2), K.
Maruthachalam (3), K. V. Subbarao (3), S. J. Klosterman (1)
(1) USDA ARS, Salinas, CA, U.S.A.; (2) University of California
Cooperative Extension, Monterey and Santa Cruz counties, Davis, CA,
U.S.A.; (3) UC Davis, Davis, CA, U.S.A.
Phytopathology 100:S107
The fungus Verticillium dahliae is the causal agent of Verticillium wilt of
lettuce and other specialty crops in the Salinas Valley of California. Spinach,
another major specialty crop in California, is not affected by Verticillium wilt
in commercial production. However, spinach seed infected with V. dahliae
and planted in the Salinas Valley increases inoculum density and introduces
exotic strains that may contribute to Verticillium wilt epidemics. The goal of
this work is to develop a real-time quantitative PCR (qPCR) assay for the
detection and quantification of V. dahliae in spinach seed. The assay is based
on the use of SYBR Green methodology with previously published primer
sequences specific for the -tubulin gene of V. dahliae. Parallel plating and
qPCR assays revealed that the qPCR assay can be used for reliable detection
Vol. 100, No. 6 (Supplement), 2010
S107
of V. dahliae in seed infected at the 10 percent level. Because the qPCR assay
enables rapid and reliable detection of V. dahliae, the assay has implications
as a useful tool to limit the spread of the pathogen.
Detection and quantification of the sugar beet cyst nematode, Heterodera
schachtii, through qPCR
G. RAUSCHER (1), K. Richardson (1)
(1) USDA ARS, Salinas, CA, U.S.A.
Phytopathology 100:S108
Heterodera schachtii Schm. is a major parasite of sugar beet (Beta vulgaris
L.) worldwide. As few as two eggs per cubic centimeter are enough to cause
significant economic damage. Classical diagnostic methods determine the
number of cysts in soil samples, but are time consuming. The goal of this
study was to design a quantitative PCR (qPCR) assay to more rapidly detect
and quantify H. schachtii and facilitate pre-planting field detection. Primers
for a multiple copy sequence (ITS2) and a single copy gene ( -tubulin) were
designed and qPCR standardized using SYBR Green. Specificity and
sensitivity of the reactions were tested using commonly found soil
microorganisms. ITS2 primers amplify H. schachtii from different origins
including New York and California, detecting as little as 10pg of nematode
DNA per gram of soil and do not show cross amplification with other
commonly found soil inhabitants. The -tubulin primers show high specificity
as well, with a lower detection threshold of 14pg/g. Correlation of results
between the genes is 0.97. The described qPCR assay will provide breeders
with a more sensitive and less variable method to distinguish responses of
breeding lines to H. schachtii. Further applications of these tests have the
potential to improve pest management programs.
Incidence of bacterial spot on pumpkins in Illinois
A. RAVANLOU (1), M. Babadoost (1)
(1) University of Illinois, Urbana, IL, U.S.A.
Phytopathology 100:S108
This study was conducted to assess incidence and severity of bacterial spot,
caused by the bacterium Xanthomonas campestris pv. cucurbitae (Xcc), and
variation among Xcc isolates in Illinois. Bacterial spot has become one the
most devastating diseases of pumpkins in Illinois and other Midwest states.
Xcc infects leaves and fruit, producing angular spots on leaves and small,
circular lesions on fruits. Infected fruit with Xcc is easily colonized by
Fusarium spp. and soft rot bacteria, resulting in rapid collapse of fruit. In the
past four years, yield losses in some pumpkin fields in Illinois exceeded 60%.
Jack-o-lantern pumpkins are more susceptible to Xcc than processing
pumpkins. In 2009, 17 randomly-selected jack-o-lantern pumpkin fields were
selected and incidence and severity of bacterial spot on leaves and fruit were
assessed throughout the season. The fields were visited four times, at 3-week
intervals. In each field, leaves and fruit in 12 locations, in an M-shaped
sampling pattern, were evaluated. In each location, the incidence and severity
of the disease were assessed on 10 leaves and five fruits. Average incidence of
bacterial spot on leaves was 2.4, 4.2, and 5.0% in July, August, and
September, respectively. The incidence of fruit infection ranged from 2 to
98%, with an average of 45.5%. Ten bacterial isolates from leaves and fruit
from each field were collected and the variation in pathogenicity and genetics
of the isolates are being investigated.
Melaleuca quinquenervia plants differ in susceptibility towards fungus
Puccinia psidii infection and disease development
M. B. RAYAMAJHI (1), G. Wheeler (1), P. D. Pratt (1), T. D. Center (1)
(1) USDA-ARS, Ft. Lauderdale, FL, U.S.A.
Phytopathology 100:S108
Puccinia psidii (rust fungus) attacks immature healthy foliage of Melaleuca
quinquenervia (melaleuca), an invasive plant in southern Florida, U.S.A.
Melaleuca plants grown under same growing conditions manifest either
susceptible or resistant reactions towards this fungus. We hypothesize that the
variable reactions may be due to the differences in terpenoid contents in
melaleuca. This hypothesis was tested using screenhouse and field-grown
melaleuca plants. Melaleuca seedlings were tagged, grown to ca 45-cm height,
inoculated with uredospores, placed in screenhouse under rust-fungus infected
trees and evaluated for symptoms during a 4-wk period. Plants were also
evaluated for major terpenoid content. Crude terpenoids were tested for
effects on uredospore germination. The percentage of seedlings that developed
pustules (susceptible), halos only (resistant) and no halos or pustules
(immune) were 63.07, 33.90, and 0.03, respectively; these trends were similar
among field-grown plants. Terpenoids from neither nerolidol nor viridifloral
types of plants inhibited uredospore germination. Gas chromatography
analysis of susceptible plants showed significant increases in total terpenoid
concentrations as well as some of its constituents (myrcene, limonene and
Beta-caryophyllene) compared with resistant plants. These results suggest that
terpenoid constituents influence rust-fungus disease development in melaleuca,
though this interpretation needs to be confirmed through additional research.
S108
PHYTOPATHOLOGY
Burkholderia andropogonis from citrus appears to have a functional hrp
system and pthA and pthB from Xanthomonas citri enhance its
pathogenicity
G. REBELLO (1), D. Gabriel (1)
(1) University of Florida, Gainesville, FL, U.S.A.
Phytopathology 100:S108
Xanthomonas citri (Xc), the causal agent of citrus canker, can horizontally
transfer pthB, an avrBs3/ pthA family type III effector gene on a self
mobilizing plasmid, to other bacteria in planta (El Yacoubi et al., 2007).
Burkholderia andropogonis (Ba), which has a relatively wide host range is the
causal agent of bacterial brown leaf spot of citrus (Duan et al., 2009); Ba can
therefore occupy the same niche as Xc. Since Florida’s citrus canker
eradication program was suspended in early 2006, there may be an increased
opportunity for horizontal gene transfer involving Xc and Ba in Florida. Ba
transformants which carried either pthA or pthB exhibited increased
pathogenicity on citrus and also grew at least one log better than wild type in
all citrus varieties tested, suggesting that the PthA and PthB effectors act in
part to suppress host defenses. Type III effectors require a Type III secretion
system (T3SS) in order to be delivered into plant cells. Southern blots using
hrcC suggested the presence of a functional T3SS in Ba, and hrcV was
amplified, cloned and sequenced from Ba. A hrcV knockout mutant of Ba
failed to cause a hypersensitive response in nonhost tomato and was much less
pathogenic on citrus as compared to wild type. Complementation experiments
are in progress.
Effect of maize kernels maturation on transcriptional activity in
Aspergillus flavus
B. N. REESE (1), G. A. Payne (2), C. P. Woloshuk (1)
(1) Purdue University, Department of Botany and Plant Pathology, West
Lafayette, IN, U.S.A.; (2) North Carolina State University, Plant Pathology,
Raleigh, NC, U.S.A.
Phytopathology 100:S108
Aspergillus flavus infects maize kernels in the field and contaminates them
with aflatoxin. To better understand how the fungus responds to the kernel
environment during infection, we analyzed gene transcription after growth of
the fungus on kernels at four developmental stages (blister, milk, dough,
dent). Five days after inoculation, total RNA was isolated from kernels and
hybridized to Affymetrix Gene Chip arrays containing probes representing
14,163 A. flavus genes. Statistical comparisons of the expression profile data
revealed significant differences (P < 0.01) that included unique sets of upregulated genes for each kernel stage and six patterns of expression over the
four stages. Among the genes expressed in colonized dent kernels were a
phytase gene and six putative genes involved in zinc acquisition. A knockoutmutant of the phytase gene, PHY1, was obtained. Although growth of the
mutant on medium containing inorganic phosphate was the same as the wild
type, growth of the mutant was severely restricted when phytate was the sole
source of phosphate. Further, growth after 5 days on maize ears (based on
ergosterol content) and aflatoxin production of the mutant were reduced 50%
compared to the wild type. The results indicate that phytase in A. flavus may
have an important role in pathogenesis.
Dissecting cyst nematode CLE perception in Arabidopsis roots
A. REPLOGLE (1), J. Wang (1), J. Smeda (1), M. G. Mitchum (1)
(1) University of Missouri, Columbia, MO, U.S.A.
Phytopathology 100:S108
Cyst nematodes cause billions of dollars in crop damage by inducing enlarged,
multinucleate feeding cells in host roots that serve as the sole nutrient source
for the nematode to complete its life cycle. The feeding site, which is known
as a syncytium, is formed in response to nematode secreted effector proteins.
Heterodera species produce CLAVATA3(CLV3)/ESR (CLE) effector
proteins sharing functional similarity with plant CLE signaling peptides
involved with several aspects of plant development including maintenance of
stem cell pools in the root meristem. Previously, we identified nematode CLE
receptor candidates by screening mutants of plant CLE receptors and other
closely related LRR-RLKs for resistance to synthetic Heterodera CLE peptide
treatment. Mutants of CLV2, CORYNE (CRN) and BARELY ANY MERISTEM
(BAM1) were resistant to peptide treatment and used in nematode infection
assays and overexpression studies to reveal that CLV2 and CRN appear to be
major players in nematode CLE perception. Further characterization of the
defects in syncytium formation on the receptor mutants will help elucidate the
role CLE peptides play in nematode parasitism.
Testing for mycotoxins using LC-MS/MS
J. RICHARD (1), D. Houchins (1), C. Brewe (1), M. Prinster (1)
(1) Romer Labs, Inc., Union, MO, U.S.A.
Phytopathology 100:S108
Mycotoxins have traditionally been detected by a variety of methods,
including rapid methods (test kits) and reference methods, such as HPLC and
GC. Of the reference methods, GC can be limited due to the necessity of
derivatizing the compounds of interest. This may also be required for some
HPLC methods. However, liquid chromatography may be used in conjunction
with a variety of detectors, including fluorescence, UV-VIS, and others,
including mass spectrometers. The coupling of liquid chromatography with a
mass spectrometer (or tandem mass spectrometers, LC/MS/MS) allows for
methods which are applicable to a wide variety of analytes, with no
limitations by molecular mass, a straightforward sample preparation, and no
chemical derivatization required. These methods also have the benefit of
providing structural information on the target compound and the possibility of
testing for many analytes in one run. Matrix effects and the effects of
variations in sample preparation may be eliminated by the use of stable
isotope-labeled internal standards. These features make LC/MS/MS methods
useful and versatile in the detection of mycotoxins.
Functional characterization of a transcription factor family in Fusarium
verticillioides
J. B. RIDENOUR (1), C. P. Woloshuk (2), W. Shim (3), B. H. Bluhm (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) Purdue University,
West Lafayette, IN, U.S.A.; (3) Texas A&M University, College Station, TX,
U.S.A.
Phytopathology 100:S109
Fusarium verticillioides is a ubiquitous pathogen of maize, attacking
seedlings, kernels, and stalks. Additionally, F. verticillioides produces
fumonisin mycotoxins. Currently, little is known at the molecular level
regarding pathogenesis or mycotoxigenesis in this important fungus. The goal
of this study was to identify genes and regulatory mechanisms underlying
pathogenicity and fumonisin biosynthesis in F. verticillioides. To this end, an
entire family of transcription factors - CCAAT-binding factors (CBFs) - was
functionally characterized through reverse genetics. The genome of F.
verticillioides is predicted to contain six CBF-encoding genes, all of which
were disrupted utilizing split-marker homologous recombination. For each
mutant, morphology, pathogenesis, kernel colonization, and fumonisin
biosynthesis were analyzed. The mutants displayed a range of morphological
abnormalities, including reduced growth and conidiation. Furthermore, at least
one of the genes was required for wild-type levels of kernel colonization, and
at least two of the genes were required for wild-type levels of fumonisin
biosynthesis. This study is one of the first systematic analyses of fungal CBFs
in the context of plant pathogenesis and one of the first successful attempts to
characterize an entire family of transcription factors in F. verticillioides.
Additionally, these findings provide new insight into the regulation of
pathogenesis and fumonisin biosynthesis in F. verticillioides.
Tomato grafting to manage bacterial wilt (caused by Ralstonia
solanacearum) in the southeastern U.S.
C. L. RIVARD (1), R. M. Welker (1), S. O’Connell (1), M. M. Peet (2), F. J.
Louws (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.; (2) United States
Department of Agriculture, Washinton, DC, U.S.A.
Phytopathology 100:S109
Ralstonia solanacearum causes severe losses to tomato growers who manage
infested soils in the southeastern U.S. Commercial tomato varieties offer little
host resistance and fumigation has limited efficacy as primary inoculum can
be re-introduced through soil water movement. Bacterial wilt (BW) is
managed worldwide by grafting popular fruiting varieties onto resistant
rootstocks. However, few rootstocks are available in the U.S. and little is
known regarding their efficacy against native Ralstonia strains. Field trials
were conducted in NC from 2007–2009 to determine the utility of grafting to
manage BW and evaluate commercially-available rootstocks against endemic
populations of R. solanacearum. Four rootstocks displayed effective partial
resistance, and BW AUDPC values were significantly lower among all
rootstocks compared to non- and self-grafted controls in all trials (P < 0.05).
Fruit yield was significantly affected by grafting onto resistant rootstock (P <
0.05). Interestingly, several rootstocks showed differential efficacy across
locations, suggesting that pathogen population dynamics plays a role in
rootstock selection. ‘RST-04-105’ had complete resistance in eastern NC, but
showed intermediate resistance in western NC. Similarly, ‘Dai Honmei’ had
high resistance in western NC, but was intermediate in eastern NC. Grafting is
an effective management strategy against bacterial wilt and could be an
important component of a successful IPM program.
Anthracnose disease of annual bluegrass as affected by light-weight
rolling and equipment traffic
J. A. ROBERTS (1), J. A. Murphy (1), B. B. Clarke (1)
(1) Rutgers University, New Brunswick, NJ, U.S.A.
Phytopathology 100:S109
Light-weight rolling is a common management practice employed to smooth
the playing surface and increase ball roll distance (BRD) on golf course
putting greens. Anthracnose is a serious disease of annual bluegrass [ABG;
Poa annua L. f. reptans (Hausskn) T. Koyama] putting green turf caused by
Colletotrichum cereale Manns. Light-weight vibratory rolling has been shown
to reduce anthracnose severity on ABG, but the impact of other roller
types or location of equipment traffic on this disease is unknown. A 3-yr field
trial was established in New Brunswick, NJ in 2006 to evaluate the influence
of roller type (i.e., sidewinder, vibratory and non-rolled) and location (center
or perimeter) of equipment traffic on anthracnose severity and BRD of ABG
turf maintained at 3.2 mm. The study was established as a strip-plot design
with 8 replications. Both roller types reduced disease severity 2 to 13%
compared to non-rolled turf under moderate disease pressure in 2007 and
2008. The heavier sidewinder roller had less disease than the vibratory roller
on 4 of 13 rating dates and perimeter plots, which received increased
equipment traffic, had less disease compared to center plots on 6 of 13 rating
dates during this period. Results indicate that rolling can be used to increase
BRD and improve turf quality without intensifying, and in some cases
reducing, anthracnose severity on ABG putting green turf under moderate
disease pressure.
Impact of hail damage during early reproductive stages on ear rot and
mycotoxin contamination of maize
A. E. ROBERTSON (1), G. P. Munkvold (1), C. R. Hurburgh (1), S. M.
Ensley (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S109
During the 2009 growing season in Iowa, two severe hail storms affected over
400,000 hectares of maize during early reproductive stages. Concerns were
raised about increased ear rot disease and associated mycotoxin
contamination. To address these concerns, samples of ears were collected
within 48 hours of harvest from 57 fields damaged by hail and 25 undamaged
fields. Ears were visually assessed for kernel damage and ear rot severity.
After shelling, grain was ground and tested for deoxynivalenol (DON),
zearalenone (ZEA) and fumonisins (FUM) using commercially available
antibody-based lateral flow strip tests. Confirmation analysis on elevated
samples was performed by Gas Chromatography for DON and High Pressure
Liquid Chromatography for ZEA and FUM. Fusarium, Gibberella and
Cladosporium ear rots were the most prevalent diseases. Hail damage to
kernels increased the risk of ear rot. The most prevalent mycotoxin detected
was DON (mean 2.63 ppm), followed by ZEA (mean 0.53 ppm) and FUM
(mean 0.49 ppm). Levels of DON and ZEA in grain from hail damaged fields
were greater than those detected in grain from undamaged fields. There was a
positive relationship between ear rot severity and DON and ZEA
contamination (r = 0.645, P < 0.001 and r = 0.474, P < 0.001, respectively).
Levels of DON also were correlated with test weight (r = –0.38, P = 0.004)
and protein (r = 0.49, P < 0.001). In the future, preharvest scouting of fields
with suspected mycotoxins may be an effective strategy for targeting
postharvest inspection and marketing activities.
Serological detection and molecular analysis of Tobacco ringspot virus
and Strawberry latent ringspot virus in mint (Mentha sp.)
N. L. ROBERTSON (1), B. J. Furman (1)
(1) USDA ARS, Arctic and Subarctic Plant Genetic Resources Unit, Palmer,
AK, U.S.A.
Phytopathology 100:S109
Mint (Mentha sp.) is commercially cultivated around the world by the food,
medicinal, and landscape industries. Over 400 clonal mint accessions have
been maintained by USDA, Agricultural Research Station in Corvallis,
Oregon by the National Clonal Germplasm Respository since 1983, and more
recently (2010) in Palmer, Alaska by the Arctic and Subarctic Plant Genetic
Resources Unit. Due to an increased concern for viruses occurring in mint,
424 transplanted mint accessions in Palmer were assayed for selected viruses
previously known to naturally infect mint. Leaves were collected from plants
within four weeks of emergence and processed for ELISA and total RNA
extractions. Assays were performed according to manufacturer’s directions for
the following ELISA kits: 1) Agdia, Inc. (IN) - Alfalfa mosaic virus,
Strawberry latent ringspot virus (SLRSV), Tobacco ringspot virus (TRSV),
and universal potyvirus, and 2) AC Diagnostics, Inc. (AR) - Cucumber mosaic
virus and Cherry rasp leaf. ELISA results confirmed 13 SLRSV and nine
TRSV singly infected plants and one plant with a double infection. Infected
plants were confirmed by RT-PCR for all except one of the TRSV infected
plants, and for only three SLRSV infected plants. Natural occurrence of
TRSV in mint is limited to the United States while the distribution of SLRSV
is worldwide. Maintenance of healthy mint plants is an important aspect when
selecting plant material to send to requesting researchers.
Vol. 100, No. 6 (Supplement), 2010
S109
Association of a phytoplasma with dieback in palms in Puerto Rico
confirmed by nested-PCR assays
J. V. RODRIGUES (1), A. M. Vitoreli (2), A. L. Ramirez (3)
(1) University of Puerto Rico, San Juan, U.S.A.; (2) University of Florida,
Gainesville, FL, U.S.A.; (3) Dept. Agriculture of Puerto Rico (CAPS), San
Juan, U.S.A.
Phytopathology 100:S110
Diseases associated with phytoplasma infections are among the most serious
phytosanitary problems in palms. Those diseases have the potential to
adversely affect the landscape and tourism industry, where palms are key
species, as well as the ecosystem. In Guaynabo, Puerto Rico, dieback and
mortality of palms was observed, resembling symptoms described for lethal
yellowing associated with phytoplasma. Samples from different species of
palms were collected. DNA was extracted using DNeasy kit, and PCR was
carried out using universal primers for amplification of phytoplasma DNA P1
and P7, and P1m/LY1623Sr. PCRs were followed by nested-PCR with two
pairs of primers R16F2n/R16R2 and LY16-23Sf2/LY16-23Sr2. The amplified
products were visualized in agarose gel/UV. A single fragment of 1.2Kb was
observed in both primer combinations. Number and size of the fragment were
expected for determining the occurrence of phytoplasma. All tests were
repeated four times and confirmed by PDC-Regional Lab for the SPDN,
Gainesville, FL. DNA from nonsymptomatic plants did not result in the
amplification of PCR products. So far, detection of phytoplasma associated
with symptoms similar to lethal yellowing of palms was confirmed for three
species: Royal Palm (Roystonea sp.), Fishtail Palm (Caryota mitis), and
Carpentaria (Carpentaria acuminata). To our understanding this is the first
report of a phytoplasma occurring in palms in Puerto Rico.
Genetic diversity of barley spot blotch resistance sources by chromosome
haplotyping
S. RODRIGUEZ (1), C. Torres Tuyo (1), P. Silva (1), A. Castro (1), C.
Pritsch (1)
(1) University Uruguay, Montevideo, URUGUAY
Phytopathology 100:S110
Spot blotch, caused by Cochliobolus sativus, is a major foliar disease of barley
that may become even more severe due to global climate change. Additional,
independent, non redundant sources of resistance should therefore be
incorporated into breeding programs to improve effectiveness and durability
of new barley cultivars. Chromosome haplotyping at major QTLs for spot
blotch resistance applied to candidate barley germplasm could contribute to
identify valuable, non redundant resistant genotypes. Here, we analyzed a set
of 25 diverse spot blotch resistant genotypes and 15 elite cultivars at QTLs
Rcs-qtl-1H-5-7 (four SSR markers, cv Morex as reference genotype), Rcs-qtl2H-7-8 (four SSR markers, cv Calicuchima as reference genotype), Rcs-qtl3H-1-3 (three SSR markers, Bowman BC as reference genotype) and Rcs-qtl7H-2-4 (two SSR and two STS markers, cv Morex as reference genotype).
The number of haplotypes identified at each QTL varied between four (Rcsqtl-3H-1-3) and ten (Rcs-qtl-7H-2-4). Moreover, nine resistance sources have
no allele in common to cv Morex at Rcs-qtl-7H-2-4, while seven genotypes
shared only one allele with Morex at QTLs Rcs-qtl-1H-5-7. Also, three
genotypes shared no more than one allele with cv Calicuchima at Rcs-qtl-2H7-8 and three other genotypes shared no more than one allele to cv Bowman
BC at Rcs-qtl-3H-1-3. These results contribute to our knowledge of useful,
non redundant genetic diversity available within spot blotch resistant
germplasm and may benefit barley breeding programs.
Arabidopsis thaliana ecotypes with differential susceptibility to the
bacterial pathogen Xylella fastidiosa
E. E. ROGERS (1)
(1) USDA-ARS, Parlier, CA, U.S.A.
Phytopathology 100:S110
Pierce’s disease of grapes and almond leaf scorch are devastating diseases
caused by the bacterium Xylella fastidiosa (Xf). To date, progress in
determining the mechanisms of host plant susceptibility, tolerance or
resistance has been slow, due in large part to the long generation time and
limited available genetic resources for grape, almond and other known hosts
of Xf. The long generation time and limited genetic resources for Xylella
fastidiosa compound the problem. The model plant Arabidopsis thaliana is an
ideal system for rapid progress in genetic and pathological studies. There are
many publically available genetic resources for Arabidopsis and it has a short
generation time. Arabidopsis has been evaluated as a model host for Xf and a
pin-prick inoculation method has been developed. Following infection, Xf can
be detected by microscopy and PCR. Xf has also been re-isolated from
infected Arabidopsis tissue. Timcourses following Xf growth have revealed
Arabidopsis ecotypes with differing susceptiblity to infection. The genetic
inheritance of these differences is being investigated. Additionally, differences in gene expression between ecotypes following Xf infection will be
presented.
S110
PHYTOPATHOLOGY
Assessing relationships among isolates of Wheat streak mosaic virus using
single nucleotide polymorphisms
S. ROGERS (1), U. Melcher (1), R. Allen (2), J. Fletcher (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.; (2) Oklahoma State
University, Tulsa, OK, U.S.A.
Phytopathology 100:S110
Relationships among multiple isolates of a given plant pathogen species have
been investigated by several molecular typing methods. The ability to extend
the use of such typing strategies to assess previous spatial and temporal
pathogen movement within a geographical area would facilitate disease
management and forensic studies. We investigated the use of single nucleotide
polymorphism (SNP) typing for assessing relationships among isolates of a
plant virus within a specific geographical location. Wheat streak mosaic virus
(WMSV)-infected wheat from nine locations within the western region of
Montana was obtained from researchers at Montana State University. cDNA,
generated from total RNA from infected wheat, was used as a template in the
SNaPshot Multiplex System (Applied Biosystems). Fluorescent ddNMPs were
added to the 3′-end of SNP-specific primers and the resulting sequences were
separated by capillary electrophoresis. Each of the nine WSMV isolates
produced a unique fingerprint for the SNPs, and a weak correlation between
the fingerprints and the locations from which the isolates were collected was
noted. The use of greater numbers of samples and assessment of isolates from
additional geographical locations will allow more conclusive interpretations of
SNP typing data and whether they could be useful for characterizing patterns
of pathogen spread. This work represents the first application of SNP typing
to plant pathogens for forensic purposes.
Assessment of Strawberry mild yellow edge virus infection in different
ecotypes of the Chilean native strawberry Fragaria chiloensis (L.) Duch.
P. F. ROJAS (1), C. Sandoval (1), P. D. Caligari (1), R. R. Martin (2)
(1) Universidad de Talca, Talca, CHILE; (2) USDA-ARS, Corvallis, OR,
U.S.A.
Phytopathology 100:S110
Fragaria chiloensis ssp chiloensis (L.) Duch, is distributed naturally in Chile.
Two botanical forms have been described; the white-fruited chiloensis, which
is cultivated in coastal mountains between latitudes 35° and 39°S, and the
small-red fruited patagonica, which grows widely between latitudes 35°and
47°S. F. chiloensis, the mother of the current commercial strawberry,
produces berries with unique quality characters and has the potential to be
used as a commercial berry. However, their yields can be severely affected by
viral diseases. Aphid-borne viruses have been found in wild and cultivated F.
chiloensis, but these plants do not show symptoms and the defense response
mechanism in this species is unknown. The objective of this work was to
study the development of SMYEV infection in different ecotypes of F.
chiloensis. A SMYEV isolate from Chile was used to graft-inoculate threemonth old healthy plants and a real-time PCR detection method was used for
detecting the SMYEV RNA using CP specific primers. Our results showed
virus detection at three days post inoculation (p.i.) in most chiloensis form
ecotypes, whereas in the patagonica form the virus was not detected until six
to eight weeks p.i. This suggests a differential response to SMYEV infection
in the different genetic backgrounds. PR was supported by CONICYT and
UTAL fellowships.
Survey and characterization of viral diseases affecting tomato crops in
the north of Chile
I. ROSALES VILLAVICENCIO (1), P. Sepúlveda (1), C. Medina (1), M. P.
Muñoz (1), C. Rojas-Bertini (2), R. Mora (1), M. Madariaga (1), J. K. Brown
(3)
(1) Instituto de Investigaciones Agropecuarias, Santiago, CHILE; (2) Instituto
de Investigaciones Agropecuarias, Arica, CHILE; (3) Department of Plant
Science, University of Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S110
During the past years the phytosanitary status of tomato cultivation in the
north of Chile has declined due to the proliferation of several viral diseases.
Various reasons have favored the introduction and proliferation of previously
unrecorded viruses: the climatic conditions, the increase of the international
commercial exchanges, and the introduction of Bemisia tabaci biotype B to
the area. A survey was conducted in the Azapa Valley (Region of Arica and
Parinacota) that included the main tomato viruses: Cucumber mosaic virus
(CMV), Alfalfa mosaic virus (AMV), Pepino mosaic virus (PepMV), Tomato
moisac virus (ToMV), Tomato spotted wilt virus (TSWV), Tobacco etch virus
(TEV), Potato virus Y (PVY), Peru tomato mosaic virus (PTV) and the
presence of begomovirus. Virus identification was based on enzyme-linked
immunosorbent assay (ELISA) and PCR-based methods. After analyzing
more than 1000 plants with virus-like symptoms, we have found no presence
of CMV, AMV, ToMV, PVY, TSWV, and TEV. The more prevalent viral
agents detected were PTV (9%), PepMV (27%) and the recently described
begomovirus Tomato yellow vein streak virus (ToYVSV), detected in a 43%
of samples. ToYVSV is considered one of the most important begomovirus
species currently affecting tomato and potato production in Brazil and
Argentina (South-America), and it is the only begomovirus detected in the
Chilean territory so far.
Xanthomonas albilineans needs an OmpA family outer protein for disease
symptom development and multiplication in the sugarcane stalk
P. C. Rott (1), L. A. FLEITES (2), G. Marlow (2), M. Royer (1), D. Gabriel (2)
(1) CIRAD, Mixed Research Unit for Biology and Genetics of Plant Pathogen
Interactions, Montpellier, FRANCE; (2) University of Florida, Gainesville,
FL, U.S.A.
Phytopathology 100:S111
Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that
causes sugarcane leaf scald. Xa produces albicidin, a potent antibiotic and
phytotoxin which blocks chloroplast differentiation, thus causing the foliar
symptoms of the disease. Albicidin is the only known pathogenicity factor in
Xa, yet albicidin deficient mutants are still able to colonize the sugarcane
plant. In an attempt to identify other major pathogenicity factors, we screened
1,216 independent Tn5 insertions in Xa strain XaFL07-1 by single inoculation
onto sugarcane cultivar CP80-1743. Mutants were screened for reduced
pathogenicity (i.e., capacity to induce leaf symptoms and to multiply in the
sugarcane stalk). Five independent Tn5 insertions were found in gene
XALc_0557, which is predicted to encode an OmpA family outer membrane
protein. Each of these insertions resulted in a mutant strain that elicited very
slight to no symptoms and was not able to move as efficiently within the
sugarcane stalk, both spatially and in intensity, as wild type XaFL07-1.
Additional phenotypic studies showed that these mutants: 1) produced
albicidin, 2) were less motile and 3) were slower growing than the wild type
Xa in vitro. However, these OmpA mutants were able to multiply in sugarcane
leaf tissue to levels similar to the wild-type strain XaFL07-1.
Complementation analyses are currently underway.
Complete 3′ end genome analysis of the asymptomatic Citrus tristeza virus
isolate B192 and its eight symptomatic single aphid transmitted
subisolates
A. ROY (1), N. Choudhary (1), V. D. Damsteegt (2), J. S. Hartung (3), R. H.
Brlansky (1)
(1) University of Florida, Lake Alfred, FL, U.S.A.; (2) USDA-ARS, Foreign
Disease-Weed Science, Fort Detrick, MD, U.S.A.; (3) USDA-ARS, Beltsville,
MD, U.S.A.
Phytopathology 100:S111
The most important viral disease of citrus is caused by Citrus tristeza virus
(CTV). CTV infection often exists in field isolates as a complex of multiple
genotypes. Aphid transmission is important for CTV dispersal. The complete
3′ terminal half sequences of the asymptomatic CTV isolate B192 and its
single aphid transmitted (AT) sub-isolates were compared. Using genotype
specific primers, the CTV-B192 source was identified as mixture of T30 and
VT genotypes. However the T30 genotype was absent in 1st or 2nd level AT
sub-isolates. Moreover, two AT sub-isolates contained additional T3 genotype
along with VT in mixed infections. These two subisolates infected plants
showed symptoms of severe vein clearing, vein corking in Mexican lime and
stem pitting in sweet orange and grapefruit. The VT genotype was the minor
genotype in the source isolate but was identified as the major and most
transmissible genotype in the AT sub-isolates. Sequence analyses of p6, p23,
p27, p33, p61 and p65 genes of CTV-B192 were homologous with
asymptomatic isolate T30 while the sequence of p13, p18 and p25 genes were
phylogenetically related to the New Zealand resistance breaking isolates than
the T36 group isolates. The p20 sequence showed a 93% sequence identity
with T36 isolates. The complete 3′ terminal half sequence derived from the
source and AT sub-isolates clustered with asymptomatic T30 and
symptomatic VT genotypic isolates, respectively, in the phylogenetic tree.
Endophytic bacteria in the inhibition of the germination of spores of
Ustilago scitaminea
L. RUARO (1), S. R. de Souza (1), E. Daros (1), R. B. Fragoso (1)
(1) Universidade Federal do Parana, Curitiba, BRAZIL
Phytopathology 100:S111
Ustilago scitaminea, that cause the smut of the sugarcane if spreads and
survives mainly through the teliospores. The use of isolated bacteria of the
sugarcane, with antagonistic potential to inhibit the germination of these
espores can consist in option in the reduction of the diseases. Four isolated
bacterial ones: Herbaspirillumn seropedicae (cepa SmR1), Azospirillum
brasiliense (cepas: AbV5, AbV6, SF0) were evaluated in the inhibition of
germination of teliospores. Blades with paraffin rings were used contemning
the suspension with teliospores of the pathogen and suspension of the
antagonist, kept in B.O.D. at 28°C. The percentage of germination of the
teliospores was evaluated 4 h after inoculation. The average values of the
inhibition percentage were calculated in relation the witness. The best ones
resulted had been presented by Azospirillum brasiliense lineage SF0 and
Herbaspirillum seropedicae SmR1 lineage, which 97 and 96% of the
germination of the teliósporos had inhibited respectively. Azospirillum
brasiliense lineages: AbV5, AbV6 had not differed statistical from the witness
without application of bacteria. It is concluded that these organisms present
potential in the reduction of the diseases, having to be tested in field.
Soybean cyst nematode infects roots of sugar beet
K. RUDOLPH (1), M. D. Bolton (2), B. D. Nelson (3)
(1) Plant Pathology, North Dakota State University, Fargo, ND, U.S.A.; (2)
USDA-ARS Sugar Beet Unit, Fargo, ND, U.S.A.; (3) North Dakota State
University, Fargo, ND, U.S.A.
Phytopathology 100:S111
Soybean cyst nematode (SCN; Heterodera glycines) is the most important
pest of soybean in the world. With the increase of soybean production in the
Red River Valley of North Dakota and Minnesota over the past decades, SCN
has become a growing threat to local soybean production. The sugar beet cyst
nematode (Heterodera schachtii), a devastating pathogen of sugar beet and a
close relative to SCN, is currently not found in the Red River Valley. Because
SCN is closely related to H. schachtii, we initiated studies to determine
whether SCN is able to infect sugar beet seedlings. Five cultivars of sugar beet
were grown in soil infested with eggs of SCN HG 0 and roots were examined
within two weeks. SCN readily penetrated into the cortex of sugar beet
seedling roots, as determined by microscopy and SCN-specific PCR primers.
Studies are currently underway to ascertain whether infection by SCN
increases disease susceptibility of sugar beet seedlings to common soil borne
pathogens in the Red River Valley.
Characterization of Pythium and Fusarium species associated with
soybean seeds and seedlings
J. Rupe (1), C. Rothrock (1), M. AVANZATO (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S111
The importance of Pythium and Fusarium spp. on soybean has received
considerable attention but little is known about the spatial and temporal
dynamics of Pythium and Fusarium spp. on the scale of individual seed/roots.
The objective of this research was a) to characterize Pythium and Fusarium
spp. associated with soybean seeds/seedlings, and b) to determine
pathogenicity of both pathogens on soybean seeds/seedlings. Field soil was
collected from two Arkansas locations during 2007 and 2008. Growth
chamber experiments were set up at the average planting temperatures for
April (21°C), May (25°C) and June (28°C). Four, 12, 24, 48 and 72 h after
sowing and at the vegetative stages, Ve, Vc and V1, soybean seeds/seedlings
were collected and plated on amended water agar medium to recover
fungal/oomycete flora. Isolates of Pythium and Fusarium spp. were identified
to species by morphological and molecular techniques and evaluated initially
with an in vitro pathogenicity seed assay. Nine Pythium species, P.
sylvaticum, P. irregulare, P. ultimum, P. spinosum, P. mamillatum, P.
dissotocum, P. accanthicum, P. attrantheridium, and P. logandrum and five
Fusarium species, F. oxysporum, F. equiseti, F. tricinctum, F. solani and F.
graminearum were recovered from soybean seeds and seedlings. P.
sylvaticum, P. irregulare, P. ultimum, P. spinosum, P. mamillatum, and P.
dissotocum; and F. oxysporum, F. equiseti, and F. tricinctum were pathogenic
to varying degrees on soybean seeds.
First report of blue stain fungi associated with decline of pine trees in
Lebanon
A. T. SAAD (1), L. Hanna (1), M. Temsah (2)
(1) American University of Beirut, Beirut, LEBANON; (2) Beirut, LEBANON
Phytopathology 100:S111
Pine forests, mainly Pinus pinea, P. halepensis and P. brutia cover an area of
about 17000 ha on the sandy western slopes of the Lebanese range of
mountains and a few coastal areas. Lebanon is a small country on the Eastern
side of the Mediterranean Sea. These pine forests are valued for their
economic returns from nut and wood products, as well as recreational and
touristic sites. Recently, a new disease appeared to invade Pinus pinea trees
resulting in their quick decline and death within few years as brought to our
attention by the National Forestry Service. Affected trees in Beirut on the
coastal area and the mountainous Metn County showed chlorosis and wilting
symptoms with galleries of bark beetles in which fungal growth was observed.
Cut sapwood was blue stained. Isolations from affected trees in Beirut
revealed the occurrence of Ophiostoma teleomorph with long necked
perithecia and the Sporothrix anamorph. Isolates from samples collected from
P. pinea trees at five different locations in the Metn County had Ophiostoma
teleomorphs with perithecia lacking necks and all with Leptographium
anamorphs. Studies on characterization, identification and speciation of the
Ophiostoma isolates will be reported. This is the first report of the blue stain
fungi on pines trees in Lebanon.
Vol. 100, No. 6 (Supplement), 2010
S111
Survival of Erwinia tracheiphila on muskmelon (Cucumis melo) leaves
during wetness periods
E. SAALAU ROJAS (1), A. Owens (1), M. L. Gleason (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S112
Erwinia tracheiphila causes bacterial wilt of cucurbits. This bacterium is
vectored by cucumber beetles (Acalymma vittatum and Diabrotica undecipunctata howardi). Transmission occurs through contact of viable E. tracheiphila
infested beetle frass and fresh feeding wounds. The ability of E. tracheiphila
to survive on leaves as an epiphyte is currently unknown. To determine the
survival of E. tracheiphila on leaves, muskmelon plants were grown at 27°C
with 14 h-photoperiod in growth chambers. At first true leaf stage, plants were
spray-inoculated with a 1 × 107 CFU/ml suspension of E. tracheiphila until
runoff using a hand-triggered sprayer. Inoculated plants were maintained at
100% RH and 26°C in a dew chamber for the duration of the experiment.
Populations of E. tracheiphila were determined by leaf washings and dilution
plating at 0, 12, 24, 36, and 48 hours after inoculation. On average, population
size decreased from 106 CFU/g of fresh weight at 0 hours at inoculation to 104
CFU/g fresh weight 48 hours after inoculation. The results suggest that
epiphytic survival of this pathogen on muskmelon may have an important role
in disease transmission under favorable weather conditions.
Efficacy of extended-duration row covers in suppressing bacterial wilt on
muskmelon (Cucumis melo) in Iowa
E. SAALAU ROJAS (1), J. C. Batzer (1), M. L. Gleason (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S112
Bacterial wilt (pathogen: Erwinia tracheiphila) causes major losses on
muskmelon in the U.S. By delaying removal of spunbond row covers until 10
days after anthesis, plants are protected from cucumber beetles that vector E.
tracheiphila. Six field trials in Iowa during 2007- 2009 assessed the efficacy
of this strategy for suppressing bacterial wilt. Treatments included 1) no row
cover, 2) row covers removed at anthesis, 3) row covers removed 10 days
after anthesis, with ends opened at anthesis, and 4) row covers removed 10
days after anthesis, with bumble bee boxes inserted under row covers at
anthesis. In two trials, delaying removal of row covers until 10 days after
anthesis provided durable protection from bacterial wilt (less than 10% wilted
plants compared to 35–75% for treatments 1 and 2). When transplanting was
delayed by one month, all row cover treatments provided protection from
bacterial wilt (less than 10% incidence vs. 60% for treatment 1). In 2009 field
trials, no wilt occurred in any treatment, possibly due to low populations of
cucumber beetles. The results suggest that the protective effect of delayedremoval row covers is impacted by transplanting date and disease pressure.
Assessing genetic diversity of Erwinia tracheiphila strains isolated from
different cucurbit hosts using rep-PCR
E. SAALAU ROJAS (1), J. C. Batzer (1), M. L. Gleason (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S112
In 2008 and 2009, E. tracheiphila strains were isolated from cucurbit plants
exhibiting bacterial wilt symptoms. Colonies that matched the morphology of
E. tracheiphila were confirmed by PCR using E. tracheiphila-specific primers
(ET1-2 or ETC1-2). Purified genomic DNA of 15 strains isolated from
muskmelon (Cucumis melo), cucumber (Cucumis sativus), and squash
(Cucurbita pepo) from eight U.S. states was amplified using the rep-primer
sets ERIC1-2 and BOXA1R. DNA band patterns were observed after agarose
gel electrophoresis. The patterns obtained from Cucurbita hosts were distinct
from those of Cucumis spp. Strain profiles of C. melo and C. sativus showed
very similar banding patterns across species and states. This study is the first
to report genetic diversity among E. tracheiphila strains. The results suggest
that E. tracheiphila strains could be specific for cucurbit host genera.
Genomic sequences and simultaneous detection of two cryptic viruses
from pepper
S. Sabanadzovic (1), R. A. VALVERDE (2)
(1) Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, MS, U.S.A.; (2) Department of Plant Pathology
and Crop Physiology, Louisiana State University AgCenter, Baton Rouge,
LA, U.S.A.
Phytopathology 100:S112
Pepper (Capsicum annuum L.) has been reported to contain a range of
endogenous dsRNA molecules likely representing the genomes of distinct
cryptoviruses. Indeed, partial sequences of Pepper cryptic virus 1 (PCV-1)
have recently been generated from Jalapeno M pepper. In this work we have
concluded the study on the PCV-1 genome and completely sequenced the
genome of another cryptovirus designated as Pepper cryptic virus 2 (PCV-2).
The two viruses could be distinguished by dsRNA pattern and shared limited
S112
PHYTOPATHOLOGY
identical amino acid content in both genomic segments. Interestingly, in the
genomic molecule encoding the putative RNA-dependent RNA polymerase,
they shared a higher level of common amino acids with cryptoviruses reported
from other crops (i.e. Raphanus sativus cryptic virus 3, Black raspberry
cryptic virus) than to each other. Two sets of virus-specific primers were
successfully applied for the simultaneous and discriminative detection of these
two viruses in various pepper germplasm.
A new ilarvirus from subgroup 1 infects ligustrum
S. SABANADZOVIC (1), I. E. Tzanetakis (2)
(1) Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, MS, U.S.A.; (2) Department of Plant Pathology,
Division of Agriculture, University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S112
A mechanically transmissible virus was repeatedly isolated from young leaves
of ligustrum specimens displaying virus-like symptomatology collected in
Mississippi. Electron microscope observations of leaf dip preparations revealed the presence of quasi-isometric virions resembling the members of the
family Bromoviridae, which was in agreement with the dsRNA patterns obtained from the same plants. Shotgun cloning followed by sequence comparisons and phylogenetic analyses indicate that this virus is an as yet undescribed
member of Subgroup I in the genus Ilarvirus closely related to Strawberry
necrotic shock and Blackberry chlorotic ringspot viruses. This virus,
provisionally named Ligustrum line pattern virus (LLPV) has been found in
several ligustrum specimens showing severe line pattern and ring spot
symptoms indicating its possible involvement in the etiology of the disease.
Viruses of plants in the Great Smoky Mountains National Park
S. SABANADZOVIC (1), N. Abou Ghanem-Sabanadzovic (1)
(1) Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, MS, U.S.A.
Phytopathology 100:S112
The majority of known plant viruses have been described from cultivated
plants and/or agronomic weeds. Despite the fact that natural, non-agronomic
ecosystems represent an excellent and unexplored substrate for study on virus
ecology, taxonomy and evolution they remain understudied. For this reason,
in 2006/2007 we initiated a study on phytoviruses in the Great Smoky
Mountains National Park (GSMNP) within the framework of on-going All
Taxa Biodiversity Inventory (ATBI) activities. In a few years, we successfully
identified a number of phytoviruses, most of which represent as yet
undescribed species. Among them, several viruses belong to genera currently
represented by just one or few members (i.e. gen. Petuvirus, gen. Enamovirus,
gen. Oryzavirus), or were detected also in some cultivated crops (i.e.
blackberries, grapevines) indicating their possible economic importance.
First virus infecting Kudzu in U.S.A.
S. SABANADZOVIC (1), N. Abou Ghanem-Sabanadzovic (1), W. F. Moore
(1), T. W. Allen (2), R. C. Stephenson (1), A. Lawrence (3)
(1) Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, MS, U.S.A.; (2) Delta Research and Extension
Center, Mississippi State University, Stoneville, MS, U.S.A.; (3) Electron
Microscope Center, Mississippi State University, Mississippi State, MS, U.S.A.
Phytopathology 100:S112
Kudzu (Pueraria montana var. lobata), a plant native to the Southeastern
Asia, was originally introduced into the southeastern United States for forage
and erosion containment. Adapting extremely well to the local climate, kudzu
has since become a major noxious weed covering millions of acres. Curiously,
despite intensive study on methods of its control, no viral diseases have been
reported on kudzu in the U.S. Virus-like symptoms consisting of mottling and
ring spots were observed in October 2009 on kudzu leaves in Northeastern
Mississippi. Laboratory analyses showed the presence of high molecular
weight dsRNA molecules in infected tissue, but not in the controls, indicating
virus involvement in the disease. Electron microscope observations of
partially purified extracts showed the presence of flexuous virions. Partial
sequencing of the genome showed that the virus from kudzu is a potyvirus
belonging to the Bean common virus subgroup. This is the first virus reported
to infect kudzu in the U.S.
Another marafivirus infecting blackberries
S. SABANADZOVIC (1), N. Abou Ghanem-Sabanadzovic (1)
(1) Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, MS, U.S.A.
Phytopathology 100:S112
In the course of a study on viruses of cultivated blackberries, a symptomatic
specimen BB-R-1, collected from a backyard in Northern Mississippi, resulted
positive in RT-PCR for tymo/marafiviruses using a general primer set.
However, no products were observed in RT-PCR with specific primers for the
recently reported Blackberry virus S (the only virus belonging to the family
Tymoviridae known to infect small fruits), which prompted further work on
this virus. Complete sequencing showed that the genome of this virus shares
characteristics with members of the genus Marafivirus. Pairwise comparisons
of the complete polyprotein encoded by this virus with related viruses showed
limited levels of amino acid identities indicating that this virus is likely a new
species in this taxon. This virus was found in several other locations in
Mississippi, suggesting that it may be common in blackberry production fields
in the Southern United States. Its effect on the host is yet to be understood.
ITS-RFLP as a criterion to study identification of Rhizoctonia solani
M. SAFFARIAN ABBAS ZADEH (1), S. Soltani Nejad (2), R. Farrokhi
Nejad (1), B. Mahmoudi (3)
(1) Department of Plant Protection, College of Agriculture, Shahid Chamran
University-Ahvaz, Ahvaz, IRAN; (2) Ahvaz, IRAN; (3) Sugar Beet Seed
Institute, Karaj, IRAN
Phytopathology 100:S113
Rhizoctonia solani is a world wide soil-borne pathogenic fungus showing the
tremendous variation in characteristics such as morphology and host
specificity. Traditional identification of R. solani based on anastomosis
behavior did not provide adequate evidence for identification of different AGs
and sub groups. In this study, for finding valid and rapid method for
identification, hyphal anastomosis reaction and ITS-RFLP of isolates
representing R. solani recovered from sugar beet root rot and potato tuber with
black scurf were compared. Entirely, three anastomosis groups AG3, AG4 and
AG5 recovered from potato and four anastomosis groups AG2, AG3, AG4
and AG5 and 8 unknown isolates (from dry rot symptom) obtained from sugar
beet. In molecular studies, a DNA fragment of 700-750 bp in size was
amplified from rDNA preparations of all isolates with the ITS5&4. The
amplified products of DNA for all the isolates digested with the enzymes
Taq1, Bsur1, EcoR1, and Tru91. ITS-RFLP showed that isolates of different
AGs generated very distinct patterns and were separated based on AGs as well
as sub AGs. For instance, Tru91 was sufficient to discriminate among
different AGs and subgroups. Also, in the present study, the polymorphism
existing among different isolates permitted the characterization of dry rot
isolates for the first time. According to our results, Dry rot isolates are
probably placed in AG1. Generally, it seems that ITS-RFLP technique is a
precise method for study of identification of R. solani isolates.
Resistance to Maize streak virus in testcrosses of early generation lines of
maize
M. T. SALAUDEEN (1), A. Menkir (2), G. I. Atiri (3), S. Hearne (2), P. Lava
Kumar (2)
(1) International Institute of Tropical Agriculture (IITA) and Department of
Crop Protection and Environmental Biology, University of Ibadan, Ibadan,
NIGERIA; (2) International Institute of Tropical Agriculture (IITA), Ibadan,
NIGERIA; (3) Department of Crop Protection and Environmental Biology,
University of Ibadan, Ibadan, NIGERIA
Phytopathology 100:S113
Maize streak disease caused by Maize streak virus (MSV, genus
Masterevirus) is a major disease of maize (Zea mays L.) unique to African
continent. MSV is mainly controlled through host plant resistance. In this
study, 250 testcrosses of S2 lines of maize were evaluated for MSV resistance
under field conditions during 2009 wet season in Nigeria. Plants were
inoculated at seedling stage using experimentally reared viruliferous
leafhoppers, Cicadulina triangula. There was no immunity to MSV, however,
substantial differences were observed in host response to the virus among
testcross lines (P < 0.01). Six testcrosses were highly resistant, 71 were
resistant, 146 moderately resistant, 27 were susceptible. Disease severity was
negatively correlated with grain weight (-0.028), grain yield (-0.030), ear
number (-0.214), kernel weight (-0.155), and kernel number (-0.023). MSV
resistance in most genotypes was found to be recovery type, i.e., plants were
infected and showed severe symptoms at early stage and reduction in
symptoms in subsequently emerged leaves. MSV detection by enzyme-linked
immunosorbent assay indicated high virus concentration in symptomatic
leaves and low or undetectable levels in moderate or asymptomatic leaves
suggesting positive correlation between symptoms and virus titer in plants.
Inbred parents of the test crosses will be exploited to identify markers linked
to MSV resistance using single nucleotide polymorphism (SNP) markers.
Relationship between grain yield and Fusarium Head Blight in soft red
winter wheat as influenced by cultivar resistance
J. D. SALGADO (1), M. W. Wallhead (1), L. V. Madden (1), P. A. Paul (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S113
Fusarium Head Blight (FHB) of wheat is predominantly caused by Fusarium
graminearum (teleomorph: Gibberella zeae) in North America. FHB affects
wheat by reducing grain fill and kernel size, leading to low test weight and
yield loss. However, it is unclear how yield loss is influenced by relative
susceptibility of the affected cultivar to FHB. Field plots of three soft red
winter wheat cultivars with different levels of FHB resistance (Hopewell,
Truman and Cooper, moderately susceptible, moderately resistant, and
susceptible to FHB, respectively) were planted and inoculated with G. zeae/F.
graminearum, with the objective of characterizing the relationships between
FHB and grain yield. Plots were spray-inoculated at anthesis (Feekes 10.5.1)
with spore concentrations ranging from 0 to 150,000 spores/mL. FHB
intensity was estimated at soft dough and yield determined following harvest.
Averaged across inoculation treatments, mean FHB index and grain yield
ranged 1.49 to 24.64% and 5,552 to 6,311 Kg/ha for Cooper; 3.76 to 42.06%
and 3,975 to 5,298 Kg/ha for Hopewell; and 0.67 to 9.61% and 5,473 to 5,907
Kg/ha for Truman. Based on regression slopes, yield reduction per unit
increase in index varied among cultivars, being highest for moderately
susceptible Hopewell, intermediate for moderately resistant Truman and
lowest for susceptible Cooper, with estimated losses of 27.77, 23.91 and 20.26
Kg/ha per unit increase of disease index, respectively, for the three cultivars.
New and re-emerging rust diseases from Idaho and Oregon
R. SAMPANGI (1), M. C. Aime (2), K. Mohan (1), C. Shock (3)
(1) University of Idaho, Parma, ID, U.S.A.; (2) Louisiana State University,
Baton Rouge, LA, U.S.A.; (3) Oregon State University, Ontario, OR, U.S.A.
Phytopathology 100:S113
New host–pathogen records contribute to vital information that aids in quick
and accurate diagnosis of plant diseases. New pathogen reports help in
establishing baseline data about pre-existing and emerging plant pathogens
and the epidemiology of the diseases they cause, thus furthering the
understanding of pathogen biology, host ranges, and the geographic range of
pathogens. First reports contribute to data on disease occurrences and
resulting host-fungus indices can serve as primary resources for information to
plant disease diagnosticians, extension educators, plant health professionals,
and regulatory officials. Rust diseases, caused by fungi in the basidiomycete
order Pucciniales, are one of the most important agents of agricultural losses.
Presented herein are examples of new and reemerging rust diseases on
regional crops turf grass, forbs and forages from Idaho and Oregon, all noted
between 2006–2009: Puccinia graminis Pers.:Pers on Poa pratensis L,
Puccinia similis Ellis & Everh on Artemisia tridentata Nutt., Puccinia jonesii
Peck on Lomatium dissectum (Nutt.) Mathias & Constance, Puccinia
sherardiana Körn on Sphaeralcea grossulariifolia (Hook. & Arn.) Rydb and
Uromyces intricatus Cooke on Erigonum umbellatum Torr.
Nematode and bacterial associates of the invasive Brown Garden Snail:
Helix aspersa
K. SANCHEZ (1), C. Pagan (1), S. A. Nadler (1), E. P. Caswell-Chen (1)
(1) University of California, Davis, CA, U.S.A.
Phytopathology 100:S113
Helix aspersa (Brown Garden Snail) is an invasive terrestrial mollusk. It is a
pest of plants and can damage young seedlings and foliage. We collected 350
snails in California (San Francisco, Sacramento, Davis, Woodland, San Jose,
and Tulare) to identify associated nematodes and bacteria, and to determine
snail tissues where nematodes and bacteria occur. Snails were dissected, and
nematodes and bacteria were recovered from surface rinsate, the foot muscle,
shell, digestive gland, stomach, heart, mantle, and feces yielding 500
individual nematodes. Nematodes and bacterial isolates were subject to PCR
amplification using primers for ITS, 16S, 28S, 18S, and rpo . Nematodes
were recovered from ca. 91% of snails and included Caenorhabditis elegans
(in 60% of snails), Rhabditis terricola (32%), Aphelenchoides fragariae
(44%), Xiphinema index (24%), Heterodera spp. (28%), and Aphelenchus
avenae (45%). There were 25 distinct bacterial colonies isolated from organ
tissues, with Serratia proteamaculans, Klebsiella terrigena, and
Stenotrophomonas maltophilia recovered, and five bacteria isolated from snail
slime,
including
Psuedomonas
putida
and
Sphingobacterium
kitahinoshimense. These associations establish the brown garden snail’s role
as an important phoretic host for plant pathogens, and lead us to consider the
possible use of snails as sentinels for the detection of pathogens in the
environment.
Effect of fungicide dip treatments on pink root disease and yield of
transplanted sweet onions in Georgia
F. SANDERS, JR. (1)
(1) University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S113
Pink root disease of onion caused by the fungus, Phoma terrestris, is a
common soil borne disease in the onion growing region of Georgia and
growers experience yield reductions due to pink root every year. The fumigant
Vapam (metam sodium) is used to control pink root in onion seed beds
however, this treatment is too expensive to apply to production fields. In this
investigation, dip treatments with the fungicides Endura (boscalid), Topsin
Vol. 100, No. 6 (Supplement), 2010
S113
(thiophanate methyl) Switch (cyprodinil + fludioxinil) were evaluated for pink
root suppression. Trials were conducted in two consecutive growing seasons
at the Vidalia onion and Vegetable Research and Educational Center in Lyons,
Georgia. The experimental design for both trials was a 2 × 2 factorial with
variety and fungicide treatment as factors with four replications. Onion
transplants were treated with fungicide dips at plant and compared to foliar
fungicide sprays and an untreated control. Pink root incidence was taken prior
to harvest in both trials by examining the roots of 10 plants per plot for
disease symptoms, and yields were taken. Endura dip treatments reduced pink
root incidence and increased yield when compared to the untreated control,
and there was no difference in yield between plots treated with Endura dips
and plots treated with three foliar sprays of Pristine (boscalid +
pyraclostrobin) in both trials. Transplant dip treatments with Endura have the
potential to increase onion yields and provide suppression of pink root.
Etiology of pod rot of Valencia peanut in New Mexico
S. SANOGO (1), N. Puppala (2)
(1) New Mexico State University, Las Cruces, NM, U.S.A.; (2) Clovis, NM,
U.S.A.
Phytopathology 100:S114
Soilborne pathogens typically causing pod rot on peanut (Arachis hypogaea)
include Rhizoctonia solani, Sclerotium rolfsii, and Pythium species. Although
pod rot is known to occur in New Mexico, no etiological study has been
conducted on this disease. In 2005 and 2006, Valencia peanut fields were
surveyed in eastern New Mexico in order to characterize mycelial
microorganisms associated with pod rot. Peanut plants were collected and
processed for isolation of microorganisms by plating seeds, pieces of shell,
root, and stem on acidified potato dextrose agar. In both years, a diverse group
of mycelial microorganisms were recovered from all plant part tissues, with
Rhizoctonia solani as the most predominantly isolated microorganism. In
2005, R. solani was found in all fields with frequency of isolation varying
from approximately 7% to 43% across all plant part types. Frequency of
isolation was approximately 12 to 32% for root, 7 to 43% for seed, 8 to 38%
for shell, and 8 to 27% for stem. In 2006, R. solani was found in all fields with
frequency of isolation varying from approximately 5% to 45% across all plant
part types. Frequency of isolation was approximately 32 to 45% for root, 5 to
22% for seed, 25 to 28% for shell, and 20 to 27% for stem. In controlled
environment studies, all isolates of R. solani from different plant parts were
shown to be pathogenic to Valencia peanut. Based on the findings from this
study, pod rot management in New Mexico should include steps to target R.
solani.
Colletotrichum capsici and Colletotrichum coccodes: Predominant causal
agents of anthracnose of chile pepper in New Mexico
S. SANOGO (1)
(1) New Mexico State University, Las Cruces, NM, U.S.A.
Phytopathology 100:S114
The occurrence of anthracnose of chile pepper (Capsicum annuum) has been
reported in various extension publications in New Mexico. However, the
causal agents of this disease have not been identified. Symptoms are typically
expressed as depressed sunken lesions on ripe fruit. In 2008 and 2009, chile
pepper fruit with symptoms of anthracnose were collected from several fields
in southern New Mexico. Acervuli were observed on calyces and on surface
of lesions. Chile pepper plants with blighted stems covered with acervuli were
found. Microscopic observations of acervuli revealed the presence of two
major types of conidia characteristic of Colletotrichum capsici and C.
coccodes. Fruit and stem tissue plated on acidified potato dextrose agar
yielded predominantly colonies of C. capsici and C. coccodes. Both species
were isolated from the same fruit and stems of several samples. On potato
dextrose agar, C. coccodes produced abundant sclerotia whereas C. capsici
produced none. Radial growth of two isolates, one of each species, was
compared at 25 and 30°C. Whereas radial growth of both isolates was similar
at 25°C, radial growth of C. capsici isolate was over 30% greater than that of
C. coccodes isolate at 30°C. Both isolates caused typical anthracnose
symptoms on ripe chile pepper fruit inoculated with conidia of each isolate.
This etiological study indicates that at least two species of Colletotrichum are
primarily associated with anthracnose of chile pepper in New Mexico.
Field evaluation and genetic characterization of a quantitative trait locus
conferring resistance to Southern leaf blight
J. SANTA-CRUZ (1), A. Belcher (2), J. Zwonitzer (3), C. Arellano (4), M.
Krakowsky (5), P. Balint-Kurti (6)
(1) Dept. of Plant Pathology, North Carolina State University, Raleigh, NC,
U.S.A.; (2) Research and Extension Center, University of Idaho, Aberdeen,
ID, U.S.A.; (3) Dow Agrosciences, Huxley, IA, U.S.A.; (4) Dept. of Statistics,
North Carolina State University, Raleigh, NC, U.S.A.; (5) Dept. of Crop
Science, North Carolina State University, Raleigh, NC, U.S.A.; (6) Dept. of
S114
PHYTOPATHOLOGY
Plant Pathology, North Carolina State University, USDA-ARS, Plant Science
Research Unit, Raleigh, NC, U.S.A.
Phytopathology 100:S114
Southern leaf blight (SLB) caused by the fungal pathogen Cochliobolus
heterostrophus (anamorph = Bipolaris maydis), is a common disease of maize
in southeastern U.S., as well as many hot and humid tropical and subtropical
areas in the world. Most of the disease resistance used in maize is quantitative
in nature; however, quantitative disease resistance remains poorly understood.
To investigate quantitative resistance to SLB, we used the highly resistant
inbred maize line NC292. This line is derived from crossing NC250, an elite
source of SLB resistance, to the highly susceptible line B73 followed by three
further backcrosses to B73 and several rounds of selfing. At each stage in this
process the plants were selected for SLB resistance. Using genome-wide
marker analysis of NC292, we detected 12 NC250-specific introgressions.
Furthermore, 9 disease QTLs associated with SLB resistance were mapped on
a related population, from which 4 colocalized with NC250-specific
introgressions. We identified a strong QTL for SLB resistance at the tip of the
short arm of chromosome 6 of maize which colocalized with a NC250
introgression in NC292, namely introgression 6A. Specific objectives of this
research include fine-mapping introgression 6A, cloning of the gene that
accounts for this effect, and evaluating yield and fitness effects of this
introgression under both high and low disease pressure for possible future use
of this resistance. Preliminary growth chamber phenotyping experiments
showed that introgression 6A segregates as a single recessive resistance gene,
and can be scored in growth chamber experiments on a single plant basis.
Over 168 F2 individuals and over 300 F2:3 families were phenotyped, and
genotyped with SNPs markers in order to narrow down the region of interest
(< 1.5Mb). Currently, candidate genes are being analyzed. To evaluate fitness
and yield, we have developed isohybrid pairs by crossing B73 with/without
introgression 6A to several inbred lines (testers). We have found significant
differences between the treatments in some pedigrees. Summer yield
experiments are being designed to study the influence of the presence or
absence of 6A introgression on agronomic traits and disease.
Detection of reniform nematode by conventional and real-time PCR
R. J. SAYLER (1), T. L. Kirkpatrick (1), R. T. Robbins (1), R. D. Cartwright
(1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S114
Reniform nematode Rotylenchulus reniformis is a recent introduction into the
continental U.S. This plant parasite has a wide host range including
economically important crops such as cotton and soybeans corn. Worldwide,
the reniform nematode typically occupies tropical and subtropical
environments, and appears to be restricted to the southern U.S. from Texas
east to the Atlantic ocean and as far north as the Missouri Bootheel. The goal
of this project is to optimize protocols for both conventional and real-time
PCR that identify the reniform nematode on the basis of the sequence of the
internal transcribed spacer (ITS) region of this species. A PCR-based
detection and quantification method for this nematode may provide a more
rapid and less labor-intensive alternative to visual identification for
diagnosticians and extension specialists, which in turn should yield more
timely management recommendations for growers. These primers have
successfully amplified a 240 base pair fragment of the ITS region from this
nematode.
Improved extraction of DNA of Ca. Liberibacter species from plants and
cultivated cells using pressure cycling technology (PCT)
N. Schaad (1), A. SECHLER (2), A. Marques (3), N. Lawrence (4), R.
Schumacher (4)
(1) USDA ARS NAA FDWSRU, Ft. Detrick, MD, U.S.A.; (2) FDWSRU,
USDA-ARS, Ft. Detrick, MD, U.S.A.; (3) EMBRAPA, Brasilia, BRAZIL; (4)
Pressure BioSciences Inc, South Easton, MA, U.S.A.
Phytopathology 100:S114
Huanglongbing, one of the most destructive diseases of citrus, is caused by
three species of Ca. Liberibacter. Diagnosis of the disease is reliant on realtime PCR (RT-PCR). Detection of the pathogen is complicated, especially
from small survey samples, because of low titer and uneven distribution of the
bacterium throughout the infected plant and the complex nature of the plant
tissue from which it is extracted. Cultured cells of Ca. Liberibacter species are
also difficult to disrupt for efficient DNA extraction. Pressure cycling
technology (PCT) is a dynamic technique that can be used for the highlyefficient extraction of protein and nucleic acids from simple and complex
samples. Here we compare DNA extraction methods on the three known citrus
species of Ca. Liberibacter from infected plants and cultured cells using PCT
with the PCT Shredder™ and NEP 2320 Barocycler (Pressure BioScience,
South Easton, MA) and commercially available DNA extraction kits.
Combinations of these techniques and methods were tested and the resulting
samples were used with RT-PCR. Preliminary results from pressure cycling
together with The PCT Shredder™ found an increase in RT-PCR positive
samples of Ca. Liberibacter species by 12–42% from infected plants over
other methods and an increase in DNA yield from cultured cells.
Moderate temperature fluctuations rapidly reduce viability of Ralstonia
solanacearum Race 3 biovar 2 in infected geranium, tomato, and potato
J. M. Scherf (1), A. Milling (1), C. ALLEN (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S115
Ralstonia solanacearum Race 3 biovar 2 (R3bv2) causes bacterial wilt of
potato, tomato, and geranium plants in the highland tropics and temperate
zones where more typical tropical R. solanacearum strains don’t cause
disease. R3bv2 is a high-concern quarantine pathogen in Europe and Canada
and a U.S. Select Agent pathogen because it could threaten the potato industry
if it became established in cool temperate zones. Previous experiments
revealed that R3bv2 did not survive as well as subtropical US Race 1 (R1bv1)
strains in water at 4°C, but that R3bv2 survived longer than R1bv1 in potato
tubers at 4°C. To better understand this key epidemiological trait, we
measured survival of R3bv2 and R1bv1 in infected tomato and geranium
plants, and in infected potato tubers following typical temperate winter
temperature cycles of 2 days at –5°C followed by 2 days at +10°C. Population
sizes of both strains were initially above 10e7 cfu/gm, but they declined
rapidly under these conditions in all three plant hosts. No culturable R.
solanacearum cells could be detected after 6 to 7 cycles. The temperature
fluctuations are critical for loss of bacterial viability since at a constant
temperature of –20°C, large populations of both tested strains survived in
infected plant tissue for at least 6 months. These results suggest that even
when sheltered in infected plant tissue, R3bv2 is unlikely to survive the
temperature fluctuations that commonly occur during a northern U.S. winter.
Investigating the ironwood tree (Casuarina equisetifolia) decline on Guam
using applied multinomial modeling
K. A. SCHLUB (1), B. D. Marx (1), Z. Mersha (2), R. L. Schlub (2)
(1) Department of Experimental Statistics, Louisiana State University, Baton
Rouge, LA, U.S.A.; (2) Guam Cooperative Extension Service, University of
Guam, Mangilao, GUAM
Phytopathology 100:S115
The ironwood tree (Casuarina equisetifolia), a protector of coastlines of the
sub-tropical and tropical western Pacific, is in decline on the small island of
Guam, where aggressive data collection and efforts to mitigate the problem
are underway. For each sampled tree, the level of decline was measured on an
ordinal scale consisting of five categories, ranging from healthy to near dead.
Several predictors were also measured including tree diameter, fire damage,
typhoon damage, presence or absence of termites, presence or absence of
basidiocarps, and various geographical or cultural factors. The five decline
response levels can be viewed as categories of a multinomial distribution,
where the multinomial probability profile depends on the levels of these
various predictors. Such data structure is well suited to a proportional odds
model, thereby leading to odds ratios, involving cumulative probabilities
which can be estimated and summarized using information from the predictor
coefficient. Various modeling techniques were applied to address data set
issues: reduced logistic models, spatial relationships of residuals using latitude
and longitude coordinates, and correlation structure induced by the fact that
trees were sampled in clusters at various sites. Among our findings, factors
related to ironwood decline were found to be latitude, basidiocarps, termites,
and level of human management.
affected roots. Previous culture-based (CB) examinations of the SV and SJV
samples (RDA of morphological OTUs) associated Cylindrocarpon spp.,
Fusarium spp., other fungi, and Pythium spp. with PRD at both locations (P =
0.002 for ordinations). Both in healthy and diseased roots, some organisms
detected by CI methods were not discriminated by CD methods, and vice
versa. Therefore, both methods appear essential to resolve PRD etiology.
Efficacy of phosphonate treatments against Sudden Oak Death in
Tanoaks
D. SCHMIDT (1), M. Garbelotto (1)
(1) University of California Berkeley, Berkeley, CA, U.S.A.
Phytopathology 100:S115
Pytophthora ramorum, the causal agent of Sudden Oak Death (SOD), has
killed hundreds of thousands of trees in California and Oregon. Tanoaks
(Lithocarpus densiflorus) are both stem and foliar hosts and, as such, die from
SOD and help spread the disease. Phosphonate treatments are routinely used
in agricultural and orchard crops affected by Phytophthora diseases. We have
developed a detached-leaf bioassay for studying the effectiveness of
phosphonate treatments for SOD in tanoaks. The assay involves infecting the
petioles of tanoak leaves with agar plugs of P. ramorum in culture. SOD
infection is analyzed by examining the spread of P. ramorum down the midrib
of the leaf. This assay has shown that tanoaks in wildland settings, treated
with phosphonates, are resistant to SOD infection. In addition, we are
maintaining long-term studies of tanoaks treated with phosphonates in SOD
infected forest areas. Paired 20mx20m treatment and control plots were
established near existing SOD infections. The trees were evaluated for disease
symptoms and general health prior to the initial treatment and each subsequent
year. The results show that phosphonate treatments are effective at slowing
and preventing the spread of the disease in the treated areas. Treatments at the
leading edge of SOD infected areas were less effective, confirming that
phosphonate treatments are significantly more effective as preventative rather
than curative treatments.
Factors affecting the development of the green stem malady in soybean
R. W. SCHNEIDER (1), G. B. Padgett (2), D. J. Boquet (2), R. A. Valverde (1)
(1) Louisiana State University Agricultural Center, Baton Rouge, LA, U.S.A.;
(2) Louisiana State University Agricultural Center, Winnsboro, LA, U.S.A.
Phytopathology 100:S115
The soybean disorder known as the “green stem malady” has recently become
one of the primary constraints to soybean productivity in the Mid South.
Symptoms may assume several guises, but the common diagnostic symptom
is stems and often leaves remain green while the pods mature. This causes the
stems to remain supple, and the thrasher on the harvester becomes congested
with stems. A multi-disciplinary field study was conducted in 2008 and 2009
at three locations in Louisiana in an attempt to determine the causes of this
disorder. The following factors were investigated in multifactorial
experiments: cultivars, the fungicide pyraclostrobin (Headline), the herbicide
glyphosate (Roundup), presence of specific viruses, and irrigation. Cultivars
were the dominant statistical factor in symptom development. There was a
significant interaction between cultivar and Headline such that symptom
expression was more severe in sensitive cultivars following the application of
the fungicide. Likewise there were significant interactions between Headline
and irrigation, between Roundup and irrigation, and between cultivar and
irrigation. There was no interaction between Headline and Roundup. None of
the viruses appeared to be related to the disorder.
Culture-independent examination of microbial community shifts
associated with replant disease of almond
L. S. SCHMIDT (1), R. G. Bhat (2), G. T. Browne (1)
(1) USDA ARS CPGRU, Davis, CA, U.S.A.; (2) University of Californa,
Davis, CA, U.S.A.
Phytopathology 100:S115
Phylogenetic analysis and population identification of the phyopathogen
Xylella fastidiosa using zot and gyrB genes
H. L. SCHREIBER (1), J. M. Repshare (1), C. E. Skipper (1), L. D. Morano
(2), B. R. Bextine (1)
(1) University of Texas at Tyler, Tyler, TX, U.S.A.; (2) University of Houston Downtown, Houston, TX, U.S.A.
Phytopathology 100:S115
Growth and productivity of successive almond and stone fruit plantings can be
severely suppressed by Prunus replant disease (PRD). PRD occurs in absence
of plant parasitic nematodes, is associated with poor root health, and is
prevented by soil fumigation, but its etiology is poorly resolved. We used
culture-independent (CI) methods to examine microbial communities
associated with PRD in two California almond orchards [Sacramento Valley
(SV), San Joaquin Valley (SJV)]. Total DNA was extracted from roots (≤1
mm diam.) of healthy and PRD-affected trees and used for PCR amplification
of rDNA from bacteria, stramenopiles, and fungi. The amplicons were cloned,
sequenced, and grouped into operational taxonomic units (OTUs). At the SJV
site, redundancy analysis (RDA) discriminated shifts in bacterial, fungal, and
stramenopile populations associated with PRD (P = 0.02, 0.05, and 0.06,
respectively). Similar trends were observed at the SV site, but the ordinations
were not significant (P = 0.13 to 0.30). At both sites, Cylindrocarpon
destructans and Phaeonectriella lignicola were found predominately in PRD-
The phytopathogenic bacterium Xylella fastidiosa has been separated into four
subspecies, fastidiosa, sandyi, multiplex, and pauca, through analysis of
genetic sequence similarity. These subspecies cause disease in distinct plant
hosts, including economically important crops, such as Pierce’s Disease of
grapevines, oleander leaf scorch, phony peach disease, and citrus variegated
chlorosis. In this study, 40 Texas strains of X. fastidiosa subsp. fastidiosa,
sandyi, and multiplex were classified using DNA sequence comparisons of
orthologous Zonula Occludens Toxin (zot) and gyrase B (gyrB) genes.
Previous phylogenetic analyses have used the highly conserved gyrB gene to
categorize X. fastidiosa strains into subspecies. The zot gene in X. fastidiosa is
homologous to zot genes in other gammaproteobacteria which produce the
exotoxic protein Zot, and orthologous and paralogous zot genes have been
identified in each subspecies of X. fastidiosa. Classification of X. fastidiosa
strains using the zot gene produced accurate cladistical groupings into
subspecies similar to those produced by analysis of the gyrB gene.
Vol. 100, No. 6 (Supplement), 2010
S115
Additionally, zot genes displayed genetic differences between Texas and
California strains of X. fastidiosa, establishing the zot gene as a target to
differentiate X. fastidiosa populations. The zot gene offers a novel target for
phylogenetic analysis, and may offer additional disease tracking and
identification opportunities for disease management protocols.
Expression rate of the Zonula Occludens Toxin (zot) gene in two growth
states and two media types of Xylella fastidiosa
H. L. SCHREIBER (1), C. E. Skipper (1), J. M. Repshare (1), L. D. Morano
(2), B. R. Bextine (1)
(1) University of Texas at Tyler, Tyler, TX, U.S.A.; (2) University of Houston
- Downtown, Houston, TX, U.S.A.
Phytopathology 100:S116
was used to confirm the presence or absence of these contigs in 8 culture and
34 diseased tissue samples. The presence of Rhizobiaceae-like sequence in all
samples confirmed the successful sequencing of the genome from cultured
cells of Liberibacter. Ralstonia plasmid sequence was found in 25% of Asian
samples, but not in North and South American samples. Unique sequences
showed two patterns; sequence present in all samples suggesting bacterial
chromosomal DNA and sequence present in a percentage of the samples
suggesting plasmid DNA.
Host-derived RNAi targeted to a novel root-knot parasitism gene in
tobacco
K. K. SCHWERI (1), G. Huang (2), B. Xue (1), M. G. Mitchum (3), T. J.
Baum (4), R. S. Hussey (2), R. Lewis (1), E. L. Davis (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.; (2) University of
Georgia, Athens, GA, U.S.A.; (3) University of Missouri, Columbia, MO,
U.S.A.; (4) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S116
Xylella fastidiosa is the causal agent of many plant diseases, inclduing
Pierce’s Disease of grapevines, citrus variegated chlorosis in orange trees, and
leaf scorch in almonds, alfalfa, oleander, and coffee. A detailed pathogenic
mechanism has not been described, though a combination of factors leading to
xylem occlusion seems likely. Recently the Zonula Occludens Toxin (Zot)
was noted as a possible virulence factor in X. fastidiosa. Zot is an exotoxin
produced by the prophage gene zot which is homologous to the zot gene found
in other phytopathogens, such as Xanthomonas campestris and Ralstonia
solanacearum. Many pathogenic bacteria, including X. fastidiosa, lose
virulency after serial passages through axenic media as the expression of
virulence genes is downregulated. Using quanititative, real-time polymerase
chain reactions (qRT-PCR), this study quantified the expression rate of the zot
gene relative to a conserved housekeeping gene gyrase B (gyrB) in newly
harvested (NH) and serially passed (SP) cultures of X. fastidiosa grown in
axenic media or media augmented with extracted xylem sap from Vitis
vinifera. This comparison revealed significant differences in zot expression
rate between NH and SP cultures of X. fastidiosa as well as differences
between rates zot expression in X. fastidiosa grown in axenic media and X.
fastidiosa cultured in augmented media. These results support the hypothesis
that the Zot exotoxin is a virulence factor in X. fastidiosa.
Root-knot nematodes (RKN), genus Meloidogyne, have multiple crop host
species and cause severe economic losses worldwide. Proteins encoded by
RKN parasitism genes are secreted into host plant root cells to form elaborate
and essential feeding cells. Previous studies have shown that silencing the
novel 16D10 RKN parasitism gene transcript using host-derived RNA
interference (RNAi) makes Arabidopsis thaliana plants highly resistant to all
four major RKN species. The 16D10-RNAi construct that was used in the
above study was used to transform two haploid lines of Nicotiana tabacum,
TN90, a burley tobacco, and Hicks, a flue-cured tobacco. Double-haploids
were recovered through midvein tissue culture of mature leaves, and T1
progeny were produced through self-fertilization. Nematode infection assays
of T1 plants have shown a significant reduction in the number of eggs
produced by females of M. arenaria when compared with wild-type. No offtarget effects of 16D10 RNAi have been observed in the regenerated tobacco
lines. Attempts to correlate RNA expression with the severity of the nematode
infection are underway.
Attempting to transmit citrus canker from diseased ripe grapefruit to
healthy grapefruit saplings under field conditions
T. SCHUBERT (1), G. Bonn (1)
(1) Florida Dept. of Agriculture & Consumer Services, Gainesville, FL, U.S.A.
Phytopathology 100:S116
Movement, germination and production of Puccinia pelargonii-zonalis
urediniospores on greenhouse-grown geraniums
E. A. SCOCCO (1), J. Buck (1)
(1) University of Georgia, Griffin, GA, U.S.A.
Phytopathology 100:S116
Xanthomonas citri subsp. citri (Xcc) causing citrus canker is an important
foliar and fruit pathogen of citrus in the sub-tropical agricultural regions of the
world. In order to meet quality standards and phytosanitary regulations, citrus
fruit packed and shipped from Florida for consumption must be free of canker
lesions. Recent changes to the regulations allow fruit from cankered groves to
be shipped domestically, leaving open the possibility that fruit with lesions
might slip by graders to enter into existing canker-free production areas. To
determine whether lesions on ripe fruit could lead to canker symptoms on
susceptible citrus we laid out field plots to attempt transmission of Xcc from
infected commercially-packed grapefruit. Diseased fruit surfaces as well as
surfaces of adjacent susceptible grapefruit saplings were assayed for Xcc
periodically during the growing seasons of Florida. Dilution plating of fruit
and leaf swabbing onto a semi-selective medium and bioassays in greenhousegrown grapefruit saplings indicated the occasional detection of Xcc on the
surface of infected fruit following a wetting period (dew, rain and overhead
watering). However, Xcc did not move from the diseased fruit to the adjacent
healthy saplings. The low number of viable canker bacteria associated with
peel lesions in ripe grapefruit, especially during wet warm periods, suggests
that the risk of canker transmission from them is exceptionally low.
Geranium rust caused by Puccinia pelargonii-zonalis can result in significant
monetary losses to greenhouse operators. Inoculum production and movement
of urediniospores throughout the greenhouse can affect management options.
The purpose of this research was to determine urediniospore production per
pustule over a 24 h period and to track airborne movement of inoculum in a
greenhouse. Geraniums cv. Maverick Red in 3.8 L pots were spray-inoculated
with 105 urediniospores/mL until leaf wetness and then bagged for 24 h to
initiate infection. After 12–14 days, urediniospores were vacuumed-collected
from lesions every 24 h for 3 days. Urediniospore samples were suspended in
0.05% Tween 20, enumerated by hemacytometer, and four 50 µl aliquots from
each sample were placed on water agar. Germination was assessed after 24 h
(21°C) at 200X magnification. Movement of urediniospores along a
greenhouse bench was assessed using three 3.8 L geraniums cv. Maverick Red
with sporulating lesions. Rotorods were placed 0.3, 0.9, 1.5 and 2.1 m away
from the inoculated plants at pot height and glass slides coated with petroleum
jelly were placed on the bench at 0.3 m intervals. Glass slides and rotorods
were collected after 8 h and observed at 100X magnification. An average of
1580 urediniospores were produced per pustule every 24 h and germination
ranged from 37–59%. After 8 h, movement of urediniospores was detected
from infected plants up to 1.82 m at bench level and 2.1 m at pot height.
Sequencing Candidatus Liberibacter asiaticus from cultivated cells
E. Schuenzel (1), J. Crow (2), A. Sechler (3), R. Kim (2), N. SCHAAD (3)
(1) USDA ARS NAA FDWSRU, Ft. Detrick, MD, U.S.A.; (2) National
Center for Genome Resources, Santa Fe, NM, U.S.A.; (3) FDWSRU, USDAARS, Ft. Detrick, MD, U.S.A.
Phytopathology 100:S116
Using amplified DNA from cultured cells and Illumina Solexa second
generation sequencing technology, we sequenced L. asiaticus strain China1
previously shown to be pathogenic. A collection of 21M trimmed paired-end
90mer reads was aligned to all published bacterial genomes at NCBI using
GSnap. 2,220 reads aligned to the current reference for L. asiaticus
CP001677.2, primarily to regions identified as rDNA. Alignment to
chromosome 1 and the plasmid of Ralstonia pickettii was observed. Reads
also aligned to the family Rhizobiaceae. Over 90 percent of reads were novel,
not aligning to any bacterial genome at NCBI. A draft Phase 1 assembly was
constructed (contigs unordered and unoriented) resulting in 481 contigs,
indicating a genome size of 3.8M bp. These contigs demonstrate local
similarity to bacterial sequences at NCBI, containing novel material and ORFs
similar to known genes or conserved domains. Sequence-derived evidence
S116
PHYTOPATHOLOGY
Biocontrol & functional properties of pseudomonads isolated from
different ecological niches & diversity of phlD a key gene in the 2, 4DAPG biosynthesis
J. SEKAR (1), V. R. Prabavathy (1), S. Nair (1)
(1) M.S. Swaminathan Research Foundation, Chennai, INDIA
Phytopathology 100:S116
2, 4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic exhibiting
biocontrol activity against plant pathogens. 1500 strains isolated from the
rhizosphere region of different ecosystems viz., agricultural fields, coastal,
hills & mangroves were screened for pseudomonads using rpoD primer. 350
isolates amplifying 760bp amplicon were considered as pseudomonads
positive. On screening further for phlD gene, 45 strains amplifying a 745bp
amplicon were selected as DAPG positive. Of these 120 positive
pseudomonads from the agriculture field have 23 DAPG positive isolates,
from 42 pseudomonads strains 5 codes for DAPG from the mangrove and 152
pseudomonads 17 DAPG positives from the saline coastal ecosystems. All the
DAPG positives were partially sequenced & analyzed for their genetic
diversity. Antagonistic activity of pseudomonads were assayed against plant
pathogens resulted in 27% suppressing M. grisea growth and 35% inhibit the
growth of X. oryzae & R. solani. Isolates negative for DAPG also exhibited
antagonistic activity and also isolates positive for DAPG coding strains failed
to exhibit antagonistic activity against the plant pathogens. In that 55% of
strains having coding genes for HCN, 37% for chitinase production, 8% for
quorum sensing, 33% for biofilm formation, 15% for phosphate solubilizing,
63% for protease & 46% cellulase producers. Functional properties of
pseudomonads were comparatively good in agricultural ecosystem and the
role of other metabolite coding genes is in progress.
slower symptom development of clubroot in Brassica crops. Sectioning and
staining to assess the impact of temperature on each stage of the pathogen’s
life cycle are in progress.
RPG1-B derived resistance to AvrB expressing Pseudomonas syringae
requires RIN4-like proteins in soybean
D. SELOTE (1), A. Kachroo (1)
(1) University of Kentucky, Lexington, KY, U.S.A.
Phytopathology 100:S117
Grey leaf spot (GLS) is a destructive disease of maize in many parts of Africa
and the Americas. The causal agent of GLS was generally regarded as
Cercospora zeae-maydis. However, recent studies demonstrated that two
distinct species, C. zeae-maydis (previously Group I) in the Americas and C.
zeina, (referred previously as C. zeae-maydis Group II) in Southern Africa,
U.S.A. and Brazil. GLS in Nigeria was first observed in 1996 and C. zeaemaydis was considered to be the causal organism based on morphological and
growth characteristics of the single-conidial cultures recovered from infected
maize leaves in 2002–2004 epidemics. We assessed genetic relatedness of
these isolates by nucleotide sequencing and cluster analysis of the nuclear
ribosomal internal transcribed spacer region (ITS1, ITS2, and the 5.8S gene)
and partial gene sequences of elongation factor 1-α, actin and beta tubulin.
The nucleotide sequences of Nigerian isolates are highly homogenous and
closely (96%) related to C. apii, C. beticola and C. sorghi f. maydis (ex.
maize, Kenya) but less similar (85% to 89%) with C. zeae-maydis and C.
zeina. Cluster analysis based on the four gene sequences, the Nigerian isolates
formed a new phylogenetic lineage in maize infecting Cercospora species
complex. Our results suggest that GLS in Nigeria is caused by a unique
species of Cercospora, and C. zeae-maydis and C. zeina, were not present. A
polymerase chain reaction based diagnostic assay was developed to
distinguish all three species.
Soybean RPG1-B mediates species-specific resistance to AvrB expressing
Pseudomonas syringae, similar to the non-orthologous RPM1 in Arabidopsis.
RPM1-derived signaling is presumably induced upon AvrB-derived
modification of the RPM1-interacting protein, RIN4. Similar to RPM1,
RPG1-B does not directly interact with AvrB. However, RPG1-B associates
with RIN4-like proteins from soybean. Unlike Arabidopsis, soybean contains
at least four RIN4-like proteins (GmRIN4a-d), all of which bind AvrB. In
contrast, GmRIN4b, c, and d bind RPG1-B, but GmRIN4a does not. Silencing
either GmRIN4a or b abrogates RPG1-B-derived resistance to P. syringae
expressing AvrB. Binding studies show that the various GmRIN4 isoforms
interact with each other. The lack of functional redundancy amongst the
GmRIN4 proteins and their abilities to interact with each other, suggests that
these proteins might function as a heteromeric complex in mediating RPG1-Bderived resistance. The GmRIN4 proteins also participate in soybean basal
defense, since silencing GmRIN4a or b enhances basal resistance to virulent
strains of P. syringae and the oomycete Phytophthtora sojae.
Comparison of transient expression vectors for production of
recombinant proteins in plants
K. H. SHAH (1), H. Bohlmann (1)
(1) Institute of Plant Protection, Department of Applied Plant Sciences and
Plant Biotechnology, University of Natural Resources and Applied Life
Sciences, Vienna, AUSTRIA
Phytopathology 100:S117
Production of recombinant proteins in plants is getting more and more
importance not only in plants research field but also in the field of medicines.
Transient expression vectors are efficient tools for this purpose. To date, a
large number of such vectors have been constructed. Each of these is reported
to be highly efficient, robust and cost effective which makes it difficult to
choose the best vector. We have therefore, undertaken a comparative analysis
of a variety of available transient expression vectors. These included the
vectors pJLTRBO, pPZP3425, pEAQ-HT, and pBY030-2R. In addition, we
transferred the TMV expression cassette from pJLTRBO, which is a single
copy vector, into the pPZP vector backbone. This vector was called
pPZP5000. We compared the expression of GUS and GFP in Nicotiana
benthamiana by Agrobacterium-mediated transformation. We found that
pJLTRBO and pPZP5000 had a comparable expression level without RNAi
inhibitor. The other vectors needed co-infiltration of an RNAi inhibitor
expression construct to give good expression levels. The only vector which is
not based on a virus genome, pPZP3425, gave also satisfactory results.
Effect of temperature on clubroot (Plasmodiophora brassicae) symptom
initiation on Shanghai pak choy
K. Sharma (1), B. D. Gossen (2), M. MCDONALD (3)
(1) University of Kassel, Witzenhausen, GERMANY; (2) Agriculture & AgriFood Canada, Saskatoon, SK, CANADA; (3) University of Guelph, Guelph,
ON, CANADA
Phytopathology 100:S117
Clubroot, caused by Plasmodiophora brassicae Woronin, is an important
disease of Brassica crops worldwide. Studies were conducted to assess the
effect of temperature on initiation of visible clubroot symptoms on Shanghai
pak choy (Brassica rapa L. subsp. Chinensis (Rupr.) var. communis Tsen and
Lee). Three-day-old seedlings were transplanted into small plastic pots (roottrainers) containing soil-less growing media, kept at 20°C for 1 wk, and
inoculated by pipetting 600 µL of resting spore suspension (108 spores of P.
brassicae /mL) onto the base of each seedling. After inoculation, the seedlings
were transferred to growth cabinets at 10, 15, 20, 25 and 30°C (14-h
photoperiod, 65% RH). Each day from 8 to 36 days after inoculation (DAI),
the roots of 12 plants per treatment were collected, washed, and assessed for
symptom development. No symptoms were observed at 36 DAI in plants kept
at 10°C. Swelling of the tap root was visible at 28 DAI in plants at 15°C, 14
DAI at 20° and 30°C, and 10 DAI at 25°C. This result supports the results
from companion studies, including field trials, that cool temperatures result in
Genetically distinct Cercospora species cause grey leaf spot of maize (Zea
mays L.) in Nigeria
K. SHARMA (1), M. Ayodele (1), R. Bandyopadhyay (1), A. E. Anu (1), A.
Menkir (1), P. Lava Kumar (1)
(1) International Institute of Tropical Agriculture (IITA), Ibadan, NIGERIA
Phytopathology 100:S117
Genetic characterization of Fusarium verticillioides associated with ear
rot of maize in Nigeria
K. SHARMA (1), M. Ayodele (1), P. Lava Kumar (1)
(1) International Institute of Tropical Agriculture (IITA), Ibadan, NIGERIA
Phytopathology 100:S117
Fusarium verticillioides (sexual stage Gibberella moniliformis) is a common
fungal pathogen associated with diseases such as ear and kernel rot and also
responsible for mycotoxin contamination in maize (Zea mays L.) in Nigeria.
Fusarium isolates from symptomatic and asymptomatic ears of maize
obtained from different maize growing regions in Nigeria had similar
morphological and growth characteristics. Genetic diversity was assessed by
sequencing the nuclear ribosomal DNA internal transcribed spacers (ITS-1
and ITS-2), and partial gene sequences of elongation factor 1-α, actin and
histone. Cluster analysis grouped these isolates into two, one group aligned
with F. verticillioides and the other with G. moniliformis. However, data of
histone gene was not useful to distinguish teleomorph and anamorph stages of
these fungi. Nucleotide base composition between the two forms differed by
2%. Nucleotide diversity based on elongation factor, ITS and Actin is π =
0.023387, 0.008547 and 0.002769 with number of segregating sites S = 27, 26
and 11, respectively, observed based on Tajima’s neutrality test confirming
the differences between the two stages.
A strain differentiating macro array for Plum Pox Virus detection
D. SHERMAN (1), D. Suciu (2), R. Vigaya Satya (3), W. Schneider (1)
(1) USDA-ARS-FDWSRU, Ft. Detrick, MD, U.S.A.; (2) CombiMatrix
Corporation, Mukilteo, WA, U.S.A.; (3) Biotechnology HPC Software
Applications Institute, TATRC, USAMRMC, Frederick, MD, U.S.A.
Phytopathology 100:S117
A series of oligonucleotide probes were identified for strain differentiation
and detection of Plum Pox Virus (PPV). Probes were designed using two
different identification programs; Probe Design Suite and Tool for
Oligonucleotide Fingerprint Identification (TOFI). The probes were selected
to have melting temperatures (Tm) of 70–75°C, lengths of 35 – 40 bases, and
GC content of 45 – 50%. The Probe Design Suite identified 10 general PPV
probes and 10 Prunus sp. probes that met these criteria were used for further
study. TOFI identified 67 strain specific probes and 13 multi-strain probes.
Specificity was tested using healthy Prunus persica tissue and four PPV
isolates: Penn4 (D), European D, M and EA. Oligonucleotide probe testing
started with an open-ended SYBR green PCR array to determine probe
specificity. Batch first strand-DNA was prepared using PPV specific primers
that amplify across the entire genome for all the strains. Probes that were
found to be specific by PCR array were then tested in a nylon macroarray
format. Initial tests using the macroarray demonstrated low background signal
and adequate sample labeling using a colorimetric detection method. The long
term goal is to generate an optimized strain-differentiating PPV pathogen
macroarray for plant diagnostic centers and/or nursery certification programs.
Vol. 100, No. 6 (Supplement), 2010
S117
The virulence mechanisms of Xylella fastidiosa in xylem fluid from
resistant and susceptible grapevines
X. SHI (1), Z. Liang (2), J. Bi (3), J. G. Morse (4), D. A. Cooksey (5)
(1) Department of Plant Pathology, NYSAES, Cornell University, Geneva,
NY, U.S.A.; (2) Horticulture Department, Cornell University, Ithaca, NY,
U.S.A.; (3) University of California Cooperative Extension, Salinas, CA,
U.S.A.; (4) Department of Entomology, University of California–Riverside,
Riverside, CA, U.S.A.; (5) Plant Pathology Department, University of
California-Riverside, Riverside, CA, U.S.A.
Phytopathology 100:S118
Xylella fastidiosa (Xf) is a fastidious, xylem-limited, non-flagellated, insecttransmitted, Gram-negative bacterium that causes many plant diseases,
including Pierce’s disease (PD). It was investigate that while grapevine V.
vinifera is susceptible to the PD strain of Xf, V. champinii and V. smalliana
are tolerant or resistant to the PD strain. The virulence mechanisms of Xf PD
strain between resistant and susceptible grapevines were investigated by
examining the in vitro effect of pure xylem fluid from resistant and
susceptible grapevines on Xf multiplication, aggregation, and attachment of
PD strain. The aggregations of large clumps were formed in pure xylem fluid
from susceptible grapevine, whereas small clumps were observed in resistance
grapevines xylem fluid. Macroarray was being applied for analysis of the
differential gene expression files of Xf PD strain in differential xylem fluids
of grapevines. Xylem fluid was also be analyzed to determine the chemical
compounds or elements that control the virulence of Xf.
Identification and characterization of Fusarium oxysporum, the causal
agent of koa wilt in Hawaii
A. SHIRAISHI (1), J. Uchida (1)
(1) University of Hawaii at Manoa, Honolulu, HI, U.S.A.
Phytopathology 100:S118
Koa (Acacia koa) is endemic to Hawaii and plays extremely important roles
from economical, cultural, and ecological standpoints. However, serious wilt
and dieback have been caused by a fungal pathogen, Fusarium oxysporum,
and making the establishment of koa plantations extremely difficult. The
objectives of this study are to develop a DNA-based system for rapid
detection of F. oxysporum causing koa wilt, and to determine genetic diversity
of F. oxysporum isolated from wilt koa. The genetic diversity within the
species F. oxysporum includes the diversity in pathogenicity to koa. Pathogens
were isolated from surface sterilized specimens collected from wilt koa trees
in the Hamakua research station, the island of Hawaii. Out of 157 isolates
obtained, 78 were Fusarium species. Pathogenicity tests were conducted with
the Fusarium isolates by adding 10 ml of 105 spores/ml of each isolate to 10
koa seedlings under greenhouse conditions. After 3 months, koa mortalities
varied from 0% to 70%. Also, seedlings showed various symptoms. DNA
sequencing confirmed that 10 isolates, including the strongest pathogen and
moderate ones, used in the test were F. oxysporum. This study has confirmed
that pathogenicity varies within the species. In future works, AFLP analysis
and VCG test will be conducted with the isolates to develop molecular
markers that will allow koa growers/researchers to test if soils are
contaminated with pathogenic F. oxysporum before planting and help
establishing healthy koa plantations.
Screening of Medicago Tnt1 lines identifies genes involved in molecular
interactions between Macrophomina phaseolina and its plant host
B. SHUAI (1), A. Reyes Gaige (1)
(1) Wichita State University, Wichita, KS, U.S.A.
Phytopathology 100:S118
Macrophomina phaseolina is a soil born necrotrophic fungus that causes
charcoal rot disease in a wild range of plant hosts. Unlike most fungal
pathogens, M. phaseolina prefers hot and dry conditions. Therefore, charcoal
rot disease is most prominent in places with hot and dry summers. Although
charcoal rot disease is one of the leading causes of reduced crop yield in the
U.S. and around the world, there is no effective method for preventing and
treating the disease. Moreover, very little is known about the molecular
mechanisms involved in host-pathogen interactions. Using Medicago
truncatula as a model, we established a genetic screen to identify genes that
are involved in disease development. In our initial screen of 250 Medicago
Tnt1 transposon insertion lines, we have identified 7 lines that have shown
altered susceptibility to M. phaseolina. Comparing to the wild type plant, 6
out of the 7 lines were more resistant and 1 line was more susceptible to the
fungal pathogen. Molecular techniques are applied to identify genes that are
responsible for the changes. The knowledge gained from this study will
become valuable for crop improvement in the future.
Disease survey of commercial soybean fields in Alabama in 2009
E. J. SIKORA (1), J. F. Murphy (1), K. S. Lawrence (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S118
S118
PHYTOPATHOLOGY
A statewide survey of commercial soybeans was conducted in 2009.
Observations on incidence and severity of foliar diseases, viruses, and
nematodes were taken from 40 fields located in major soybean production
areas in Alabama. A one acre section of each field was used for disease
evaluation of foliar diseases; 50 trifoliate leaves were collected for virus
testing [Bean pod mottle virus (BPMV), Soybean mosaic virus (SMV) and
Tomato spotted wilt virus (TSWV)]; and soil samples were collected for
nematode screening. Cercospora leaf blight (Cercospora kikuchii) was the
most common foliar disease, observed in 77% of fields with disease severity
in the moderate-to-high range in 45%. Downy mildew (Peronospora
manshurica), target spot (Corynespora cassiicola) and soybean rust
(Phakopsora pachyrhizi) occurred in over 40% of fields with disease severity
typically at low levels. Stem canker (Diaporthe phaseolorum) occurred in
22% of fields with incidence between 5–35%. BPMV was detected in 65% of
fields with incidence between 2–54%. SMV was detected in 48% of fields
with incidence between 2–88% and TSWV was found in 12% of fields with
incidence between 5–11%. Reniform (Rotylenchus reniformis), root-knot
(Meloidogyne spp.) and soybean cyst (Heterodera glycines) were found in 30,
16 and 8% of the fields. This was the second year of a two-year study and
showed that soybeans in Alabama are exposed to multiple plant pathogens
with incidence and severity depending on prevailing weather conditions as
well as other factors.
Histochemical detection of H2O2 and O2- in barley leaves infected with
Cochliobolus sativus
P. Silva (1), S. RODRIGUEZ (1), C. Torres Puyo (1), F. Gamba (1), C.
Pritsch (1)
(1) University Uruguay, Montevideo, URUGUAY
Phytopathology 100:S118
Spatiotemporal accumulation of H2O2 in barley leaves has been reported as an
early response to infection by Cochliobolus sativus. However, these analyses
have included a limited number of barley genotypes. Also, little is known
whether O2- follows the same accumulation pattern as H2O2. The aim of this
study is to describe in planta H2O2 and O2- accumulation in relation to C.
sativus infection process. Additionally, we aim to compare the temporal and
spatial pattern of H2O2 accumulation among NDB112 (resistant), cv. Daymán
(intermediately resistant) and line CLE253 (highly susceptible). Primary
leaves spray inoculated with C. sativus spores were sampled at 24 and 48 hai
for H2O2 detection by pre-treatment with 3, 3-diaminobenzidine (DAB) and at
48 hai for O2- detection by reduction of nitrotetrazolium blue chloride (NBT).
Hyphae were visualized by staining with Calcofluor. At pre-penetrated
epidermal cells (cv Dayman), both H2O2 and O2- localized exclusively under
appresoria. In contrast, whole cell accumulation of H2O2 was observed at
uninfected, underlying mesophyll cells while O2- was restricted to chloroplasts
of neighbouring cells. At later stages, whole cell staining for H2O2
accumulation was evident at both single epidermal cells exhibiting
intracellular hyphae and infected mesophyll cells. Similar spatiotemporal
distribution of H2O2 was observed for cv CLE253. In contrast, H2O2
accumulation was not detected in underlying, uninfected mesophyll cells in
NDB112-inoculated tissues.
Evaluation of effectiveness of carbendazim comparatively to
trifloxystrobin + tebuconazole for control of citrus postbloom fruit drop
in Brazil
G. J. SILVA JUNIOR (1), M. B. Spósito (2), D. R. Marin (2), L. Amorim (1)
(1) Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP),
Piracicaba-SP, BRAZIL; (2) Fundo de Defesa da Citricultura (Fundecitrus),
Araraquara-SP, BRAZIL
Phytopathology 100:S118
Postbloom fruit drop of citrus, caused by Colletotrichum acutatum, is
controlled by fungicide sprays during flowering. Nowadays carbendazim is
most commonly used fungicide in the state of São Paulo, Brazil. In this study
the effectiveness of carbendazim was assessed in vitro and in orchards and
compared to trifloxystrobin + tebuconazole. Mycelial growth inhibition by
carbendazim (1 to 1000 g/mL) was tested in vitro. Field experiments were
carried out in two sweet orange orchards in the state of Sao Paulo in 2009.
Citrus trees received 2, 3 or 4 sprays of carbendazim (1000 g/ha) or
trifloxystrobin + tebuconazole (80 + 160 g/ha) at 7-days interval, starting at
the green bud stage. The percentage of symptomatic petals, number of
persistent calyces and fruits per branch were determined. Carbendazim
reduced the colony diameter by about 50 to 60% at all concentrations, without
complete inhibition. In the field, the PFD incidence and the fruit set on
carbendazim sprayed trees did not differed from disease incidence and fruit
set in control trees. Four sprays of trifloxystrobin + tebuconazole mixture
significantly reduced the proportion of symptomatic flowers by 94 and 71%
and persistent calyces by 86 and 40%, in two orchards. Fruit set in control
trees were 85 and 98% lower than fruit set in trees treated with four sprays of
fungicide mixture. The data suggest that trifloxystrobin + tebuconazole can be
used to control the disease, replacing carbendazim which showed to be
ineffective. Supported by FAPESP.
Spatial and temporal dynamics of postbloom fruit drop in sweet orange
orchards in Sao Paulo State, Brazil
G. J. SILVA JUNIOR (1), M. B. Spósito (2), D. R. Marin (2), L. Amorim (1)
(1) Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP),
Piracicaba-SP, BRAZIL; (2) Fundo de Defesa da Citricultura (Fundecitrus),
Araraquara-SP, BRAZIL
Phytopathology 100:S119
Epidemics of postbloom fruit drop (PFD), caused by Colletotrichum
acutatum, occur suddenly and can cause losses of up to 100%. The objective
of this study was to analyze the spatial and temporal dynamics of PFD in
young orchards, in order to understand the spread mechanisms of the disease.
Symptoms on petals were assessed in 2008 and 2009 bloom seasons in two
orchards of 2- and 3-yr-old Pera sweet orange with 500 trees each (20 rows
with 25 trees). Population dynamics models were fit to PFD progress curves
by non linear regression. The index of dispersion for binomial distribution
(ID) as well as the Taylor’s power law relationship between variances were
calculated to determine the disease pattern. The model that best fit the data
was the logistic, with initial inoculum ranging from 0.001 to 0.02 and progress
rates from 0.11 to 0.45. PFD progress rates were similar to those reported for
Phytophthora infestans in potato and considered high for a pathogen that
depends on rain for dispersal. The ID was equal to 1.0 in most of the cases and
the regression of the variance for the Taylor’s power law had the parameters
log(A)=0 and b=1 (p < 0.05), suggesting random distribution. There are two
possibilities to justify this behavior: (i) the pathogen has additional
mechanisms of dispersal, such as bees, for example, (ii) the inoculum is
present uniformly in the orchards, due to pathogen survival in seedlings, soil
or weeds. Both possibilities are under investigation. (Supported by FAPESP,
Brazil).
Pantoea agglomerans, a maize seed transmitted bacterium in Mexico
H. V. SILVA-ROJAS (1), G. Mahuku (2), P. D. Esker (3)
(1) Colegio de Postgraduados, Montecillo, MEXICO; (2) Centro Internacional
de Mejoramiento de Maiz y Trigo, Texcoco, MEXICO; (3) University of
Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S119
Pantoea agglomerans has been reported to cause leaf blight and vascular wilt
of maize in the Central Highland Valley of Mexico. Symptoms were
consistently observed in maize from commercial and seed production fields at
elevations ranging from 100 to 2,700-m. An experiment was conducted under
greenhouse conditions to determine whether P. agglomerans was seed
transmitted, and also the transmission efficiency using seed from different
cultivars and with different disease incidences. Three cultivars, HS2 and
Triunfo (three-way) hybrids, and Cacahuacintle, a landrace, were planted in a
split plot design with three replications, and inoculated with three strains of P.
agglomerans. A total of 1,200 seeds of each treatment were planted in trays
containing sterilized soil. Initial symptoms of chlorotic streaks were observed
in seedlings 15 days after emergence, and evaluations were conducted for a
period of five weeks. To confirm the presence of P. agglomerans in chlorotic
streaks, small portions of tissue were taken from the edge of the lesions, and
placed on CPG medium. Sequencing of the 16S rDNA was conducted for
specific identification of P. agglomerans isolated from chlorotic streaks.
Results revealed a strong strain x cultivar interaction, and the rate of
seed transmission ranged from 6 to 26% for the cultivars Triunfo and
HS2, respectively. These results indicate that P. agglomerans is seed
transmitted and at a higher rate than what has been reported for other species
of Pantoea.
Volatile hexanal to postharvest control of brown rot of peach caused by
Monilinia fructicola and M. laxa
J. SILVEIRA BAGGIO (1), S. de Afonseca Lourenço (1), L. Amorim (1)
(1) Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ/USP),
Piracicaba, BRAZIL
Phytopathology 100:S119
Brown rot, caused by Monilinia fructicola, M. laxa and M. fructigena, is one
of the most important peach diseases. In Brazil, peaches are affected by the
first two species, and the second one had been reported recently. Currently,
alternative methods for postharvest diseases have been studied, like the
volatiles use. Tests were made to determine the effects of volatile hexanal in
the development of the disease in peaches inoculated with both fungi species.
Experiments were carried out with two different peach cultivars: Chiripá and
O’Henry. Hexanal concentration at 50 µL/L was applied as a single dose at
the moment of fruit inoculation (eradication treatment) or 24 hours after
inoculation (curative treatment). Wounded and unwounded fruit were
inoculated with 30 µL of conidia suspensions of each pathogen. The peaches
were put inside a sealed recipient with a Petri dish containing the volatile for
24 hours at 20°C. The control treatment did not receive the volatile. Brown rot
incidence and brown rot lesion diameter were assessed daily during 7 days.
Overall, hexanal was more efficient in cv. O’Henry than in cv. Chiripá. Brown
rot disease was reduced in average by 30% and 72% in eradication treatments
for cv. Chiripá and O’Henry, respectively. In curative treatments, disease was
reduced in average by 43% and 72% respectively in cv. Chiripá and O’Henry.
Hexanal provides a promising alternative to chemical fungicides and can be
used in postharvest handling systems. Supported by FAPESP.
GmFAD3 genes mediate developmental and defense-related physiology in
soybean
A. SINGH (1), M. El-Habbak (1), S. Ghabrial (1), A. Kachroo (1)
(1) University of Kentucky, Lexington, KY, U.S.A.
Phytopathology 100:S119
Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid
(18:2) to linolenic acid (18:3) is an important step in fatty acid (FA)
biosynthesis. Silencing three microsomal isoforms of GmFAD3 using a bean
pod mottle virus (BPMV)-based vector, increased 18:2 levels, and lowered
18:3 levels in soybean vegetative tissues. Greenhouse grown GmFAD3silenced plants produced seeds that were significantly larger in size and
greater in weight. The average number of seeds obtained from the GmFAD3silenced plants did not differ significantly from control plants. Thus, silencing
GmFAD3 in soybean resulted in an ~ 60% increase in seed yield.
Furthermore, seeds from GmFAD3-silenced plants contained similar amounts
of proteins, carbohydrates, FAs and oil as those from control plants.
Interestingly, the GmFAD3-silenced plants exhibited altered defense-related
phenotypes, accumulating higher levels of the phytohormones salicylic acid
(SA) and jasmonic acid (JA), and increased expression of pathogenesis-related
genes. Consequently, these plants exhibited enhanced resistance to an
avirulent strain of Pseudomonas syringae and a virulent strain of
Phytophthora sojae. Our results show that the GmFAD3 genes modulate seed
development and phytohormone-derived defense signaling in soybean.
Epidemiology of almond leaf scorch disease in the San Joaquin Valley of
California
M. SISTERSON (1), J. Chen (2), K. Daane (3), R. Groves (4), B. Higbee (5),
C. Ledbetter (1)
(1) USDA ARS, Parlier, CA, U.S.A.; (2) USDA ARS PWA, Parlier, CA,
U.S.A.; (3) University of California, Berkeley, CA, U.S.A.; (4) University of
Wisconsin, Madison, WI, U.S.A.; (5) Paramount Farming Co., Bakersfield,
CA, U.S.A.
Phytopathology 100:S119
Almond leaf scorch (ALS) disease has been present in California for more
than 60 years. This disease is caused by the bacterium Xylella fastidiosa,
which causes several other important plant diseases, including Pierce’s disease
of grapes. The epidemiology of ALS in the San Joaquin Valley of California
was investigated to determine: 1) effects of ALS on tree yield and longevity,
2) regional incidence, and 3) disease progress curves in select orchards. Yields
of ALS-affected trees were significantly lower than yields of unaffected trees.
Yield loss varied with cultivar and tree death due to ALS over a 5–6 year
period was rare. Almond leaf scorch disease was common in the San Joaquin
Valley and at least one infected tree was found in 34 of 61 (56%) orchards
containing the cultivar Sonora. Incidence in surveyed orchards was typically
low (<2%). Multi-year surveys in two severely affected orchards found that
incidence varied with cultivar and appeared to increase at a steady rate. For
example, in one orchard incidence in the cultivar Sonora increased from 5.8%
in 2003 to 8.5% in 2009. Incidence in the cultivar Nonpareil in the same
orchard was lower with 1.3% of trees affected in 2003 and 2.7% of trees
affected in 2009. The results indicate that ALS is present in orchards
throughout the San Joaquin Valley, but that incidence and yield effects vary
with cultivar.
Exploring the diversity of Phytophthora and related genera in aquatic
environments in Maryland, U.S.A.
D. SKALTSAS (1)
(1) University of Maryland, College Park, MD, U.S.A.
Phytopathology 100:S119
In an attempt to discover the species diversity in streams of Maryland we have
conducted an intensive survey in 2009. Sites were located throughout the State
including different ecosystems such as oak forests, urban parks, agricultural
sites, and brackish water in Eastern Shore around Chesapeake Bay. In total 27
streams were surveyed and baited from May to August. In each site, four
rhododendron leaves in a mesh bag was deployed and collected after 1–3
weeks. Due to the water temperature fluctuations, sites in lower elevation in
eastern Maryland were baited up to 11 times in weekly intervals. Watersoaked or necrotic tissue samples from baited leaves were plated on selective
agar (PARPNH) for Phytophthora, and any outgrowing colonies sub-cultures
after 3–5 days. In total 1,600 isolates were identified as Phytophthora and 450
Vol. 100, No. 6 (Supplement), 2010
S119
as Pythium or an unidentified Oomycete. Isolates were initially identified
based on ITS sequencing. This poster presents one of the most comprehensive
analysis of species assemblage of Phytophthora that exists in diverse aquatic
environments in Maryland.
Interaction between pattern, process and scale in plant disease epidemics
P. SKELSEY (1), K. A. Garrett (1), K. A. With (1)
(1) KSU, Manhattan, KS, U.S.A.
Phytopathology 100:S120
Optimal arrangement of crops and crop varieties could make agricultural
landscapes more resilient to plant pathogen invasions, but is there a ‘correct’
scale at which to introduce artificial heterogeneities in plant communities? For
example, aggregation of host fields into clusters could serve to hamper disease
spread by increasing the separation distances between host areas, but
aggregation of clusters into super-clusters could lead to large crop losses if
just one or two areas became infected. Here we present a spatiotemporal
simulation framework to investigate the scale-dependence of pattern-process
relationships in plant disease epidemics. Epidemics that result from
transmission of both vectorborne and airborne infectious agents are simulated using models such as a standard SEIR model and Levy flights to
describe vector movement, and a previously published model for potato
late blight linked to an atmospheric dispersion model. We introduce a
new class of neutral landscape model that facilitates the simultaneous study
of host pattern-epidemic process relationships across a continuum of
spatial scales, i.e., from plant-scale through to intercontinental-scale. In some
model scenarios, landscape designs that reduced disease at one spatial scale
led to increased disease at other spatial scales. These initial results indicate
potential for the development of new scale-appropriate management
strategies.
A metabolic fingerprinting technique for functional genomics in
Fusarium verticillioides
J. E. SMITH (1), B. H. Bluhm (2)
(1) University of Arkansas, Garfield, AR, U.S.A.; (2) University of Arkansas,
Fayetteville, AR, U.S.A.
Phytopathology 100:S120
The maize pathogen Fusarium verticillioides infects roots, stalks, ears, and
kernels. In addition, the fungus produces fumonisin mycotoxins during kernel
colonization. Although the regulation of fumonisin biosynthesis is
increasingly well understood, little is known about broad changes in the
metabolome underlying pathogenesis. The objective of this study was to
develop a metabolic fingerprinting technique to simultaneously detect and
quantify structurally diverse metabolites produced by F. verticillioides. To
create a metabolic fingerprint of the fungus, a workflow based on gas
chromatography-mass spectrometry (GC-MS) was optimized to detect over
70 metabolites in the wild type strain, nearly half of which were
putatively identified based on matches with characterized compounds in
structural databases. Then, metabolic fingerprints were obtained from
genetically defined mutants of the fungus and compared to the fingerprint of
the wild type. Statistically significant differences in the production of
characterized and uncharacterized metabolites were reliably detected in
several mutants. Additionally, changes in the metabolome of the wild-type
strain in response to environmental conditions such as pH and nitrogen
availability were consistently detected. This metabolic fingerprinting
technique provides a novel tool for functional genomics and reverse genetics
in F. verticillioides, in that a wide range of “hidden” metabolic phenotypes
can be identified rapidly.
Resistance breakdown in Rz2 containing sugar beet cultivars to Beet
necrotic yellow vein virus
M. J. Smith (1), R. Acosta-Leal (1), C. M. RUSH (1)
(1) Texas AgriLife Research, Amarillo, TX, U.S.A.
Phytopathology 100:S120
Beet necrotic yellow vein virus (BNYVV) causes the economically important
disease, Rhizomania, in sugar beets. Genetic resistance to rhizomania,
conferred by the dominant Rz1 gene, has been overcome by resistance
breaking (RB) strains of BNYVV. As a result, a second resistance gene, Rz2,
recently was introduced into commercial cultivars. In 2009, isolated plants,
from fields in southern Minnesota planted to cultivars with Rz2 resistance,
exhibited typical rhizomania symptoms. Diseased plants had a high virus titer,
as measured by real time RT-PCR, and tested positive for the presence of the
Rz2 gene. Subsequently, alleleic discrimination based on the p25 region of
BNYVV RNA 3, showed that the majority of the diseased plants contained an
A67, typical of wild type BNYVV, instead of a V67, previously associated with
Rz1 RB strains of BNYVV. This suggests that the molecular determinant(s),
which allows isolates of BNYVV to overcome Rz2 genetic resistance, differs
from that reported for Rz1 resistance breaking isolates.
S120
PHYTOPATHOLOGY
Using weather variables to predict the probability of dollar spot
development
D. L. SMITH (1), J. P. Kerns (2)
(1) Oklahoma State University, Stillwater, OK, U.S.A.; (2) University of
Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S120
Dollar spot, caused by Sclerotinia homoeocarpa, is the most damaging disease
of cool-season turfgrasses throughout the U.S. A reliable dollar spot
prediction model would be useful for timing fungicide applications for highvalue turfgrasses. Logistic regression was used to develop a model to predict
the probability of dollar spot development on creeping bentgrass using
weather variables as inputs at sites in Oklahoma (2008 and 2009) and
Wisconsin (2009). Numbers of dollar spot foci were counted daily in plots
receiving no fungicide or treated with fungicide. Various on-site weather
variables were recorded hourly. Weather data were transformed to 5-day
moving averages. Disease severity/plot was converted to a binomial variable
where 1 was average severity ≥ 1 spot and 0 was average severity < 1 spot.
Transformed weather data and the class variables season and fungicide, were
used as independent variables with average disease severity (DS) as the
dependent variable in model development. The best model included the class
variable fungicide, and 5-day moving averages of daily relative humidity and
minimum daily air temperature (Max-rescaled R-square=0.46; C=0.89).
Minimum temperature thresholds to activate the model were set at 14°C based
on field and controlled environment chamber studies. Independent validation
exercises demonstrated that the model accurately recommended fungicide
sprays when an action threshold of 30% was used in the model at both
locations.
Complete genome sequence of Pantoea vagans biocontrol strain C9-1
T. H. Smits (1), F. Rezzonico (1), T. Kamber (1), C. A. ISHIMARU (2), J. E.
Frey (1), A. Goesmann (3), V. O. Stockwell (4), B. Duffy (1)
(1) Agroscope Changins-Wädenswil ACW, Wädenswil, SWITZERLAND; (2)
University of Minnesota, St. Paul, MN, U.S.A.; (3) Universität Bielefeld,
Bielefeld, GERMANY; (4) Oregon State University, Corvallis, OR, U.S.A.
Phytopathology 100:S120
Pantoea vagans strain C9-1 is an effective, reliable biocontrol agent registered
in the U.S.A. and Canada for fire blight control. We sequenced the complete
genome of C9-1 (4.88 Mb) using 454 technology with 25x coverage yielding
1,224,924 reads and 207 contigs. Gap closure was done with Sanger
sequencing and assembly was done using Lasergene software. The genome
includes a 4.025 Mb chromosome (55.5% GC ratio, 3693 predicted CDS, 7
rRNA operons, 78 tRNAs), and 3 circular, non-self-transmissible plasmids
(lacking tra, mob mobility genes). Plasmids pPag1 (168 kb), pPag2 (166 kb)
and pPag3 (530 kb) have 162, 229 and 535 predicted CDS, respectively.
Annotation identified a complete set of enterobacterial metabolic pathways
and biosynthetic pathways for all amino acids and most cofactors. Four large
regions containing phage-related genes, a single genomic island, ampicillin
and tellurite resistance genes, and 41 genes encoding putative multidrug
exporters were identified. The complete beta-carotenoid yellow-pigment
biosynthetic pathway was identified on pPag3. Metabolic versatility is a
distinctive feature of the C9-1 genome, particularly efficient utilization of
sugars contributing to competitiveness in flower infection courts. Biosynthetic
genes for multiple antibiotics (pantocin A, and dapdiamide) were
characterized. Additional features of potential biocontrol/ecological fitness
relevance were identified. Their potential role in biocontrol is under study.
White pine blister rust resistance in a seven year old field trial of 28
western white pine (Pinus monticola) families in the Coast Range of
Oregon
R. A. SNIEZKO (1), J. Hill (2), R. S. Danchok (2), A. J. Kegley (2), S. Long
(2), J. B. Mayo (2), A. J. Smith (3)
(1) USDA FS Dorena Genetic Resource Center, Cottage Grove, OR, U.S.A.;
(2) USDA Forest Service/Dorena Genetic Resource Center, Cottage Grove,
OR, U.S.A.; (3) Plum Creek Timberlands, Cottage Grove, OR, U.S.A.
Phytopathology 100:S120
Western white pine (WWP) is highly susceptible to the non-native pathogen
Cronartium ribicola, cause of white pine blister rust. There are few reports
from field trials to verify rust resistance from artificial inoculation programs
of WWP. This is the first trial reported for the Coast Range of Oregon. WWP
families from two resistance programs were planted in 2003. Families
exhibiting several types of resistance, including a hypersensitive reaction
(HR) that occurs in the needles, are represented. Infection events had occurred
in several years, and both old cankers and recent stem infections were present;
however, little mortality has occurred to this point. As of March 2010, 65% of
trees were infected, with families ranging from 24 to 100% infected. The
number of stem infections per tree ranged from 0 to 91. The susceptible
control family had the highest percentage of trees infected and the most severe
infections. The families with HR resistance showed relatively high infection,
indicating that a strain of the rust virulent to HR in WWP is present, the
northernmost documented occurrence of vcr2 to date. Most infections were
recent and low in severity at this point. The incidence of rust suggests this site
may be a moderate rust hazard and that large differences among families in
rust resistance are present. This trial will continue to be monitored to assess
the range and durability of blister rust resistance and the growth performance
of the families.
Exploration of further sequence data for unknown regions of Ambrossia
asymptomatic virus 1
N. SOKHANDAN BASHIR (1), U. Melcher (2)
(1) The University of Tabriz, Tabriz, IRAN; (2) Oklahoma State University,
Stillwater, OK, U.S.A.
Phytopathology 100:S121
Ambrossia asymptomatic virus 1 (AAV-1) is a novel virus discovered from
Tallgrass Prairie Preserve (TPP) in northeastern Oklahoma. Sequences of four
genome stretches of the virus have previously been determined from a clone
library of the virus. However, there were two gaps in the genome with
unknown sequences. The purpose of the present study was to complete the
genome sequence by exploring sequences data for these gaps. Accordingly, a
number of ten primers were designed corresponding to the known regions of
the virus. Then, polymerase chain reaction (PCR) was performed by the use of
different combination of the new primers or one of these primers coupled with
a universal primer M13F or M13R. As a result, amplification with M13R/ R3
and F1/ R5 ended in ~600 and ~550 bp fragments, respectively. These
sequences were subcloned in pCR2.1-TOPO vector (Invitrogen) and subjected
to sequencing. Analysis of sequence data revealed that M13R/ R3-amplified
fragment covers most of gap2 between Flex 4 and Flex 5 fragments. The
fragment amplified by primers F1 and R5 covered whole of gap1 region,
between Flex 2 and 3. This research has been a part of a major study to
discover viruses of wild plants from TPP.
Characterization of Phytophthora infestans from Northern Thailand
based on their mating type, metalaxyl sensitivity, and mtDNA haplotypes
J. SOPEE (1), S. Sangchote (1), P. Chiampiriyakul (2)
(1) Department of Plant Pathology, Kasetsart University, Bangkok,
THAILAND; (2) Department of Plant Protection, Maejo University,
ChiangMai, THAILAND
Phytopathology 100:S121
Isolates of Phytophthora infestans, causal agent of potato late blight, was
collected in northern Thailand from 2006 to 2009. Thirty-three isolates were
analyzed for mating type, metalaxyl sensitivity and mitochondrial DNA
haplotypes. Metalaxyl sensitivity testing of these isolates by on the agar
amended with metalaxyl at different levels of concentration. It indicated that
90 percent isolates distributed in 6 locations were intermediate sensitive to
this fungicide and 10 percent located in 2 locations were sensitive. All of these
isolates were A1 mating type and mitochondrial DNA haplotype IIa. This
investigation on characteristics variable of P. infestans isolates obtained in
Thailand which imports potato seed tubers from the major potato producing
countries.
Identification and disruption of a cercosporin polyketide synthase and
ABC transporter in Cercospora coffeicola
A. G. SOUZA (1), S. Herrero (2), L. A. Maffia (3), M. E. Daub (2)
(1) North Carolina State University / Universidade Federal de Vicosa,
Raleigh, NC, U.S.A.; (2) North Carolina State University, Raleigh, NC,
U.S.A.; (3) Universidade Federal de Vicosa, Vicosa, BRAZIL
Phytopathology 100:S121
Brown eye spot of coffee, caused by Cercospora coffeicola, causes significant
losses due to a decrease in quality and quantity of coffee produced. Many
Cercospora species produce cercosporin, a photoactivated toxin thought to be
involved in pathogenesis. The objective of this study is to determine the role
of cercosporin in C. coffeicola pathogenesis by creating disrupted strains
unable to produce the toxin. We first evaluated six Brazilian C. coffeicola
isolates from fields representing organic and conventional production systems
in the Minas Gerais state for their ability to produce cercosporin in vitro.
Production varied among isolates, ranging from 3.5 - 25.3 µM/ 5 mm plug;
production was undetectable in one isolate. The highest producing isolate is
being used to identify homologs of a polyketide synthase (CTB1-2196aa) and
ABC transporter (ATR1-1456aa) genes involved, respectively, in production
and sensitivity to cercosporin in C. nicotianae. The C. coffeicola CTB1 and
ATR1 genes were amplified using degenerate and standard PCR primers. We
are presently sequencing these genes and analyzing them against available
sequences. In addition, we have successfully transformed this isolate with a
GFP construct using PEG-mediated protoplast transformation, and are
currently using a CTB1 disruption construct to generate disrupted strains. In
the future, these strains will be tested for their ability to parasitize coffee
plants in Brazil. Sponsor: CNPq.
Mapping the future: Metamodels for scaling potato late blight risk
analysis in climate change scenarios
A. SPARKS (1), G. Forbes (2), R. Hijmans (3), K. Garrett (1)
(1) Kansas State University, Manhattan, KS, U.S.A.; (2) International Potato
Center, Lima, PERU; (3) University of California, Davis, CA, U.S.A.
Phytopathology 100:S121
The general problem of how epidemiological processes scale is a topic of
great interest in ecology. For example, while many disease forecasting models
predict in-season disease risk using hourly or finer resolution weather data,
global weather or climate change model datasets are often only available as
monthly values. We created metamodels to adapt an established potato late
blight risk model, Simcast, for use with daily and monthly forms of data using
generalized additive models. The daily and monthly weather metamodels have
R-squared values of 0.62 and 0.78 respectively. The metamodels were used to
create global late blight risk maps under current and climate change scenarios
for resistant and susceptible varieties. Changes in global late blight risk for
areas where wild potato species are indigenous and countries where chronic
malnutrition is a problem were also evaluated. While geographic regions tend
to maintain their level of risk relative to other regions, some areas experience
greater increases (such as Nepal) and decreases (such as Malawi) in risk.
Areas of high wild potato species richness show an increased late blight risk.
This metamodel framework was useful for evaluating the relative risk of late
blight under climate change scenarios and can be adapted to other
pathosystems.
Fungicide management strategies for the control of Fusarium head blight
in southern Brazil
P. SPOLTI (1), L. Simon (1), J. dos Santos (1), N. Barros (1), D. S. Guerra
(2), E. M. Del Ponte (1)
(1) UFRGS, Porto Alegre, BRAZIL; (2) BASF, Passo Fundo, BRAZIL
Phytopathology 100:S121
Effective management of Fusarium head blight (FHB) epidemics in wheat
(caused by Fusarium graminearum) in Brazil may be achieved by using
genetic and chemical control. Four non-inoculated field trials were conducted
in 2009 at different locations with the aim to assess and compare the effect of
two fungicides (mixture = metconazole + pyraclostrobin (BAS556) x
metconazole (Caramba)); two dosages (0.5L/ha x 0.75L/ha) of the mixture;
and number of applications (1 = flowering x 2 = flowering + 10 days later) on
disease control and yield. A single and different wheat cultivar was used per
location. FHB index varied from 5.1 to 7.3 in the two locations (with
moderate resistant varieties) and from 10.1 to 31.2 in two locations
(susceptible varieties). All treatments significantly reduced disease related to
the unsprayed check. The use of two applications gave better results, and the
use of mixture in highest dosage reduced around 92% and 76% of FHB index
in susceptible and moderate resistant genotypes, respectively, and did not
differ from the triazol. Fungicides had both neutral and positive effect in
reducing infection by F. graminearum in grains. The better performance in
disease control by using two applications of the mixture is possibly related to
extended protection, which resulted in an average increase of 27% in test
weight compared to metconazole. Mycotoxin analyses are underway and
results will be presented together with other effects on yield components.
Evaluation of selective media and selective chemicals on the isolation of
Rhizoctonia spp. from soil
T. N. SPURLOCK (1), C. S. Rothrock (2), W. S. Monfort (3)
(1) University of Arkansas, Cave Springs, AR, U.S.A.; (2) University of
Arkansas, Fayetteville, AR, U.S.A.; (3) University of Arkansas, Lonoke, AR,
U.S.A.
Phytopathology 100:S121
Characterizing Rhizoctonia spp. in field soil has been performed many times
using methods such as baiting, elutriation, direct plating or direct observation.
In most cases, selective media are used to suppress bacteria, antagonistic
fungi, such as Trichoderma spp., and other fast growing fungi and oomycetes
that could limit the ability to properly assess populations. Assaying
populations of Rhizoctonia spp. by several techniques using different selective
media resulted in poor suppression of Trichderma spp. from soils and
selection of specific Rhizoctonia species or anastomosis groups (AGs) of R.
solani. Thus, research was initiated to evaluate these selective media and
existing or additional components in these media using isolates representing
new or important anastomosis groups, species, or intraspecific groups to
improve characterization of soil populations of Rhizoctonia spp. in a variety of
cropping systems and soils in Arkansas. Preliminary testing indicated that
thiophanate-methyl provided suppression of fungi similar to benomyl, but
both favored isolation of R. oryzae. Ethanol potassium nitrate medium
amended with 8 ppm prochloraz suppressed growth of Trichoderma spp., but
not fast growing Zygomycetes. The value of existing and amended selective
media using the toothpick and multiple-soil pellet methods for these soils with
a history of soybean and rice production will be discussed.
Vol. 100, No. 6 (Supplement), 2010
S121
Identification of potential virulence genes of Candidatus Liberibacter
asiaticus, the pathogen associated with citrus greening (Huanglongbing)
A. SREEDHARAN (1), N. Wang (1)
(1) University of Florida, Lake Alfred, FL, U.S.A.
Phytopathology 100:S122
Ulvan inhibits the appressorium differentiation of Colletotrichum
gloeosporioides on apple leaves
M. J. STADNIK (1), L. Araujo (1)
(1) Universidade Federal de Santa Catarina, Florianópolis, BRAZIL
Phytopathology 100:S122
Citrus greening or huanglongbing (HLB) is a devastating disease of citrus, and
poses a major threat to the citrus industry in the United States. Candidatus
Liberibacter asiaticus is the strain associated with HLB in the U.S. The
bacterium was introduced to Florida in 2005. Unsuccessful attempts to culture
Ca. L. asiaticus have notably hampered efforts to understand its biology and
pathogenesis mechanism despite some limited progresses in culturing. In
order to identify the potential virulence genes, the genome sequence of Ca. L.
asiaticus was screened, and about 30 candidate genes were identified. Selected
genes were then tested on Nicotiana benthamiana for symptom expression
using transient assays, and two of the candidate genes were chosen for further
characterization. The leaves, petiole and roots of these infected plants were
observed under the light microscope, to determine the effect of these proteins
on the plants. Reverse-transcriptase PCR was conducted to confirm the
expression of the genes in planta. The ability of the proteins to cause
symptoms will be further confirmed by another transient assay, using a
different vector. Real-time quantitative PCR will also be used to quantify the
expression of these potential virulence genes. Identification and
characterization of the various virulence genes in Ca. L. asiaticus will be the
first step towards understanding the biology, and the basis of interaction of
this pathogen with its host.
The aim of this study was to monitor the development of infection structures
of C. gloeosporioides, the causal agent of Glomerella leaf spot (GLS), on
resistant and ulvan-treated susceptible apple plants. For this, the resistant
seedlings were sprayed with distillated water, while susceptible ones were
treated with the algal polysaccharide ulvan (10 mg/mL) or water. All
seedlings were inoculated 6 days after treatment. GLS severity was assessed
daily between 4 and 10 days after inoculation, based on the visual estimation
of the necrotic tissue percentage. Foliar discs from fully developed leaves
were collected at 24, 48 and 72 hour intervals after inoculation and
microscopically examined for determining the conidial germination and
appressorium formation of fungus. Ulvan significantly reduced disease
severity in seedlings. Conidial germination was not changed by neither innate
nor ulvan-induced resistance. The development of fungal infection structures
in resistant seedlings was similar to those observed in susceptible ones. In
contrast, in ulvan-treated plants the appressorium formation was strongly
inhibited and germ tube was longer. The results indicate that ulvan reduces the
GLS severity by inhibiting the appressorium differentiation of C.
gloeosporioides.
Evaluating Thrips tabaci and Frankliniella fusca (Thysanoptera:
Thripridae) as vectors of Iris yellow spot virus
R. SRINIVASAN (1), H. Pappu (2), D. G. Riley (1), R. D. Gitaitis (1)
(1) University of Georgia, Tifton, GA, U.S.A.; (2) Washington State
University, Pullman, WA, U.S.A.
Phytopathology 100:S122
Thrips-transmitted Iris yellow spot virus (IYSV) (Family Bunyaviridae, Genus
Tospovirus) affects onion production in Georgia and other parts of the United
States. The presence of IYSV was first documented in Georgia in 2003. At
least three major thrips species that transmit tospoviruses, the onion thrips
Thrips tabaci (Lindeman), the tobacco thrips Frankliniella fusca (Hinds), and
the western flower thrips F. occidentalis (Pergande) are prevalent in Georgia.
Thrips tabaci is the only confirmed vector of IYSV. The transmission
efficiency of nymphs and adults of T. tabaci was evaluated by using
lisianthus, (Eustoma grandiflorum) as an indicator host. Results showed that
both nymphs and adults transmitted IYSV at 63% and 66% respectively. The
infection status of the test plants were evaluated by using IYSV-specific
antiserum through a double antibody sandwich enzyme linked immunosorbent
assay (DAS-ELISA). To assess the vector status of F. fusca, nymphs and adults
from IYSV-infected lisianthus were tested with antiserum specific to the nonstructural (NSs) protein of IYSV through an antigen-coated plate ELISA.
Both nymphs and adults tested positive at 23% and 40% respectively. These
results indicate that F. fusca can potentially transmit IYSV. Experiments with
lisianthus plants further confirmed IYSV transmission by F. fusca. However,
the transmission rate (6%) was significantly lower than that of T. tabaci.
Saccharin-induced systemic acquired resistance in soybean
P. SRIVASTAVA (1), S. George (1), J. J. Marois (1), D. L. Wright (2), D. R.
Walker (3)
(1) IFAS, University of Florida, Quincy, FL, U.S.A.; (2) Quincy, FL, U.S.A.;
(3) USDA-ARS, Urbana, IL, U.S.A.
Phytopathology 100:S122
Systemic acquired resistance (SAR) is a widely distributed plant defense
system that confers broad-spectrum disease resistance. Saccharin is known to
induce a SAR response in many plant species. To evaluate the potentiating
capability of saccharin in this respect, soybean (Glycine max) plants were
inoculated with Phakopsora pachyrhizi urediniospores after application of
saccharin, and were later rated for differences in the development of soybean
rust symptoms. Plants were grown hydroponically in half strength Hoagland’s
solution and were challenged with pathogen at 1, 5, 10 and 15 d after
treatment with 3 mM saccharin applied as either a foliar spray or a root drench
at the 2nd trifoliate (V2) and early reproductive (R1) stages. Plants were
destructively harvested and assessed for rust infection 2 wk after inoculation.
Leaf position and mode of saccharin application were significant factors in
determining the severity of rust infection. Saccharin applied as a root drench
was more effective than the leaf treatment at inducing the SAR, with
increased resistance observed 1 d after application and still apparent 15 d after
application. In contrast, foliar treatment with saccharin did not increase
systemic protection until 15 d after treatment. Application of saccharin, either
to the leaf or as a root drench, had no significant effect on fresh and dry
weight of plants which suggests that induction of systemic resistance to rust
infection using saccharin does not affect plant growth.
S122
PHYTOPATHOLOGY
Tomato spotted wilt virus (Bunyaviridae, Tospovirus) infection alters
feeding behavior of its vector Frankliniella occidentalis (Pergande)
C. A. STAFFORD (1), G. P. Walker (2), D. E. Ullman (1)
(1) University of California, Davis, Davis, CA, U.S.A.; (2) University of
California, Riverside, Riverside, CA, U.S.A.
Phytopathology 100:S122
Specific components of insect feeding behavior are critical to their
competency in transmission of plant viruses. Although most feeding behaviors
are not readily visible because they occur within plant tissues, the electrical
penetration graph technique allows examination of insect feeding in real time
and has been used extensively for aphids, leafhoppers and whiteflies. Very
few studies have been done with thrips due to their minute size. We show that
not only do male and female Western flower thrips (Frankliniella
occidentalis) differ dramatically in their feeding behavior, as has been
previously shown, but infection with Tomato spotted wilt virus (TSWV), a
persistent/propagative virus, results in significant changes to their interactions
with the plant host. Specifically, our data reveal that viruliferous males make
more feeding and exploratory probes, and spend more time ingesting
individual cells than non-viruliferous males over an eight hour period.
Conversely no change in behavior was observed for females. Furthermore,
due to the changes in male feeding behavior, viruliferous male feeding is more
similar to females than to non-viruliferous males. While several studies show
that infected plants alter vector feeding preference and survival, this is the first
study that we are aware of to report a change in the feeding behavior of a plant
virus vector due to viral infection of the vector. The implications of these
finding to transmission of TSWV will be discussed.
A unique microbe-microbe and host-specific rhizosphere interaction that
is detrimental to plant health
M. STANGHELLINI (1), I. Misaghi (1)
(1) University of California, Riverside, CA, U.S.A.
Phytopathology 100:S122
Monosporascus cannonballus, a host-specific root-infecting ascomycete, is the
causal agent of a destructive disease of melons known as vine-decline. Ascospores, which function as the sole survival propagules and primary inoculum
for this soilborne fungus, germinate only in the rhizosphere of melons growing
in field soil. However, no ascospore germination occurs in the rhizosphere of
melons if the field soil is heated to temperatures greater that 50°C prior to
infestation with ascospores. This observation indicates (i) that melon root
exudates alone are not the stimulant and (ii) suggests that germination is
mediated by one or more heat-sensitive members of the soil microflora. Although bacteria or actinomycetes were heretofore suspected as the germinationinducing microbe(s), our recent data demonstrate that the culprit is an obligate,
holocarpic, root-infecting zoosporic fungus known as Olpidium bornovanus.
Ascospore germination in sterile field soil occurred only in the rhizosphere of
melon roots that were colonized by a host-specific melon strain of O.
bornovanus.
Characterization of Sclerotinia sclerotiorum in common bean white mold
resistance screening locations across the U.S.A.
J. STEADMAN (1), S. McCoy (1), B. Higgins (1), L. K. Otto-Hanson (2)
(1) University of Nebraska, Lincoln, NE, U.S.A.; (2) University of Minnesota,
St. Paul, MN, U.S.A.
Phytopathology 100:S122
There are currently no sources of complete resistance to Sclerotinia
sclerotiorum in common bean and other hosts. Repeatability of resistance
expression in bean lines with putative sources of white mold resistance is a
constraint in multi-site screening nurseries. Pathogen variability in these
nurseries is unknown. To assess pathogen variation, isolates were collected
from white mold field screening nurseries in eight states and 2 countries over
4 years and analyzed using mycelial compatibility tests for genotype and
straw test for measuring aggressiveness. From 146 screening nursery isolates
and 9 greenhouse screening isolates, 64 mycelial compatibility groupings
(MCGs) were identified. An additional 84 isolates collected from bean grower
fields were tested against isolates in the previous 64 MCGs and an additional
22 MCGs were found, for a total of 86 MCGs from 240 isolates. Isolates
within an MCG did not differ significantly in aggressiveness, however,
isolates in different MCGs were significantly different in aggressiveness. Four
microsatellite primers were used to characterize the 240 isolates and they
formed 67 haplotypes defined by the number of alleles at each locus. The
molecular variance (AMOVA) results confirmed the earlier findings from the
MCGs that there is more variation within populations (76%) than between
populations (24%). Of the 86 MCGs, 68 have one allelic haplotype and of the
remaining 17, 10 differed by one repeat at a locus that was found to have 20
different alleles.
Incorporation of edible brassicacious greens did not control nematode
populations
K. STEDDOM (1), K. Ong (2), J. Starr (3)
(1) Texas AgriLife Extension Service, Overton, TX, U.S.A.; (2) Texas
AgriLife Extension Service, College Station, TX, U.S.A.; (3) Texas A&M
University, College Station, TX, U.S.A.
Phytopathology 100:S123
Nematodes cause significant damage on a wide range of vegetable crops
including many grown in home gardens. Many studies have shown that
incorporation of brassicacious green manures can reduce populations of
nematodes, fungi, and other soil-borne pests. The purpose of this study was to
determine if edible greens commonly grown by home gardeners could also
function as effective green manure crops. Seven different edible greens crops
were planted in the fall of 2007 and incorporated in the spring of 2008 and
pumpkins were seeded four weeks later. Plots with Dwarf Blue Kale had
higher total populations of root knot, ring, and stunt nematodes prior to
incorporation of the crop. This was not correlated with populations from the
previous fall suggesting an increase on either the kale or weeds present due to
poor stand establishment. At the end of the trial, populations of root knot,
ring, and stunt nematodes were not significantly different between any
treatments. The total number of small pumpkins was greater with Vates
collards or dwarf blue kale than dwarf Siberian improved kale, purple top
turnip, or florida broadleaf mustard. The numbers of medium and large
pumpkins or the total number of pumpkins per plot were not significantly
different between treatments. An outbreak of southern blight, another soilborn pest, was widespread and not effected by treatment. Overall,
incorporation of edible greens were not effective at managing populations of
nematodes or S. rolfsii.
Investigations on population and hosts of Bean pod mottle virus in
Mississippi
R. C. STEPHENSON (1), S. Sabanadzovic (1)
(1) Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, MS, U.S.A.
Phytopathology 100:S123
Bean pod mottle virus (BPMV) is a viral pathogen found in virtually all
soybean fields in Mississippi during recent surveys. A study was undertaken
to better understand the local population of this virus and to investigate
sources of primary inoculum. Investigation of the local BPMV population was
carried out by sequencing and comparison of four genomic regions of
numerous isolates collected from research and production fields throughout
Mississippi. All studied isolates showed great molecular uniformity with the
exception of two, which appeared distinct in the RNA-dependent RNA
polymerase region. Sequencing of the complete RNA-1 segments for these
two isolates is currently on-going. In a study on sources of early infections,
several plant species were identified as new hosts with possible roles in the
epidemiology of this virus in the Southeastern United States.
A modified method to screen for partial resistance to Phytophthora sojae
in soybean
S. M. STEWART (1), A. E. Robertson (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S123
Phytophthora sojae, an economically important pathogen of soybean, has
been managed mainly through the use varieties with single gene (Rps)
resistance. The pathogen, however, has the ability to rapidly adapt and
overcome this type of resistance, resulting in populations that are able to
infect soybean plants with the deployed Rps genes. Partial resistance (PR) is
effective against all physiological races of the pathogen. Few cultivars with
high levels of PR are currently available because feasible methods that enable
effective incorporation of PR into desired germplasm are lacking. The layer
test is presently used to screen for PR in breeding programs. In this method,
soybean roots are forced to grow through a P. sojae-inoculated agar layer, and
assessment of PR is based on root rot evaluations that require experience and
can be subjective. The layer test was modified by replacing the agar layer with
P. sojae-inoculated rice. Dry root weight was used to screen for PR. Six
varieties of soybean with known levels of PR were evaluated using the layer
test and modified method. No differences between methods were observed for
PR, and PR ratings correlated well with corrected dry root weight (r = –0.965,
P < 0.001). The modified method has the advantage that handling agar plates
is not required, no training is needed for evaluations and more than one
pathotype can be easily used to screen for PR by mixing rice inoculum of
different isolates.
Using microsatellite markers to assess diversity of Phytophthora sojae in
Iowa
S. M. STEWART (1), A. E. Dorrance (2), A. E. Robertson (1)
(1) Iowa State University, Ames, IA, U.S.A.; (2) OARDC, Wooster, OH,
U.S.A.
Phytopathology 100:S123
Phytophthora root rot (PRR) caused by Phytophthora sojae can infect
soybeans at all growth stages, causing pre- and post-emergence damping-off,
root and stem rot. The most effective way to manage PRR is through the use
of resistant cultivars however, the pathogen continues to diversify making
resistant genes present in cultivars ineffective. Deployment of durable R-gene
mediated resistance depends on understanding the factors that contribute to
changes in the pathogen population diversity. Twenty four single sequence
repeat (SSR) loci were used to analyze the genetic diversity of sixty seven
mono-zoosporic isolates of P. sojae, collected from diseased plants and soil
throughout Iowa. Fifty percent of the loci assessed were polymorphic, and the
maximum number of alleles per locus was 2, with an average of 1.5. Isolates
were predominantly homozygous. The highest observed heterozygosity for an
individual locus was 0.045 with an average 0.005. Selfing was estimated in s
= 0.985, which agrees with previous reports of low levels of outcrossing due
to the homothallic nature of the pathogen. The diversity of the population of
P. sojae in Iowa will be compared to the diversity of P. sojae populations
from Ohio, Missouri and South Dakota.
Cultivar selection for sugar beet root rot resistance
C. A. STRAUSBAUGH (1), I. A. Eujayl (1), P. Foote (2)
(1) USDA ARS NWISRL, Kimberly, ID, U.S.A.; (2) Amalgamated Sugar
Company, Paul, ID, U.S.A.
Phytopathology 100:S123
Fungal and bacterial root rots in sugar beet caused by Rhizoctonia solani (Rs)
and Leuconostoc mesenteroides subsp. dextranicum (Lm) can lead to root
yield losses greater than 50%. To reduce the impact of these root rots on
sucrose loss in the field, storage, and factories, studies were conducted to
establish a faster and more accurate screening method. In 2009, 22
commercial cultivars were grown in a commercial field and mechanically
harvested, and then inoculated. In each root a cork borer hole at the widest
portion of the root was inoculated with Rs and another with Lm, while a third
hole was inoculated with both (RsLm). The roots were then incubated in the
greenhouse for 3 weeks out of direct sunlight, cross sectioned, and evaluated
for rot. The study was repeated with roots that had been stored for 60 days.
All roots suffered some rot with the Rs or the RsLm inoculations and the most
susceptible cultivar had 3.9 and 2.8 times more rot than the most resistant
cultivar, respectively. Only 15% of the roots developed rot with the Lm
inoculation. Similar rot results for all three inoculations were obtained with
stored roots. With the RsLm inoculation, cultivar ranking at harvest and after
storage were correlated (r = 0.6608, P = 0.0008). The RsLm inoculation may
prove to be a faster and more precise method to screen for bacterial rot
resistance but screening for fungal rot resistance will likely need to be done
using other methods.
Synergistic effects between Regalia® and other fungicides in controlling
cucumber powdery mildew and lettuce downy mildew
H. SU (1)
(1) Marrone Bio Innovations, Inc., Davis, CA, U.S.A.
Phytopathology 100:S123
Regalia® is a biofungicide formulated from extract of giant knotweed
(Reynoutria sachalinensis) and effectively controls common fungal and
bacterial diseases. Combination (tank mix) of Regalia® and other commercial
fungicides can be an effective and efficient measure in increasing pathogen
control efficacy and managing fungicide resistance in pathogens. Greenhouse
Vol. 100, No. 6 (Supplement), 2010
S123
and growth chamber experiments were conducted to evaluate the efficacy of
Regalia® in combination with commonly used chemical and biological
fungicides in controlling cucumber powdery mildew (Sphaerotheca fuliginea)
and lettuce downy mildew (Bremia lactucae). The results show a statistically
significant synergistic effect with Regalia® in a tank mix with azoxystrobin
(Quadris®), myclobutanil (Rally® 40W), quinoxyfen (Quintec®), or
triflumizole (Procure®) in controlling powdery mildew on cucumber in
repeated tests. Only an additive effect with no synergy was found with tank
mixes of Regalia® with Bacillus subtilis (Serenade®), or Bacillus pumilus
(Sonata®), cyprodinil (Vangard®), or kresoxim-methyl (Sovran®) in nonrepeated tests. Synergistic effect was also found when Regalia® was applied in
combination with acibenzolar-S-methyl (Actigard®) to control lettuce downy
mildew. The results clearly show that Regalia® can be effectively applied with
other fungicides either in a tank mix or a rotation to increase efficacy and/or
reduce the risk of fungicide resistance.
Regulation of genes involved in the interaction of tomato, Trichoderma
hamatum 382 and Xanthomonas euvesicatoria
N. Subedi (1), F. BAYSAL-GUREL (1), H. Hoitink (1), M. Ivey (1), S. A.
Miller (1)
(1) Ohio State University, Wooster, OH, U.S.A.
Phytopathology 100:S124
Trichoderma hamatum 382 (T382), a bio-control fungus, suppresses diseases
in tomato by induction of systemic resistance. T382 applied as an amendment
of planting mix colonized tomato roots and significantly reduced bacterial leaf
spot (Xanthomonas euvesicatoria 110C) development without colonizing
above-ground plant parts. The expression of certain tomato genes potentially
involved in the interaction of tomato plants with T382 and X. euvesicatoria
110C was investigated using real time quantitative PCR. The genes for
extensin and expansin, both cell wall proteins, and osmotin, a member of the
PR-5 protein family, were not consistently activated in tomato leaves by T382
before X. euvesicatoria inoculation. After X. euvesicatoria inoculation,
extensin was up-regulated in plants colonized by T382. Expansin was initially
down-regulated in X. euvesicatoria-inoculated plants, regardless of T382
treatment; however, expression of the gene remained low over time only in X.
euvesicatoria inoculated, T382-colonized plants. This suggests that disease
suppression in tomato by T382 may involve priming plants to down-regulate
expansin gene expression upon pathogen attack. Osmotin was up-regulated
only in X. euvesicatoria-inoculated plants. T382 had no effect on expression
of this PR protein by tomato plants.
Development of a biological sensor for powdery mildew (Erysiphales)
infections via monitoring of the proboscis extension reflex in
honeybees
A. M. SUTHERLAND (1), R. M. Wingo (2), K. J. McCabe (3), W. D. Gubler
(1)
(1) Department of Plant Pathology, University of California, Davis, Davis,
CA, U.S.A.; (2) Chemical Diagnostics and Engineering, Los Alamos National
Laboratory, Los Alamos, NM, U.S.A.; (3) Bioscience Dision B, Los Alamos
National Laboratory, Los Alamos, NM, U.S.A.
Phytopathology 100:S124
Organisms with superior sensory abilities can be developed into biological
sensors, able to detect and respond to stimuli of human interest. Such sensors
could be employed to provide early detection of important agricultural plant
pathogens, reducing fungicide application volume and frequency, and
therefore alleviating the economic and environmental costs involved therein.
Through classical conditioning, domestic honeybees, Apis mellifera, were
trained to associate olfactory cues from grape, Vitis vinifera “Carignane”,
leaves infected with powdery mildew (PM), Erysiphe necator, with a sucrose
reward, as evident through the proboscis extension reflex (PER), an
observable and unambiguous extension of the insect’s proboscis. This
conditioned response, however, was also elicited when bees were later
presented with uninfected grape leaves. In order to reduce the incidence of
such false positive responses, uninfected grape leaf material was added to the
conditioning process as a continuous olfactory stimulus, serving as a constant
background for the target stimulus: PM-infected leaf material. This new
method has resulted in negligible levels of false positive responses while
retaining acceptable levels of true positive responses. This work represents an
important preliminary investigation into the feasibility of using PER
observation as a biological sensor and indicator for early-season PM
infections in commercial vineyards.
A comparison of soil and corn kernel Aspergillus flavus populations:
Evidence for niche specialization
R. R. SWEANY (1), K. E. Damann (1)
(1) Louisiana State University Ag Center, Baton Rouge, LA, U.S.A.
Phytopathology 100:S124
S124
PHYTOPATHOLOGY
Aspergillus flavus is a fungal, opportunistic, soil-borne pathogen of corn
which may produce carcinogenic, acutely-toxic aflatoxins. The purpose of this
study was to determine if there are two ecotypes of A. flavus: facultative
parasites and saprophytes. Mature corn ears and soil samples were collected
from eleven Louisiana fields in August, 2007. A. flavus was isolated from
kernels (612 isolates) and soil (255 isolates). Sixteen vegetative compatibility
groups (VCGs) were identified from soil isolates and six of these VCGs were
found in corn isolates. Eighty-eight percent of corn isolates belonged to 2
VCGs (found in all fields) whereas only 5% of soil isolates were in the same 2
VCGs (found in 3 fields). Haplotypes were generated for a random subsample
of 99 corn and 91 soil isolates from polymorphisms at 8 simple sequence
repeat (SSR) loci. SSR fingerprints revealed 102 different haplotypes with 26
from corn isolates and 78 from soil isolates. One haplotype was shared
between corn and soil isolates. Both VCG assemblages and SSR fingerprints
differed significantly between the soil and the corn kernel populations.
Differences between the populations indicate not all soil isolates are effective
corn pathogens whereas some isolates have become effective at occupying the
corn niche. Understanding the pathogenicity of A. flavus is important for
developing atoxigenic biological control against toxigenic A. flavus and for
resistance screening.
Forward and reverse genetic approaches for functional analyses of
Clavibacter michiganensis subsp. sepedonicus
R. L. SYVERSON (1), C. A. Ishimaru (1)
(1) University of Minnesota, St. Paul, MN, U.S.A.
Phytopathology 100:S124
The Gram-positive plant pathogen Clavibacter michiganensis subsp.
sepedonicus (Cms) causes bacterial ring rot of potato. The complete genome
sequence of Cms (ATCC33113) became available in 2008, making broadscale identification of genes affecting pathogenesis possible. To fully
capitalize on the genome sequence, forward and reverse genetic approaches
for functional analysis of Cms were developed. In each case, the sequenced
strain of Cms was transformed via electroporation under optimized conditions.
For site-directed mutagenesis, full-length chp7, a known pathogenicity gene in
Cms, was cloned into pGEM®-T Easy. A chloramphenicol resistance marker
originating from Corynebacterium striatum was inserted at a SmaI restriction
site in chp7. The resulting construct was used to transform Cms by
homologous recombination. Mutations were confirmed by PCR, sequence
analysis, and plant assays to validate the utility of the approach. This same
strategy was applied to other annotated virulence genes. For reverse genetics,
Cms was transformed via transposon mutagenesis using Ez-Tn5, which
inserts randomly into Gram-negative and Gram-positive bacteria. In
contrast, Tn1409C, used previously for mutagenesis of Cms, inserts nonrandomly into low GC regions. About 5000 Ez-Tn5 mutants of Cms were
generated. Further library characterization will be presented. Site-directed and
transposon mutagenesis can be adapted for future high-throughput functional
studies.
Detection and distribution of Longidoridae and Trichodoridae nematodes
from Golestan National park of Iran
A. TALEZARI (1), F. Khozeini (2), S. Barouti (3), H. Zamanizadeh (1)
(1) Dept. of Plant Pathology, Science and Research Branch, Islamic Azad
University, Tehran, IRAN; (2) Iran Plant Protection Organization, Tehran,
IRAN; (3) Laboratory Complex, Science and Research Branch, Islamic Azad
University, Tehran, IRAN
Phytopathology 100:S124
Nematodes belonging to the families Longidoridae and Trichodoridae are
always able to transmit plant viruses and for that they are of most important
important plant parasitic nematodes. During the years 2008 to 2010, a total
number of 50 soil and root samples were collected from Golestan National
park located in North of Iran. The nematodes were extracted from roots by
Coolen and De’ Herde, 1972 and from soil by Jenkins, 1964 methodes. They
were fixed and transfered to glycerin. The permanent slides were prepared
from specimens, morphological and morphometric characters were studied by
light microscope. In this study 6 species belonging to 3 genera of
Longidoridae and Trichodoridae families were identified which reported for
the first time from Golestan National park. The identified nematodes are as
follow: Longidorus iranicus collected from Raspberry shrub and Nettle and
Walnut tree; Xiphinema americanum from Oak tree ; Xiphinema
diversicaudatum and X. index from Fig trees; Xiphinema pachtaicum from
Common alder, Fig, Persimmon, Walnut, and Wild plum trees and
Trichodorus primitivus from Maple, Nettle, Oak, Persimmon trees and
Raspberry shrub. Results showed that three species Longidorus iranicus,
Xiphinema pachtaicum and Trichodorus primitivus are dominat species with
high population density through visited regions. According to our knowledge
this the first report of these nematodes from Golestan National park of Iran
with 92000 hectare wide.
Description of two genotypes of Phytomonas associated to oil palm
diseases in Peru: Marchites Sorpresiva and a new disease manifestationMarchites Lenta
M. J. TALLEDO ALBUJAR (1), S. S. Morales Ruiz (2), E. Trinidad Chipana
(3), J. Arevalo Zelada (2), A. Trelles Di Lucca (3), Y. Montoya Piedra (1)
(1) Bio Links S.A., Lima, PERU; (2) Institute of Tropical Medicine
“Alexander von Humboldt” Universidad Peruana Cayetano Heredia, Lima,
PERU; (3) Palmas del Espino SA, Uchiza, PERU
Phytopathology 100:S125
Demand of oil palm products is worldwide increasing. In Peru the oil palm
plantations are expanding, however, palm diseases represent a risk to
plantations productivity. Among them, Marchites Sorpresiva (MS) was
detected in Palmas del Espino plantation since 1983. Like in other LatinAmerican regions, Phytomonas were associated to MS. Furthermore, a second
clinical manifestation appeared in 2002, which was also a vascular disease.
Nevertheless, the former was characterized by a very fast process (palms died
in 2 to 3 weeks) compared with the second one (palms died between 16 and
32 weeks). This fact leaded to name the latter Marchites Lenta (slow
progression). Interestingly, palms affected by M. Lenta presented scarce
number of Phytomonas microorganisms compared with MS. To determine if
these two different disease manifestations are due to Phytomonas or
correspond to two different Phytomonas, polymorphic DNA sequences were
analyzed. DNA was extracted from root saps containing Phytomonas from
MS (n = 22) and M. Lenta (n = 131) diseases. Specific PCR analysis of
kinetoplast DNA (1000 bp and 800 bp for MS and M. Lenta oil palms,
respectively) and miniexon PCR sequencing (≈100 bp for both diseases, 1
transition and 1 deletion nucleotide changes was observed in MS)
demonstrated that differences were directly correlated to each disease
manifestation. These findings support the hypothesis that we would be
dealing with two different diseases, associated to two discrete Phytomonas
groups.
High-Fidelity PCR as a sensitive molecular diagnostic tool to detect
Phytophthora nicotianae on spathiphyllum
T. L. Tarnowski (1), A. J. PALMATEER (1)
(1) University of Florida, Homestead, FL, U.S.A.
Phytopathology 100:S125
To compare the sensitivity of High-Fidelity (Hi-Fi) and standard PCR in
detecting Phytophthora nicotianae in symptomatic spathiphyllum
(Spathiphyllum wallisii) plants, four DNA extraction methods were tested in
conjunction with a standard and two Hi-Fi PCR protocols. The DNA
extraction methods were: 1) Extract-N-Amp Plant Kit (Sigma-Aldrich), 2)
DNeasy Plant Mini Kit (Qiagen), 3) CTAB buffer, and 4) lithium chloride
Shorty buffer. Symptomatic leaf, petiole and root tissue from four plants were
submitted to each extraction method. DNA samples were then used for each
PCR protocol using P. nicotianae -specific primers: 1) Standard PCR, 2) HiFi PCR using LongAmp enzyme, and 3) Hi-Fi PCR Taq+Accuzyme. DNA
quantification using spectrophotometry indicated Extract-N-Amp and
Shorty methods yielded the highest DNA amounts with lower purity. Both
Hi-Fi PCR protocols were more sensitive than standard PCR. The
Accuzyme protocol detected the pathogen in all samples using the DNeasy
and Extract-n-Amp methods, whereas the standard protocol detected the
pathogen only in leaf samples using the DNeasy kit. This study demonstrates that Hi-Fi PCR provides a highly sensitive tool for molecular
diagnostics in planta, and that the DNA extraction method influences PCR
sensitivity.
A new Bipolaris leaf spot of cordyline and disease response on five
cordyline varieties
T. L. Tarnowski (1), A. J. PALMATEER (1)
(1) University of Florida, Homestead, FL, U.S.A.
Phytopathology 100:S125
A new leaf spot disease of cordyline (Cordyline spp.) was submitted to the
Extension Plant Diagnostic Clinic in Homestead, FL. Lesions began as small
pinpoint chlorotic flecks and enlarged longitudinally up to 1 cm. Lesions
became necrotic with red margins and areas of dark sporulation in margin
centers. A Bipolaris sp. was consistently isolated from lesions and confirmed
as the causal agent. The species produces velvety and dark, blackish brown
colonies. Conidiophores were pale golden brown, straight to flexuous, 130285 µm × 5-9 µm. Conidia were pale brown pale to medium golden brown,
smooth and clavate with a protuberant hilum, 43-86 × 11-18 m, 3-11
distoseptate. The susceptibility of five cordyline varieties, Cordyline fruticosa
var. ‘Chocolate Queen’ and ‘Auntie Lou’, and Cordyline australis var. ‘Dark
Star’, ‘Redstar’ and ‘Sundance’, was determined. Each variety was inoculated
with a conidial suspension in a randomized complete block design and
incubated at 27°C for 7 days. The number of lesions per leaf and total per
plant were recorded. An analysis of variance indicated a significant variety
effect (P < 0.05) and ‘Chocolate Queen’ had greater number of lesions than
the other varieties (41 lesions compared to 10–16 lesions per plant). This is
the first study that attempts to characterize the etiology and disease response
of a new Bipolaris leaf spot affecting cordyline.
GFP is efficiently expressed by Wheat streak mosaic virus using a range
of Tritimovirus NIa cleavage sites and forms dense aggregates in cereal
hosts
S. TATINENI (1), R. French (1)
(1) USDA, ARS, University of Nebraska, Lincoln, NE, U.S.A.
Phytopathology 100:S125
Wheat streak mosaic virus (WSMV)-based transient expression vector was
developed to express GFP as a marker protein. The GFP cistron was
engineered between the P1 and HC-Pro cistrons in an infectious cDNA clone
of WSMV. The cleavage sites, P3/6KI, 6KI/CI, NIa/NIb, or NIb/CP, from
WSMV were fused to the C-terminus of GFP such that free GFP will be
released after proteolytic processing of viral polyprotein. WSMV-GFP
constructs infected wheat similar to that of the wild-type virus and expressed
GFP mostly as aggregated structures even though proteolytically processed
free GFP was detected by immuno-blots. GFP was similarly expressed as
aggregates with heterologous cleavage sites from Brome streak mosaic virus
or by mutating the -1 amino acid (aa) position of WSMV cleavage sites to
either alanine or arginine. Binary vectors with GFP cistron containing aa that
would result from cleavage sites (GFP-CS) or co-infiltration of GFP-CS with
WSMV NIa cistron failed to form such aggregates in Agrobacteriuminfiltrated Nicotiana benthamiana leaves, suggesting that neither aa that result
from cleavage nor possible interactions between the NIa-pro and GFP-CS are
involved in the formation of aggregated GFP. However, GFP was expressed
mostly as free protein from WSMV-GFP with Foot-and-mouth disease virus
2A peptide (33 aa) at the C-terminus of GFP cistron. WSMV-GFP vectors
were relatively stable in wheat plants and expressed GFP beyond five serial
passages at 14-day intervals.
Infection of rice by Ustilaginoidea virens
D. TE BEEST (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S125
False smut, caused by Ustilaginoidea virens, has emerged as an important
disease of rice in Arkansas since it was found in a single field in 1998. Little
is known about the disease cycle of false smut. Experiments were conducted
in the laboratory, field and greenhouse to examine infection of rice by U.
virens. Our histological results show that rice is infected by spores
germinating on root surfaces. Amplification of fungal rDNA isolated
from rice tissue samples shows that infection of roots leads to the
asymptomatic colonization of the entire plant including panicles and seeds.
Germinating rice seeds in pasteurized soils infested with spores in
concentrations ranging from 0 to 250,000 spores/gm resulted in seedling
infections ranging from 0% to 100% infection. Field and greenhouse
experiments showed that selected fungicides protected seedlings from
infection and subsequent colonization of plants. Taken together, these findings
clarify the disease cycle, disease development and epidemiology of false smut
in Arkansas and may suggest new approaches to the control of U. virens on
rice world-wide.
Baseline sensitivity of Ascochyta rabiei to penthiopryad, a new SDHI
fungicide
N. H. Thaher (1), B. D. GOSSEN (2), M. McDonald (1)
(1) University of Guelph, Guelph, ON, CANADA; (2) Agriculture & AgriFood Canada, Saskatoon, SK, CANADA
Phytopathology 100:S125
Ascochyta blight, caused by Ascochyta rabiei (Pass.) Labr., is a destructive
disease of chickpea (Cicer arietinum L.). Repeated application of fungicide is
required almost every year for blight management, and insensitivity to
strobilurin fungicides as become widespread in North America since 2006.
The sensitivity of A. rabiei to penthiopryad, an efficacious new succinate
dehydrogenase inhibitor (SDHI) fungicide not yet in commercial use, was
assessed using 50 isolates collected in 2008 in Saskatchewan, Canada. The
effective concentration to inhibit mycelium growth by 50% (EC50) was
estimated for each isolate using a radial growth assay on PDA amended with
technical grade penthiopryad at 0.01, 0.1, and 1 µg/mL with three replicates
per treatment. EC50 values ranged from 0.002 to 0.30 µg/mL with a mean of
0.10 µg/mL. A discriminatory dose of 0.3 µg/mL was selected for assessment
of 32 additional isolates collected in 2008 and 47 isolates collected in 2009
(never exposed to penthiopryad); 12 of the 79 isolates exhibited < 50%
growth inhibition. This study will provide the basis for monitoring sensitivity
in A. rabiei populations to this new fungicide. Also, cross-resistance between
penthiopyrad and boscalid (a SDHI used for blight management on chickpea)
is being assessed.
Vol. 100, No. 6 (Supplement), 2010
S125
Sensitivity of Didymella bryoniae to DMI and carboxamide fungicides
A. THOMAS (1), K. L. Stevenson (1), D. B. Langston (1)
(1) University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S126
Didymella bryoniae, the causal agent of gummy stem blight (GSB) of
watermelon, has a history of developing resistance to fungicides, most
recently the carboxamide fungicide boscalid. To facilitate fungicide resistance
monitoring, baseline sensitivity distributions were established for DMI
fungicides tebuconazole and difenoconazole and the carboxamide fungicide
penthiopyrad that were recently introduced or are being evaluated for GSB
control. In all, 77 isolates with no prior exposure to carboxamides or DMIs
were tested using a mycelial growth assay to determine the effective
concentration at which mycelial growth was inhibited by 50% (EC50). EC50
values for boscalid, penthiopyrad, tebuconazole and difenoconazole ranged
from 0.007 to 0.127, 0.009 to 0.189, 0.073 to 0.388 and 0.012 to 0.135 µg/ml
with median values of 0.037, 0.030, 0.135 and 0.046 µg/ml. There was a
significant positive correlation between the sensitivity to penthiopyrad and
boscalid (P < 0.0001, r = 0.63), indicating a significant potential for crossresistance between these compounds. In 2009, 104 isolates collected from
fungicide-treated watermelon fields were tested for resistance to boscalid and
penthiopyrad using a discriminatory concentration of 3.0 µg/ml. Of the
isolates tested, 86 were resistant and 12 were sensitive to both fungicides.
Because of the significant potential for cross-resistance, growers will be
advised not to use boscalid and penthiopyrad in the same fungicide spray
program.
IR-4 Project fungicide registration update
D. C. THOMPSON (1), J. Corley (1), W. Barney (1), D. Carpenter (1)
(1) Rutgers University, Princeton, NJ, U.S.A.
Phytopathology 100:S126
In 2009, the IR-4 Project obtained new uses of 29 chemicals on many
specialty crops with a total of 917 new chemical uses being registered. New
fungicide registrations on food crops included uses of cyazofamid,
dimethomorph, fenamidone, famoxadone, propiconazole, tebuconazole, and
triflumizole. Residue studies of kasugamycin on pear, apple, tomato, pepper,
and walnut were completed and provided to the registrant for submission to
EPA. Downy mildew of basil was identified as a problem in 2008 and
residues studies for cyazofamid and mandipropamid initiated in 2009 and
fluopicolide in 2010. A mandipropamid residue study was initiated for control
of Phytophthora capsici in snap beans. IR-4 is exploring use of quarternary
ammonium products directly on crops vs present use on non-porous surfaces
only. The chlorothalonil risk cup was expanded and residue studies on citrus,
guava and lychee were initiated in 2009 followed by almonds, mustard greens
and radish in 2010. IR-4 initiated residue studies with metrafenone, a new
powdery mildew fungicide from BASF. IR-4 petitioned EPA for registrations
on new berry and onion subgroups adding many new minor crops. Fruiting
vegetable subgroups will be established in 2010 and will include okra. Citrus,
pome and stone fruit crop groups will also be expanded in 2010. An
international residue program with mandipropamid and difenoconazole on
tomato is underway to help determine if data from around the world can be
used to establish national residue tolerances.
Mustard cover crop for management
(Phytophthora capsici) in cucurbit fields
S. B. THRU PPOYIL (1), M. Babadoost (1)
(1) University of Illinois, Urbana, IL, U.S.A.
Phytopathology 100:S126
of
Phytophthora
blight
This study was conducted to determine the effectiveness of two mustard
species, Brassica alba, ‘Tilney’ (T), and Brassica juncea, ‘Florida Broadleaf’
(FB), as short-cycle cover crops, for managing Phytophthora blight
(Phytophthora capsici) of cucurbits. Experiments were conducted during
2008–2009 in a P. capsici-infested field with a history of Phytophthora blight.
Mustards were grown in the field for 45 days and were incorporated into the
top 10-cm layer of the soil. The seeds of cucumber (‘Eureka’), jack-o-lantern
pumpkin (‘Magic Lantern’), and processing pumpkin (‘Dickinson’) were
sown after the incorporation of mustard into the soil. Average density of P.
capsici oospores per gram of dry soil was 2.67 prior to incorporation of
mustard plants. The density of oospores was 1, 1.66, 2.33, and 0 in control, T,
FB, and T+FB plots, respectively, 14 days after the incorporation of mustard
plants into the soil. There was no vine infection in cucumber plots. Incidence
of vine infection on jack-o-lantern pumpkin plots was 52.92, 43.13, 32.70, and
51.04%; and on processing pumpkin the incidence was 45.00, 52.71, 48.33,
and 47.29% in control, T, FB, and T+FB plots, respectively. Incidence of
Phytophthora fruit rot on cucumber was 26.91, 20.83, 12.93 and 13.44%; on
jack-o-lantern pumpkin, it was 24.43, 31.63, 22.75, and 28.80%; and on
processing pumpkin, the incidence was 26.20, 18.80, 22.69, and 45.37% in the
control, T, FB, and T+FB plots, respectively.
S126
PHYTOPATHOLOGY
The effect of new aphid vectors on the evolution of Soybean dwarf virus
B. TIAN (1), W. L. Schneider (2), F. E. Gildow (1)
(1) Department of Plant Pathology, Pennsylvania State University, State
College, PA, U.S.A.; (2) Agricultural Research Service, United States
Department of Agriculture, Fort Detrick, MD, U.S.A.
Phytopathology 100:S126
Soybean Dwarf Virus (SbDV) commonly occurs in clover in North America;
and prior to 2000 was not known to cause disease in soybean, presumably due
to the lack of aphid vector species capable of colonizing soybean in N.
America. Due to the introduction of the soybean aphid, Aphis glycines, into N.
America, there is a critical need for evaluating the risk of aphid transmission
and SbDV outbreaks in U.S. soybean fields. To determine the probability of a
SbDV clover strain being selected for both efficient A. glycines transmission
and adaptation to soybean, we passaged SbDV on soybeans using A. glycines
and Nearctaphis bakeri, an effective vector of SbDV. SbDV was transmitted
first from clover to soybean by N. Bakeri, and then serially passaged on
soybean by either N. bakeri or A. glycines. Transmission efficiency for N.
bakeri decreased from 33% to 20% over 6 serial transmissions on soybeans,
and then transmission ceased. Transmission efficiency by A. glycines varied
from 6–20%, ceasing after one or two transmissions. Although
transmissibility was lost, real time RT-PCR indicated that virus titers
increased in soybeans with each sequential transmission by both vectors.
Sequence analysis following the last passages of SbDV in soybeans identified
5 non-synonymous mutations in the non-transmissible isolates compared with
A. glycines and N. bakeri transmissible isolates. These mutations are likely
related to SbDV adaptation to soybeans and the loss of aphid transmissibility.
Viruses infecting soybean (Glycine max L. Merill) in Nigeria
I. TIME (1), G. I. Atiri (2), P. Lava Kumar (3)
(1) International Institute of Agriculture and University of Ibadan, Ibadan,
NIGERIA; (2) Department of Crop Protection and Environmental Biology,
University of Ibadan, Ibadan, NIGERIA; (3) International Institute of Tropical
Agriculture (IITA), Ibadan, NIGERIA
Phytopathology 100:S126
Nigeria is the largest producer of soybean in Africa with over 600,000 ha
devoted to the crop production. A survey for soybean virus diseases was
conducted in all the major soybean producing regions in mid altitude, guinea
savanna and derived savanna agroecological zones of Nigeria. A total of 1017
soybean leaf samples were collected from 135 locations and they were
analyzed for Bean pod mottle virus (BPMV), Black eye cowpea mosaic virus
(BlCMV), Cowpea aphid-borne mosaic (CABMV), Cowpea mottle virus
(CoMV), Cowpea mosaic virus (CoMV), Cucumber mosaic virus (CMV),
Cowpea severe mosaic virus (CPSMV), Cowpea mild mottle virus (CPMMV),
Southern bean mosaic virus (SBMV), Tobacco streak virus (TSV) and
Soybean mosaic virus (SBMV) using enzyme-linked immunosorbent assay
(ELISA). Viruses were detected in 77% of the samples and all the eleven
viruses were detected in soybean. Viruses were detected in all the
agroecological zones and the frequency of infection was highest in derived
savanna (83%), followed by mid-altitude (78%) and guinea savanna (72%).
CPMMV was the most prevalent virus detected in 72% of the total samples.
SMV, BICMV, CMV and BPMV were detected in 20%, 21%, 7% and 2% of
the samples, respectively. Fifteen combinations of mixed virus infections were
detected. CPMMV was common in every combination and it was also
detected in 17% of the asymptomatic samples. CPSMV, detected in two
locations, was the first record of its occurrence in soybean.
Centipedegrass: A new host of Colletotrichum sublineola
M. TOMASO-PETERSON (1)
(1) Mississippi State University, Mississippi State, MS, U.S.A.
Phytopathology 100:S126
Centipedegrass (Eremochloa ophiuroides) is rapidly becoming the home lawn
turf of choice in Mississippi. It is a medium-textured, slow-growing warmseason turfgrass that requires low input, is well-adapted to a wide range of soil
conditions but grows best in sandy, acidic soils of low fertility and can
withstand some shade. Diseases arise when centipedegrass is over-managed
with fertility and irrigation inputs and low height of cut. In spring of 2007,
centipedegrass home lawns exhibited symptoms of leaf chlorosis and sheath
blight, and irregular, chlorotic patches associated with thinning turf. Hyphal
appressoria, diagnostic of Colletotrichum, were observed in leaf sheath tissue
of diseased plants. Pure cultures of the fungus on potato dextrose agar
produced grey mycelia surrounded by a bright yellow exudate; falcate conidia
and globose to irregular hyphal appressoria were abundant. Phylogenetic
analysis using Apn2, Mat1, Sod2 and ITS sequence data identified the fungus
as a distinct lineage of C. sublineola, with centipedegrass isolates clustering
separately from isolates from sorghum and johnsongrass. Koch’s postulates
were performed in the growth chamber by inoculating seedlings with a spore
solution of C. sublineola. Chlorosis was observed 10 dpi; hyphal appressoria
were observed 21 dpi. The pathogen was successfully re-isolated from
diseased tissue. A new host of C. sublineola and a new disease of
centipedegrass have been identified.
Transcriptome analysis of a susceptible Glycine max-Phakopsora
pachyrhizi interaction using next generation sequencing
A. TREMBLAY (1), P. Hosseini (2), N. W. Alkharouf (2), S. Li (3), D. G.
Luster (4), B. F. Matthews (1)
(1) USDA ARS PSI, Beltsville, MD, U.S.A.; (2) Towson University, Towson,
MD, U.S.A.; (3) USDA ARS CGPRU, Stoneville, MS, U.S.A.; (4) USDA
ARS FDWSR, Frederick, MD, U.S.A.
Phytopathology 100:S127
Soybean is in the top five agricultural products in the United States. Soybean
rust (SR) is caused by an exotic obligate fungus. We want to analyze the
expression pattern of SR and its soybean host genes during the infection.
Thus, libraries were constructed from different soybean cells infected by SR at
different time-points and sequenced using a Solexa platform. Infection sites
were visualized by immunofluorescence and isolated by laser capture
microdissection. DNA sequences were aligned to the soybean genome and
homology searches were conducted to identify genes. From sequences without
similarity to soybean genome, contigs were formed and homology searches
were conducted. All time-points give us a limited number of sequences
aligning to the soybean genome (3,330 sequences/time-point). However, we
found much more sequences (9,000,000) without any homology to the
soybean genome. These are expected to be SR sequences. Contigs build from
those sequences had homology with genes involved in fungal development,
lignin degradation, signal transduction and intracellular communication (chitin
deacetylase, glyoxal oxidase, serine threonine protein phosphatase,
transthyretin). We also found contigs containing signal peptide which are
common to fungal virulence factor and others containing catalase and
peroxidase domain which are involved in defense. Target pathogen as well as
some relevant host genes will be studied to determine if they can be used to
control SR in soybean.
Sequence data of Xanthomonas strains isolated from U.S. rice fields
reveals substantial divergence from Xanthomonas oryzae pvs. oryzae and
oryzicola
L. R. TRIPLETT (1), J. P. Hamilton (2), N. A. Tisserat (1), C. R. Buell (2), J.
E. Leach (1)
(1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Michigan State
University, East Lansing, MI, U.S.A.
Phytopathology 100:S127
Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) are the
causal agents of bacterial leaf blight and bacterial leaf streak of rice,
respectively. Xoo causes significant losses in Asia, and is a select agent
organism subject to international quarantines. Two decades ago, Xanthomonas
spp. were isolated in the southern United States from rice plants displaying
water-soaked, chlorotic lesions. Serological studies suggested that these
isolates were Xoo, despite a weak virulence phenotype and distinct RFLP
profiles. In this study, short-read sequencing technology was used to sequence
the genomes of two Xanthomonas strains isolated from rice in Texas and
Louisiana. Sequence reads were assembled into contigs for comparative
genome analysis. Phylogenetic analysis based on hrp, gum, and rpf clusters
and housekeeping genes revealed a close relatedness among the U.S. strains,
but substantial genetic divergence between the U.S. strains and Asian strains
of Xoo and Xoc. Reciprocal Blast alignment of predicted coding sequences
supported this observation. The genome sequence revealed a lack of TAL
effectors and Xoo8-family transposons in the U.S. strains, as well as the
presence of novel putative gene clusters. These results support the hypothesis
that U.S. strains of Xanthomonas belong to a distinct subgroup or subspecies
of X. oryzae, likely introduced in a single event prior to the divergence of Xoo
and Xoc. Diagnostic primers were developed to distinguish the U.S. strains
from Xoo and Xoc.
Characterization of salicylate hydroxylase of “Candidatus Liberibacter
asiaticus” and its role in plant defense suppression
P. TRIVEDI (1), N. Wang (1)
(1) Citrus Research and Education Center, University of Florida/IFAS, Lake
Alfred, FL, U.S.A.
Phytopathology 100:S127
Citrus huanglongbing (HLB), associated with pathogen Candidatus
Liberibacter asiaticus (Las), is a devastating disease to the U.S. citrus
industry. It is intriguing to gain knowledge on the mechanism(s) by which Las
evades host defense responses. One orf encoding a homolog of salicylate
hydroxylase (sahA) is present in the genome of Las. Salicylate hydroxylase is
responsible for salicylic acid (SA) breakdown. SA is important for basal
defense, hypersensitive response, and systemic acquired resistance. We
postulate that salicylate hydroxylase of Las is involved in suppressing host
defenses against pathogen infection. To test this hypothesis, two set of
experiments were performed to determine (a) function analysis of sahA, and
(b) effect of Las infection on expression of defense related gene (PR-1) using
QRT-PCR. We first expressed sahA in Escherichia coli and found that
salicylate hydroxylase of Las is functional and can breakdown various
salicylate based substrates into catechol. To determine expression level of
defense related genes after Las infection, Xanthomonas axonopodis pv. citri
strain AW (Xac AW) was used to induce PR-1 gene expression. The PR-1 gene
expression in Xac Aw challenged plants which were inoculated previously
with Las was lower than Xac Aw challenged healthy plants. Our data suggest
that modulation of SA production and subsequent regulation of defense
related genes such as PR-1 gene could be one of the mechanisms deployed by
Las to evade plant defense responses.
Molecular diversity and population dynamics of Xanthomonas axonopodis
pv. manihotis in the Caribbean region of Colombia
C. A. TRUJILLO (1), S. Restrepo (1), C. E. López (2), J. Esquivel (3), A.
Bernal (1)
(1) Universidad de Los Andes, Bogota, COLOMBIA; (2) Universidad
Nacional de Colombia, Bogota, COLOMBIA; (3) Corporation for the
Sustainable and Participative Management of the Rural Small Growers in
Colombia, PBA, Bogota, COLOMBIA
Phytopathology 100:S127
Cassava bacterial blight is the most important bacterial disease in this crop.
Previous population studies of Xanthomonas axonopodis pv. manihotis (Xam),
the causal agent of this disease, showed a prominent diversity index, with
pathogen migration being an important determinant of diversity among
regions. Ten years later, aiming at establishing the current status of population
structure of Xam in this region, different agroecological zones were selected.
Bacterial samples were collected in five field trips from September of 2008 to
November of 2009. Bacterial haplotypes were determined by AFLPs and
clustering analysis established relationships among them, as well as their
distribution on the agroecological regions. Additionally, effector genes were
sequenced to determine their degree of variability and to assess the presence
and nature of selection exerted by the host. The results confirmed a prominent
diversity index and haplotype migration through the months in the Caribbean
region. This could be due to deficient cultural practices and/or differences in
the environmental conditions of the regions. On the other hand, a low
variability was detected on effector genes. This study shows the current
condition of populations of Xam in the Caribbean region of Colombia and it
contributes to improve the existing bacterial blight control practices.
The use of Xspecies microarray to study changes in gene expression in
phytoplasma-infected Catharanthus roseus
M. G. TUFFEN (1)
(1) University of Nottingham, Loughborough, UNITED KINGDOM
Phytopathology 100:S127
Phytoplasmas are specialised plant pathogenic bacteria that are responsible for
several economically important diseases of crops. They are spread by sap
sucking insects such as leafhoppers and within the plant are limited to the
phloem; they can not be cultured in vitro. Catharanthus roseus, or
Madagascar periwinkle, is used an experimental host for phytoplasmas as it is
susceptible to many phytoplasma strains and gives a good range of
representative symptoms. The Xspecies microarray technique allows a
microarray to be used on a species it was not designed for. In this study, gene
expression was compared between healthy and sweet potato little leaf
phytoplasma infected Catharanthus roseus, which shows symptoms of
dramatically reduced leaf size, chlorosis and increased branching. Sixty-six
differentially regulated genes were identified, including up regulation of auxin
related genes and a decrease in the expression of photosynthesis related genes.
A number of these identified genes have subsequently been isolated from C.
roseus and quantitative PCR has been used to confirm the expression results.
Investigations of crown gall in the commercial propagation of weeping fig
W. TURECHEK (1)
(1) USDA ARS SAA SPP, Fort Pierce, FL, U.S.A.
Phytopathology 100:S127
Agrobacterium larrymoorei causes galls on the trunk, branches, and stems of
weeping fig (Ficus benjamina L.). The extent to which this pathogen is
transmitted through cuttings and the extent to which it is spread through the
mother planting as a result of preparing air layers and subsequently pruning
them to produce braided plants were studied in a commercial nursery.
Branches selected for propagation were chosen from mother trees with no
visible signs of galls and tagged for future tracking. Rooted branches were cut
from the mother tree, braided with 2 to 4 other branches, planted in pots, and
then placed on ground cloth to establish. Gall formation was tracked on all
branches of each braided plant. Additional ratings were taken in the mother
tree planting 6 months after pruning. In the mother tree planting, there was
significant spatial correlation between mother trees infected before pruning
Vol. 100, No. 6 (Supplement), 2010
S127
and trees infected after pruning. There were also significant correlations
between infected mother trees and braided plants established with 1 or more
branches propagated from infected mother trees. There did not appear to be
any correlation between the time of year when plants were propagated and
gall formation. Although pruning shears are routinely soaked in sterilization
medium in commercial practice, the degree of sterilization achieved in this
nursery was not sufficient for reducing disease spread.
Supplementing nutrition with calcium and potassium silicate to control
Botrytis cinerea in poinsettia stock plants
L. TUROOP (1), J. E. Faust (2)
(1) Jomo Kenyatta University of Agric & Technology, Nairobi, KENYA; (2)
Clemson, SC, U.S.A.
Phytopathology 100:S128
Root application of silicon (Si) on poinsettia stock plants was evaluated for its
effects in reducing B. cineria and promoting cutting performance in storage
and propagation. Fertilization of two poinsettia varieties raised on soilless
media was supplemented with calcium or potassium silicates at the
concentrations of 125, 250 and 500 ppm. Control plants were drenched with
same amount of distilled water. leaf tissue were inoculated with a spore
suspension and the development of botrytis was monitored. Shelf life and
performance of cuttings in propagation was assessed. Calcium and potassium
silicate significantly reduced the severity and incidence of B. cineria on Si
treated plant tissue compared to the control. Applying 500 ppm Si reduced the
disease severity by up to 27.8% and disease incidence by 33.1%. The losses
associated with botrytis in storage were reduced by >30% in high Si fertilized
plants. Performance and incidence of botrytis in propagation was significantly
influenced by Si application. Although infection occurs on tissue treated with
high Si rate, lesion expansion was significantly reduced. The net effect of Si
on poinsettia stock plants is an overall reduction in disease development, by
eliciting a defense reaction as indicated by a significant increase in total tissue
phenolic content with increase in Si rate, thereby slowing the infection rate of
B cineria.
Influence of environmental conditions on the development of soybean
rust epidemics in soybean fields in Nigeria
M. TWIZEYIMANA (1), P. S. Ojiambo (2), R. Bandyopadhyay (3), G. L.
Hartman (1)
(1) University of Illinois, Urbana, IL, U.S.A.; (2) North Carolina State
University, Raleigh, NC, U.S.A.; (3) International Institute of Tropical
Agriculture (IITA), Ibadan, NIGERIA
Phytopathology 100:S128
Epidemics of soybean rust (Phakopsora pachyrhizi) and environmental factors
that favor rust development were studied under natural infection in Nigerian
soybean fields from 2004 to 2006 by sequentially planting early (TGx 14851D) and medium (TGx 1448-2E) maturing soybean cultivars at intervals
ranging from 1 to 1½ months. Disease onset and final disease severity varied
substantially among all planting times. Little or late infection was observed on
soybeans planted during the dry season (November to March), while disease
severity was high and rate of progress much faster on soybeans planted during
the wet season (April to October). Across years, disease severity was
consistently higher on soybeans planted in August. Infection and disease
development were significantly (P < 0.05) affected by environmental factors
with strong correlations between rust severity and evaporation (r = –0.726 and
–0.632) and wind speed (r = –0.708 and –0.634) for TGx 1485-1D and TGx
1448-2E, respectively and slight correlations between disease severity and
solar radiation, maximum and average temperatures for both cultivars.
Strong and slight correlations were observed between urediniospores
concentration (spores/m3 of air) and weather parameters. There was a
significant (P < 0.05) positive correlation (r = 0.700) between rust
severity and urediniospores concentration. Based on this study, soybean
growers in Nigeria need to modify their planting date slightly to reduce rust
epidemics.
Epidemiology of two Diodia virginiana criniviruses
I. E. TZANETAKIS (1), A. Cortez (2), J. Zhou (1), B. Poudel (1), L. Hladky
(2), R. Larsen (3), W. M. Wintermantel (2)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) USDA-ARS,
Salinas, CA, U.S.A.; (3) Vegetable and Forage Crops Research Laboratory,
USDA-ARS, Prosser, AR, U.S.A.
Phytopathology 100:S128
The genus Crinivirus, family Closteroviridae, includes several emerging
viruses that cause disease in crops around the world. Diodia virginiana,
family Rubiaceae, a widespread noxious weed of the southeastern United
States, is known to be infected by a crinivirus, Diodia vein chlorosis virus
(DVCD). During the original characterization of DVCV, herbaceous hosts
were tested as alternate hosts for the virus but none of the major crops of the
Southeast was included in that study. There are no detection tests available for
S128
PHYTOPATHOLOGY
DVCV and given the emergence of several criniviruses, we began developing
detection tests for the virus and tested the possibility that crops in the
Southeast could be infected with the virus. During the molecular
characterization process, we determined that there were two criniviruses
infecting Diodia, both transmitted by the greenhouse and banded winged
whiteflies (Trialeurodes vaporariorum and T. abutilonea, respectively). The
Diodia viruses belong to Crinivirus group 1, along with the small fruitinfecting criniviruses and Potato yellow vein virus. The similarity of the
diodia viruses with the small fruit-infecting criniviruses probed us to test the
possibility that the two diodia viruses can infect Rosaceae hosts, especially
blackberry, a crop affected by two criniviruses that can synergistically cause
blackberry yellow vein disease.
Protecting the endangered Eugenia koolauensis from further loss in the
Hawaiian Environment
J. UCHIDA (1), C. Y. Kadooka (1), R. Hauff (2), L. Loope (3)
(1) University of Hawaii, Honolulu, HI, U.S.A.; (2) State of Hawaii
Department of Land & Natural Resources, Division of Forestry & Wildlife,
Honolulu, HI, U.S.A.; (3) USGS Pacific Island Ecosystems Research Center,
Haleakala Field Station, Makawao, HI, U.S.A.
Phytopathology 100:S128
The endangered Eugenia koolauensis exists in Hawaii as only about 200
plants, scattered in small areas on two islands. The population has been at
high risk from recurrent fires, wild boars, and environmental degradation. But
with fencing and identification of the few remaining plants, conservation
biologists were hopeful that this plant could be saved. In 2005, a new invasive
rust, Puccinia psidii, the guava rust, was found attacking ohia (Metrosideros
polymorpha,) at a commercial nursery. A quick survey of the surrounding
forests, showed that it was on rose apple (Syzygium jambos,) and other trees,
as this rust attacks many in the Myrtaceae. It was also found on E.
koolauensis. Rose apple is an introduced species and is now widely distributed
on all major islands. Young leaves are severely blighted, turn black, and
defoliate. New leaves are then produced and the same cycle occurs. Soon the
reserves of the tree are used and many trees are now dying. Unfortunately, E.
koolauensis, suffers the same fate. Three fungicides were tested on ohia at a
commercial nursery and myclobutanil was identified as effective in protecting
young leaves. This product was taken to the field and tested on two plants
with good efficacy observed. Expanded efficacy and phytotoxicity testing will
be completed. Experimental studies for registration of this compound for use
in the forest, are being planned.
Biological control of Macrophomina phaseolina on sunflower in Pakistan
H. ULLAH (1)
(1) Dept. of Plant Pathology, University of Agriculture, Pakistan
Phytopathology 100:S128
In vitro sensitivity of Macrophomina phaseolina to potentially antagonistic
fungi was determined on potato dextrose agar. Aspergillus flavus was the most
effective reducing growth of M. phaseolina by 66%, followed by Aspergillus
niger (55.6%), Trichoderma viride (51.1%), Trichoderma harzianum (26.7%)
and Penicillium capsulatum (11.1%) respectively, Seeds of four sunflower
varieties (G-66, HRSB1, G-72, G-51_ were treated with cultures of A. flavus,
A. niger, T. viride and P. capsulatum and combinations.suppression by A.
niger, P. capsulatum and T. viride was 64.9, 63.8 and 31.89%, respectively.
The decrease in disease incidence compared with controls was 100% when
seed were treated with a combination of A. niger and A. flavus, whereas the A.
niger and T. viride combination reduced disease by 30.8%. Antagonist
combinations containing A. flavus and A. niger or (81.41%) A. flavus and P.
capsulatum reduced disease by 81.4 and 27.33%, respectively, compared with
controls.
Evaluation of varying sugar concentrations for growth of and aflatoxin
production by Aspergillus flavus
S. UPPALA (1), K. L. Bowen (1)
(1) Dept. Entomology and Plant Pathology, Auburn University, AL, U.S.A.
Phytopathology 100:S128
Aflatoxin contamination by Aspergillus spp. is one of the main factors
affecting peanut quality. Available carbon source is one of the nutritional
factors affecting aflatoxin production. Abundant aflatoxin production is
generally associated with substrates containing elevated levels of specific
carbohydrates. When peanuts are subjected to drought and temperature stress,
carbohydrate content of peanut seed is higher than in non-stressed seed.
Glucose, fructose and sucrose are generally accepted as the carbohydrates that
induce aflatoxin formation by A. flavus and these are primary components of
peanut seed. Our objective is to evaluate varying sugar concentrations, in the
amounts present in peanut seed, for their effect on the growth of and aflatoxin
production by A. flavus. We have grown A. flavus in media containing
different amounts of sugars for limited periods of time. Aflatoxins were
analyzed and fungal mycelial weights were determined. Mycelial weight
increased significantly with increases in sugar concentration. Other
parameters such as total aflatoxin and aflatoxin B1 are low at low sugar levels
and are slightly greater when sugar content is slightly higher as found in
drought-stressed compared to non-stressed peanut seed. Toxin per gram of
mycelium is usually high at low sugar levels.
and 1 of Pseudomonas sp. were also evaluated by their effect on plant growth
and reducing the amount of disease in planta. Their effect on germination was
not a clear one, but they increased the dry weight by 40%, and lowered the
AUDPC by about 70%. At present these 5 bacterias have been incorporated in
different schemes of evaluation with different pathogens.
Understanding the nonhost resistance mechanisms of Medicago
truncatula to Asian soybean rust and switchgrass rust
S. UPPALAPATI (1), Y. Ishiga (1), S. Mittal (1), H. Schultheiss (2), K. S.
Mysore (1)
(1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.; (2) BASF
Plant Science Company GmbH, Limburgerhof
Phytopathology 100:S129
Identification of seedling pathogens from soybean planted in field soils at
three temperatures
K. URREA (1), J. Rupe (1), C. Rothrock (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S129
Asian soybean rust (ASR) of soybeans caused by Phakopsora pachyrhizi is a
major concern and there is an urgent need for identification of durable
resistance to ASR. Switchgrass rust caused by Puccinia emaculata is a also
growing concern for bioenergy crop production. We found that Medicago
truncatula, a model legume, displays nonhost resistance to P. pachyrhizi and
P. emaculata. Initial characterization of the M. truncatula-P. pachyrhizi
incompatible interaction has shown that while the fungus forms long germtubes and directly penetrates M. truncatula epidermal cells resulting in small
necrotic lesions, it fails to sporulate on M. truncatula. Analysis of global
transcriptional changes during M. truncatula-P. pachyrhizi interactions
identified several up-regulated genes involved in phytoalexin biosynthesis and
PAMP-triggered defense responses during nonhost resistance. Interestingly,
the M. truncatula nonhost response to P. emaculata was not associated with
major transcriptional changes in phenylpropanoid pathway or pathogenesis
related genes. In addition, a reverse/forward Tnt1 insertional mutant screen
identified an enhanced penetration mutant that was more susceptible to P.
pachyrhizi, but not to P. emaculata. However, another resistant to rust (rer)
mutant showed resistance to both the nonhost pathogens. These results
suggested that M. truncatula possibly employs various nonhost resistance
mechanisms to different nonadapted rust fungi.
Etiology of branch dieback of olive trees (Olea europaea L.) in California
J. R. URBEZ-TORRES (1), F. Peduto (1), W. D. Gubler (1)
(1) University of California, Davis, Davis, CA, U.S.A.
Phytopathology 100:S129
Branch dieback of olive trees as consequence of perennial canker formation in
the vascular tissue is a major concern among growers due to significant
economical losses in mature olive orchards as a result of both yield reduction
and increase of production costs. However, the importance that branch
dieback has in olive health in California has not yet been evaluated. Therefore,
field surveys were conducted throughout the main olive-production regions to
determine the incidence of branch dieback in California. Declining branches
and trunks were collected in order to identify the fungal pathogens associated
with perennial cankers and subsequent dieback of olive trees. In all, over 700
samples were collected from 60 different olive orchards throughout
California. Olive branch dieback was observed in all mature orchards
surveyed in California. Isolation of samples showing perennial cankers
followed by morphological studies and phylogenetic analyses of three loci
allowed us to identified 16 different fungal species in the families
Botryosphaeriaceae,
Valsaceae,
Diatrypaceae,
Coriolaceae,
and
Schizophyllaceae to be associated with olive cankers in California. In order to
determine the role that these fungi play on olive health in California,
pathogenicity tests were conducted in Sevillano and Manzanillo cultivars.
Extent of vascular discoloration and percentage of recovery from inoculated
wounds showed all fungi to be pathogenic; however, virulence varied by
species.
Evaluation of rhizobacterial strains as Fusarium oxysporum biological
control agents
R. Urrea (1), L. F. Cabezas (2), S. Restrepo (2), P. JIMENEZ (1)
(1) Univ Militar Nueva Granada, Bogota, COLOMBIA; (2) Univ de Los
Andes, Bogotá, COLOMBIA
Phytopathology 100:S129
Different reasons had stimulated the development of agro products based in
microorganisms and their metabolism. This has stimulated the screening of
many sources of microorganisms, among them the rhizosphere. On the other
side, Fusarium oxysporum is an important pathogen to many crops,
demanding its control for huge amounts of fungicides that are becoming
useless controlling the fungus. Since Physalis peruviana is an important crop,
and F. oxysporum is its more limitant pathogen, rhizobacteria from P.
peruviana rhizosphere were collected in 4 production areas in Bogota plateau,
Colombia. In two screening processes 5 bacteria, out of 150, were chosen
because their ability to reduce by 50% the in vitro F. oxysporum radial
growth, and reduce the number of macro and micro conidia per colonial area.
These bacteria, 2 isolates of Bacillus subtilis, 2 of Pseudomonas flourecens
Seedling diseases are caused by a number of pathogens that can work singly
or as a complex. To identify the primary seedling pathogens of soybean in
Arkansas, soil was collected from two sites (Hope and Stuttgart) at three
planting dates (April, May and June). These soils were planted with seeds of
three soybean cultivars, each of which had one of seven fungicide seed
treatments. Tests were conducted in growth chambers at 21°C (April), 25°C
(May) or 28°C (June). After 18 to 26 days, isolations were made from the
roots and seeds on water agar and isolates were identified to species. Pythium
spp. and Fusarium spp. were the most frequently isolated pathogens at each
temperature with P. sylvaticum (65%) and F. oxysporum (45%) being the
predominate species. Rhizoctonia solani was 8% of the isolates. Pythium spp.
and R. solani were more frequently recovered from soil from Hope than
Stuttgart. Recovery of Pythium spp. and Fusarium spp. was greatest for the
May planting date followed by June and April. Recovery of R. solani was
higher in April and May and less in June. Over 90% of P. sylvaticum isolates
were highly virulent to soybean while the percentage of virulent F. oxysporum
isolates was greatest at 28°C (93%) followed by 25°C (79%) and 21°C (57%).
Only recovery of R. solani was affected by seed treatment. These results show
that P. sylvaticum and F. oxysporum are the major components of a dynamic
seedling disease complex in Arkansas.
A breakthrough in the field of agriculture
K. VADIVEL (1)
(1) Annamalai University, Chidambaram, INDIA
Phytopathology 100:S129
Mung bean (Vigna radiata L. Wilczek) commonly known as green gram is the
third important grain legume in India. Natural products like lime, hydrated
lime, turmeric powder and livestock excrements have been tested by seed
treatment and spraying them on ADT.3 green gram variety15 times at
biweekly intervals in two rabi seasons (Dec – March 2008 and 2009) and
reported. Under in vitro studies Annamalai mixture (cow urine +cow dung
+sheep dung +poultry litter+neem cake) 50% concentration recorded the
maximum vigour of 2400 followed by turmeric powder 1900 whereas in
control it was 280. Under pot trial, the Annamalai mixture recorded the
maximum yield of 11.4 g/plant whereas in field experiment T2 (cow urine
+cow dung) recorded the maximum of 3.3 tonnes/ha followed by Annamalai
mixture 3.25 tonnes whereas in control it was 193.1 kg/ha only. The fungal
diseases like cercospora leaf spot and rust infection was minimum in
Annamalai mixture whereas lime and turmeric powder recorded no powdery
mildew infection. Similarly, yellow mosaic disease was totally absent in
turmeric powder and livestock excrements applied treatments. Various
nutrients and plant growth promoters present in natural products might have
influenced the plant growth and increased the yield of green gram manifold
and the antimicrobial properties like ammonia, silica, antioxidants like
curcumin and calcium reduced the disease incidence. Thus, use of such a
simple as well as the traditional method may open up a new chapter in
agriculture.
Initial characterization of a Xanthomonas sp. causing bacterial spot of
shrub rose (Rosa spp.)
G. VALLAD (1), C. Summers (1), H. Adkison (1), E. Margenthaler (1)
(1) University of Florida, Wimauma, FL, U.S.A.
Phytopathology 100:S129
A severe bacterial spot of shrub rose (Rosa spp.) caused by a xanthomonad
was observed during summer production in Florida. Foliar symptoms
consisted of small black lesions with defined margins that were fairly vein
delimited and often located along leaf margins. Based on fatty acid
composition and 16S rRNA sequence, the strain was most closely related to
several pathovars of Xanthomonas axonopodis. The 16S-23S rRNA intergenic
spacer (ITS) and flanking portions of the 16S and 23S rRNA genes were
sequenced and compared among three rose strains and those of several
characterized strains of X. citrumelo, X. euvesicatoria, X. dieffenbachiae, X.
manihotis, and X. perforans. Sequence identity within the nearly 2 kb region
was greater than 98.3% among all strains with 100% identity among the rose
strains. The rose strains exhibited 99.9% identity with X. perforans.
Phylogenetic analyses of the ITS region consistently grouped rose strains
closest to X. perforans. Rose strains caused few symptoms when infiltrated at
Vol. 100, No. 6 (Supplement), 2010
S129
106 cfu/ml into leaves of citrus, tomato, pepper, or several members of the
Euphorbiaceae or Araceae. However, strains were clearly pathogenic on rose
and another Rosaceae, Indian Hawthorne (Rhaphiolepis indica). Results
suggest that the rose strains may represent a new species or subspecies of
Xanthomonas. Further characterization of rose strains through multi-locus
sequence typing and host testing is in progress. This is the first pathogenic
Xanthomonas sp. associated with rose.
A rapid screening method for fungicide resistance in Alternaria alternata
B. VEGA (1), P. F. Harmon (2), M. M. Dewdney (1)
(1) CREC, University of Florida, Lake Alfred, FL, U.S.A.; (2) Department of
Plant Pathology, University of Florida, Gainesville, GA, U.S.A.
Phytopathology 100:S130
Alternaria brown spot (ABS) of tangerines and tangerine hybrids is a very
important foliar disease affecting leaves, twigs and young fruit, rendering
them unsalable. Strobilurin fungicides have been used for ABS control for
many years and are the most effective products registered. In the last two
years, reduced ABS control has been observed in Florida groves with
strobilurin applications. Preliminary studies found strobilurin resistant
populations, and a larger study was initiated. The traditional method of
fungicide resistance evaluation employs media amended with logarithmicallydiluted fungicide concentrations where the mycelium growth or germ tube
length of monosporic isolates are evaluated. The incorporation of an
oxidation-reduction indicator, resazurin, to measure spore respiration in a
microtiter assay could significantly reduce cost and time. We evaluated a
minimal media, a complete media and potato dextrose broth (PDB)
amended with calcium carbonate in order to optimize this assay. The
greatest percent reduction of resazurin was observed in PDB and
complete media. At 105 spores/ml, the assay sensitivity was optimal for
fungicide resistance evaluation. Alternative respiration was suppressed with
the addition of SHAM to the strobilurin assays. Isolates had comparable ED50
values for both methods. Sensitive and baseline isolates were lower than 0.79
µg/ml of azoxystrobin while resistant isolates had values greater than 10
µg/ml.
Electroporetic Potyvirus transfection of pepper protoplasts
N. Y. VELASQUEZ (1), S. Suh (1), J. F. Murphy (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S130
Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.)
production worldwide. Three Potyviruses commonly detected in diseased
pepper plants are Potato virus Y (PVY), Pepper mottle virus (PepMoV) and
Tobacco etch virus (TEV). We have studied pepper resistance responses to
Potyvirus infection at the cellular level using freshly isolated protoplasts and a
polyethylene glycol (PEG) inoculation procedure. The PEG procedure poses
difficulties with experiment to experiment precision. We therefore developed
an electroporation inoculation procedure for Potyviruses using a Bio-Rad
Gene Pulse Xcell. Experiments evaluated voltage, number of pulses, time
interval between pulses, viral RNA concentration and number of protoplasts
inoculated. For each test, virus accumulation was determined by ELISA and
protoplast viability was monitored. Consistent infection with the highest virus
titer and protoplast viability resulted when 40 µg virus RNA was used to
inoculate 500,000 protoplasts using two 25-msec pulses of 200 volts each with
a 10-sec time interval between pulses.
Molecular characterization and ELISA based detection of Bean
leafroll virus and Pea enation mosaic virus from the Pacific Northwestern
U.S.A.
B. Vemulapati (1), K. L. DRUFFEL (1)
(1) Washington State University, Pullman, WA, U.S.A.
Phytopathology 100:S130
The complete genomic sequence of one Pea enation mosaic virus (PEMV)
isolate from Idaho (PEMV-ID) and one Bean leaf roll virus (BLRV) isolate
from Washington State (BLRV-WA) were determined. PEMV-ID contained
five ORFs. ORF4, which encodes the CP, shared a maximum identity of
98.9% with other members of PEMV while the least amino acid identity was
seen with ORF-1. Phylogeny tree based on CP sequences showed that PEMVID grouped with PEMV isolates UP58 and Germany. BLRV-WA contained
five ORFs and ORFs 1 and 3 shared a maximum identity of 99.4% with
respective ORFs of known BLRV isolates, while the least was with ORF-2.
Antigen-coated plate (ACP) ELISA assays for the detection of BLRV and
PEMV were developed using antisera raised against recombinant coat
proteins (CP) of BLRV and PEMV, respectively. Pea and alfalfa samples
collected from different fields in Washington and Idaho were tested. BLRV
antiserum detected the virus in up to 1:3200 dilution of infected samples, and
anti-PEMV serum could detect the virus in pea leaf extracts up to 1:6400
dilution.
S130
PHYTOPATHOLOGY
Recombinant antibody-mediated multiple disease tolerance in canola
S. S. VERMA (1), W. Yajima (1), M. H. Rahman (1), S. Shah (2), Y. Liang
(1), N. N. Kav (1)
(1) University of Alberta, Edmonton, AB, CANADA; (2) Alberta Innovates,
Vegreville, AB, CANADA
Phytopathology 100:S130
Plants are constantly exposed to numerous potential pathogens endowed with
diverse modes of attack and have the potential to cause significant yield
reduction, up to 15%, due to pathogen attack. Earlier research has shown that
external application of polyclonal and monoclonal antibodies in plants can
impart tolerance against certain bacteria and fungi. This approach has recently
been improved further by introducing cDNA encoding recombinant antibodies
(rAbs) into plants. We have generated transgenic lines of Brassica napus
canola expressing anti- Sclerotinia sclerotiorum ScFv and ScFv-fusion
proteins, and several of these transgenic lines expressing those constructs
exhibited increased tolerance to multiple diseases. Especially, we observed
that these transgenic B. napus were tolerant against Sclerotinia sclerotiorum,
Alternaria brassicae and Leptosphaeria maculans all of which causes major
diseases in canola. In addition, another construct containing cDNA encoding
ScFv fused with a known anti-microbial peptide increased the tolerance of
transgenic canola against these pathogens even further. Advantages of this
approach include the fact that recombinant antibodies can be engineered
against almost any target molecule, and it has been demonstrated that the
expression of functional pathogen-specific ScFv’s in plants can confer
effective pathogen protection.
Genome organization and structure of a putative member of the
Flexiviridae family infecting sweet cherries
D. V. VILLAMOR (1), K. L. Druffel (2), K. Eastwell (1)
(1) Washington State University, Prosser, WA, U.S.A.; (2) Washington State
University, Pullman, WA, U.S.A.
Phytopathology 100:S130
A novel disease was observed in an isolated sweet cherry (Prunus avium)
orchard in Washington State. The graft transmissible agent induced foliar
symptoms on cultivar ‘Bing’ that were reminiscent of cherry rusty mottle
disease, but also induced severe stem pitting on Montmorency interstock.
Because of this distinct symptomatology, the putative causal agent is herein
referred to as Montmorency stem pitting virus (MMSPV). Electrophoresis of
double-stranded RNA isolated from the infected tissue revealed a product of
ca. 8.5 kbp supporting a viral etiology. A primer pair derived from a cloned
fragment of the dsRNA yielded amplicons of the anticipated size from trees
with MMSPV symptoms, but not from trees infected with other common
viruses known to infect sweet cherry trees. Primers designed from the 3′terminus of foveaviruses yielded amplification products of 1.1 kbp that were
cloned and sequenced. The amplicon contained a putative coat protein
sequence that shared 74.2% sequence identity to the published sequence of
Cherry green ring mottle virus and 75.7% sequence identity to Cherry
necrotic rusty mottle virus, both unassigned viruses of the Betaflexiviridae
family. Primers designed from sequences obtained by 5′ and 3′ RACE allowed
the complete genome of ca. 8.7 kb to be amplified and cloned. Sequencing of
the complete virus genome is currently underway.
Disease management in strawberry production: The Philippine
experience
L. M. VILLANUEVA (1)
(1) Benguet State University, La Trinidad, Benguet, PHILIPPINES
Phytopathology 100:S130
Strawberry is grown only in Benguet Province because of its unique climatic
conditions. It has been a lucrative source of income for Benguet farmers and
adds to the revenue of Benguet Province. Diseases caused by fungi, bacteria
and nematodes are important limiting factors in strawberry production in the
area. However, researches on strawberry diseases are very nil which could be
attributed to limited funding support to the crop. To identify the major
diseases, soil and plant samples were collected from the strawberry growing
areas in Baguio City and Benguet Province. The following diseases were
observed: verticillium wilt, red stele, leaf scorch, leaf spot, leaf blight, gray
mold and other minor disorders. On the other hand, the root lesion and
strawberry crimp nematodes were the economically important nematode pests
identified. Six strawberry cultivars namely Sweet Charlie, Camarosa, Festival,
Whitney Earlibrite, and Winterdawn were evaluated for their resistance to the
major fungal pathogens and root lesion nematode. Sweet Charlie, the most
preferred strawberry cultivar seems to be resistant to some fungal pathogens
and root lesion nematode. Other potential disease management options tested
include the following: use of indigenous mulching materials, rotation with
broccoli, use of biocontrol agents and application of baking soda. Integration
of effective and compatible disease management strategies would be very
crucial in the improvement of the strawberry industry in the region.
Detection of viable Phakopsora pachyrhizi spores by indirect
immunofluorescence and propidium iodide staining
R. VITTAL (1), J. S. Haudenshield (2), G. L. Hartman (3)
(1) University of Illinois - Urbana Champaign, Urbana, IL, U.S.A.; (2)
USDA-Agricultural Research Service, Urbana, IL, U.S.A.; (3) University of
Illinois - Urbana Champaign; USDA Agricultural Research Service, Urbana,
IL, U.S.A.
Phytopathology 100:S131
We developed a rapid and reliable technique for the detection of viable
Phakopsora pachyrhizi urediniospores by integrating an indirect
immunofluorescence assay (IIFA) with direct propidium iodide (PI) staining.
Monoclonal (Pp-mAb) and polyclonal (Pp-pAb) antibodies were produced, in
mouse and rabbit respectively, in response to intact urediniospores. Spores
were exposed to Pp-mAb or Pp-pAb and any binding was detected with a
secondary antibody having a fluorescein isothiocyanate (FITC) label,
followed by vital staining with PI. The spore-antibody complex was
visualized using an epifluorescent microscope with an FITC/PI dual-emission
filter. Under these conditions, both live and dead spores stained green but the
nuclei of dead spores stained red, indicating the permeability of PI dye into
the dead spores. This method can be greatly simplified into a two-step system
by using a FITC-labeled Pp-mAb or Pp-pAb followed by incubation with PI
to detect and quantify the relative amount of non-viable to viable P.
pachyrhizi spores. This technique has been used as a research tool to confirm
viability of urediniospores based on various treatments and has the potential to
be used as an on-site system for forecasting soybean rust by monitoring the
movement of airborne spores during the soybean-growing season.
Survey of diseases of agronomic switchgrass in Tennessee
A. L. VU (1), M. M. Dee (1), T. Russell (1), O. L. Fajolu (1), K. D. Gwinn
(1), J. Zale (1), B. H. Ownley (1)
(1) University of Tennessee, Knoxville, TN, U.S.A.
Phytopathology 100:S131
Switchgrass (Panicum virgatum L.) is a perennial warm-season (C4) native
grass currently being investigated for use in biomass-based ethanol production
in Tennessee. However, little is known about diseases that occur, or the
impact of these diseases on the success of this crop. Reducing disease could
significantly increase biomass yield and overall crop quality, particularly in
the southeastern U.S. where large monocultures are being planted. The goal of
this project was to identify fungal pathogens of switchgrass to gain an
understanding of the role of disease in the overall efficiency and sustainability
of switchgrass as a biofuel crop. Naturally infected ‘Alamo’ and ‘Blackwell’
switchgrass plants were collected from growers’ fields in Vonore, TN in
summer 2009 and agronomic research plots in Knoxville, TN in winter
2007 through spring 2008. Fungi were isolated from diseased plants and
pathogenicity was confirmed with Koch’s postulates in growth chamber
studies. Several fungal pathogens were isolated also from seed. Pathogenic
species of Alternaria, Bipolaris, Curvularia, and Fusarium have been
identified; several of which have not been described previously on
switchgrass, but are known to reduce quality and yield of other crops.
Species identification was based on morphology of conidia and conidiogenous
cells, colony characteristics, colony growth at various temperatures, and
confirmed with internal transcribed spacer (ITS) sequences of ribosomal
DNA.
USDA APHIS plant pest and biocontrol permitting and regulatory policy
changes: Impacts on the stakeholder community
S. A. WAGER-PAGE’ (1)
(1) USDA APHIS, Riverdale, MD, U.S.A.
Phytopathology 100:S131
USDA Animal and Plant Health Inspection Service (APHIS) published a
proposed rule to amend 7CFR330, Plant Pest Regulations; Update of General
Provisions, for public comment in October 2001. The update included
provisions to implement permitting policy that the Plant Protection Act
clarified and expanded. A final rule was not published for several reasons
including the Agency’s response to the events of September 11, 2001 and the
Office of Inspector General (OIG) review recommending permitting policy
changes. Plus, over 1,000 comments to the 2001 rule were received. Since
2002, the Pest Permitting Branch implemented numerous permitting policy
changes based on the OIG review, Permitting Board of Advisor’s
recommendations and the development of the ePermits processing
database. Standard permit conditions were developed and established for
plant pest and regulated article movement types including interstate,
importation, and continued curation. Amendments to permitting policy
will be proposed for codification in 7CFR330 in the near future. APHIS
continues to develop and upgrade ePermits. Eighty-five percent of pest permit
applications are now received electronically, which has greatly improved
processing times. APHIS continues to modify ePermits to further improve
permit processing.
Bacillus subtilis, strain QST 713, Biofungicide II: Soil applications for
disease control, yield improvement and quality enhancement
P. J. WALGENBACH (1), D. Long (2), D. Sliva (3)
(1) AgraQuest, Inc., Davis, CA, U.S.A.; (2) AgraQuest, Inc., Demarest, GA,
U.S.A.; (3) AgraQuest, Inc., Santa Maria, CA, U.S.A.
Phytopathology 100:S131
Bacillus subtilis, QST 713, is a soil borne strain of bacteria. It is unique from
other strains of B. subtilis in its production of anti-fungal and anti-bacterial
products. These properties have previously been employed for the control of
foliage plant pathogens under the trademark Serenade ®. More recently,
research has exhibited the advantages of soil applications of QST 713 in terms
of disease suppression and beneficial plant effects leading to a product
extension, Serenade Soil ®. Soil applications, whether applied via seed
treatment or drench, result in more vigorous plants as measured by topgrowth
and rootmass. In the presence of soil borne pathogens QST 713 soil
applications suppress disease with resultant increases in plant vigor, improved
yields and, in some instances, quality. The aforementioned properties are
seemingly the result of a protective biofilm on the roots of plants, disease
suppression from an array of lipopeptides and plant modulation. The practical
implications of these new findings are discussed in regards to tomatoes,
potatoes and cucurbits.
A new fungicide for control of Oomycete diseases of vine and vegetable
crops
K. A. WALKER (1), J. S. Barnes (1), L. J. Newsom (2)
(1) BASF, Research Triangle Park, NC, U.S.A.; (2) BASF, Tifton, GA,
U.S.A.
Phytopathology 100:S131
Zampro® is a new fungicide under development by BASF Corporation for
control of Oomycete fungi including downy mildews and Phytophthora spp.
Zampro is a premix containing two modes of action, dimethomorph and
ametoctradin. Dimethomorph is a cell wall synthesis inhibitor classified as
FRAC Group 40. Ametoctradin is a strong inhibitor of mitochondrial
respiration classified as FRAC Group 45. Zampro is currently under trilateral
review with Canadian (PMRA), US EPA and Australia (APVMA). Zampro
was submitted for EPA registration for use on potato, grape, hop and
vegetable crops. Results indicate Zampro exhibits excellent crop safety and
disease control. Trial results from 2008 and 2009 and proposed directions for
use will be presented. EPA registration is expected in 2012.
Multilocus phylogeny of Ophiosphaerella species causing spring dead spot
of bermudagrass
N. WALKER (1), S. Marek (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S131
Ophiosphaerella herpotricha, O. korrae, and O. narmari are causal agents of
spring dead spot on turf-type bermudagrass (Cynodon spp. and interspecific
hybrids) in the transition zone of the United States. Growing as sterile mycelia
in culture, Ophiosphaerella spp. cannot be identified morphologically and few
DNA sequences are available for comparison. Forty O. herpotricha, twentyseven O. korrae and five O. narmari isolates collected from bermudagrass
from throughout the U.S. were selected for this study. Three O. korrae isolates
from bluegrass were also included. DNA sequences of the nuclear ribosomal
small subunit (SSU), internally transcribed spacer (ITS) region, and large
subunit (LSU), a region of the translation elongation factor 1α (EF1α) gene,
and the second largest subunit gene of RNA polymerase II (RPB2) from each
isolate were analyzed. Group I introns of various sizes often were inserted in
SSU sequences at up to four positions and corresponded with the species of
the isolate. SSU introns often disrupted primer sites around the ITS region,
precluding its use for identification. Multilocus phylogenies clustered O.
herpotricha and O. narmari into well-resolved, monophyletic clades, while O.
korrae isolates clustered more loosely due to greater sequence variation. EF1α
and RPB2 sequences were more useful than rDNA sequences for identifying
Ophiosphaerella isolates to species.
Death of epithelial cells in loblolly pine roots
C. H. WALKINSHAW (1)
(1) Columbus, GA, U.S.A.
Phytopathology 100:S131
Epithelial cells are widely distributed in biological systems where they line
tissue such as resin ducts. These extremely thin cells are located adjacent to
primary tracheids and large parenchyma cells. The objective of this study was
to describe death of the epithelial cells in relation to root necrosis and enzyme
hydrolysis. Histological and histochemical methods were used to process root
tissues from field plantings. Tissues were fixed in buffered neutral formalin
(ph 7.0) and stained with nine different schedules. Light microscopy results
show there was a gradation in damage to the roots. Epithelial cells displayed
Vol. 100, No. 6 (Supplement), 2010
S131
variation in necrosis and enzyme activity. Phenol (tannins) accumulated in the
epithelial cells providing a good marker for predicting damage to loblolly
roots. Further studies need to be conducted to better understand the nature of
the tissue damage to the roots of loblolly pines.
Influence of foliar fungicide on components of grain yield in hybrid corn
M. W. WALLHEAD (1), L. Madden (1), P. Paul (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S132
Field experiments were conducted at two locations in Ohio (Wooster and
South Charleston) to evaluate the effects of prothioconazole + trifloxystrobin,
applied at different growth stages, on components of hybrid corn grain yield.
Plots were planted with Pioneer hybrid 38A55 on 30 April at Wooster and 11
May at South Charleston, at approximately of 25,000 plants/ha at Wooster and
30,000 plants/ha at South Charleston. The experimental design was a
randomized complete block, with treatments in a split-plot arrangement.
Previous crop (corn or soybean) was the whole-plot and fungicide treatment (a
non-treated check plus treatments applied at the R1, R2, or R3 crop growth
stages) the sub-plot. All applications were made with a high-clearance sprayer
at a rate of 356 ml of the fungicide per hectare and volume of 76 L/ha. At
harvest, grain yield (YLD), test weight (TW), ear weight (EW), number of
kernel rows per ear (KRE), and number of kernels per row (KR) were
determined. Mean yield was significantly affected by location and cropping
sequence, but not by fungicide treatment. Location also had a significant
effect on TW and EW. However, other yield components were similar
between the two locations and were not significantly affected by fungicide
treatment nor cropping sequence. KRE and KR were fairly constant among
treatments, with means ranging from 14 to 16 rows/ear and 28 to 38
kernels/row, respectively.
Do climate and outbreak frequency affect levels of foliar phytochemistry
in different lodgepole pine (Pinus contorta) stands?
C. WALLIS (1), D. P. Huber (2), K. J. Lewis (2)
(1) USDA ARS, Parlier, CA, U.S.A.; (2) University of Northern British
Columbia, Prince George, BC, CANADA
Phytopathology 100:S132
Lodgepole pine (Pinus contorta Douglas ex Louden) is a widely distributed
tree in North American forests and is found in a variety of environments, each
with different levels of disease activity. We quantified the levels of defenseassociated metabolites (including soluble phenolics, lignin, and terpenes) in
the foliage of 13 distinct lodgepole pine stands scattered throughout British
Columbia to test the hypothesis that different climates would result in
different levels of these compounds. Precipitation levels were positively
correlated with soluble phenolic and terpenoid levels. Temperature was
negatively associated with foliar lignin levels, implying that this compound
affects cold hardiness. We also determined the frequency of past outbreaks of
the foliar disease Dothistroma septosporum (Dorog.) Morelet) by using
dendrochronological techniques and historical records in five of these stands,
and then correlated outbreak frequency with the levels of secondary
metabolites present in the foliage. The levels of lignin, soluble phenolics, and
monoterpenes increased in direct relationship to frequency of disease
outbreaks. Thus, disease outbreaks select for the production of defenseassociated secondary metabolites in lodgepole pine foliage.
Races of Puccinia striiformis identified in the United States in 2009
A. WAN (1), X. Chen (2)
(1) Washington State University, Pullman, WA, U.S.A.; (2) USDA ARS,
Pullman, WA, U.S.A.
Phytopathology 100:S132
Puccinia striiformis f. sp. tritici (PST) and P. striiformis f. sp. hordei (PSH)
cause stripe rust on wheat and barley, respectively. To monitor virulence
changes in the pathogen populations, stripe rust samples collected from 14
states were tested on 20 wheat and 12 barley differentials for identifying PST
and PSH races, respectively. Six previously existing PSH races were detected,
of which PSH-71 (virulent to Topper, Emir, Hiproly, Varunda, Abed Binder
12, Trumpf, Mazurka, Bigo, and Bancroft) was predominant. A total of 27
PST races were detected including new races PST-139 (virulent to Lemhi,
Chinese 166, Heines VII, Paha, Druchamp, Produra, Yamhill, Stephens, Lee,
Fielder, Tyee, Hyak, Yr8, Yr9, and Clement) and PST-140 (virulent to Lemhi,
Heines VII, Moro, Produra, Stephens, Lee, Fielder, Tres, Express, Yr8, Yr9,
Clement, and Compair). Races PST-139, PST-140, PST-114 (with all PST140 virulences plus Yamhill), PST-116 (with all PST-114 virulences plus
virulence to Paha), and PST-127 (with all PST-139 virulences, plus Moro,
Express, and Compare) were predominant. The frequency of PST-127
increased from 1.5% in 2007 and 4.3% in 2008 in California and Washington
to 9.1% in 2009 in California, Idaho, Oregon, and Washington. Twenty five of
the 27 races were detected in the western U.S. while only five (PST-78, PST80, PST-98, PST-100, and PST-102) were detected in the eastern U.S.
S132
PHYTOPATHOLOGY
Trafficking of soybean cyst nematode secreted CLE proteins in plant cells
J. WANG (1), T. Hewezi (2), T. J. Baum (2), E. L. Davis (3), X. Wang (4), M.
G. Mitchum (1)
(1) University of Missouri, Columbia, MO, U.S.A.; (2) Iowa State University,
Ames, IA, U.S.A.; (3) North Carolina State University, Raleigh, NC, U.S.A.;
(4) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S132
Soybean cyst nematodes (Heterodera glycines) produce secreted effector
proteins that function as peptide mimics of plant CLAVATA3/ESR (CLE)like peptides to promote parasitism. Similar to plant CLEs, nematode CLEs
belong to gene families encoding small proteins with N-terminal signal
peptides (SP), diverse variable domain (VD) sequences, and either a single or
multiple, conserved C-terminal CLE domain(s) that are processed to release
bioactive 12 or 13 amino acid (aa) CLE motif peptides. Previously, we
determined that the nematode 12 aa CLE motif peptide is not sufficient for
biological activity in vivo. Genetic and biochemical analysis confirmed the
requirement of the VD and revealed a novel role in trafficking
cytoplasmically-delivered CLEs to the apoplast in order to function as ligand
mimics. VD deletion studies and yeast two-hybrid analysis are underway to
identify essential motifs and/or interacting proteins required for protein
trafficking. Subcellular fractionation will provide information about how these
proteins traffic in plant cells. Together, these studies will help better
understand the trafficking mechanism of nematode secreted CLE effector
proteins in plants cells.
Numerical simulation of the long distance transports of wheat stripe rust
pathogen in China
H. WANG (1), X. Yang (2), Z. Ma (1)
(1) Department of Plant Pathology, China Agricultural University, Beijing,
PRC PEOPLES REP OF CHINA; (2) Department of Plant Pathology, Iowa
State University, Ames, IA, U.S.A.
Phytopathology 100:S132
Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an economically
important disease of wheat worldwide. The objective of this study was to
determine the connectivity between the source regions in China. Based on the
meteorological data from 1997 to 2006, numerical simulation of the long
distance transports of P. striiformis f. sp. tritici after oversummering and
overwintering was conducted using HYSPLIT (HYbrid Single-Particle
Lagrangian Integrated Trajectory) model. Total 25 and 27 source locations
were selected from oversummering regions and from overwintering regions,
respectively. The spores alive in the air for 120 hours were released at 00, 06,
12, and 18 UTC. The results indicated that in autumn the pathogen into
northwestern China was mainly from southwestern China, that into Yunnan
was mainly from Guizhou, and that into Guizhou was mainly from Yunnan. In
spring, the spores into northwestern China were mainly from Sichuan and
northern China, that into Guizhou were mainly from Yunnan and Sichuan,
that into northern HuBei were mainly from northwestern, northern and
southwestern China, and that into northeastern China were mainly from
northwestern and northern China. Either in autumn or in spring, the spores
into northern China were mainly from northwestern and southwestern China,
and that into Sichuan were mainly from northwestern China, Yunnan and
Guizhou. The inoculum sources in Xinjiang had little impact on wheat areas
outside of Xinjiang.
Molecular diversity of Sugarcane yellow leaf virus in China
M. Wang (1), D. Xu (1), G. ZHOU (2)
(1) Laboratory of Plant Virology, South China Agricultural University,
Guangzhou, PRC PEOPLES REP OF CHINA; (2) South China Agricultural
University, Guangzhou, PRC PEOPLES REP OF CHINA
Phytopathology 100:S132
Sugarcane yellow leaf virus (SCYLV, genus Polerovirus, family Luteoviride)
has become widespread in many sugarcane growing regions in China during
the past decade, and a new aphid vector, Ceratovacuna lanigera, was recently
found to transmit the virus in the field. To investigate the molecular diversity
of this virus, an RT-PCR-RFLP method was developed for genotype
discrimination based on viral CP and MP coding sequence, from which the
RT-PCR amplicons (1326 bp) displayed polymorphism between the
recognized four genotypes ie. BRA, PER, REU and CUB genotype, after the
endonuclease BamH I, Hind III and Xho I digestion. 514 samples were
collected from 53 field locations in southern China, from the year 2007 to
2009. It was revealed that 81 (15.8%) samples were SCYLV positive in the
RT-PCR detection, and 59 of them were identified as BAR genotype, whereas
other 22 displayed unexpected RFLP patterns in the RFLP analysis.
Furthermore, the amplicons from these 22 samples were cloned and
sequenced. Sequence comparison indicated that 10 of them shared high
nucleotide similarities with the recognized genotypes, 4, 4, and 2 samples
grouped into BAR, PER, and CUB genotypes, respectively, and the remaining
12 samples, based on their nuclotide identity data, could be divided into two
groups, each of which might represent a new SCYLV genotype for they had
distinct differences (less than 96.0% nt identies) with the recognized
gentotypes.
Comparative analysis of the RcsC sensor kinase from Erwinia amylovora
and other enterobacteria
D. WANG (1), Y. Zhao (1)
(1) University of Illinois, Urbana, IL, U.S.A.
Phytopathology 100:S133
RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine
kinase domain and a phosphoreceiver domain. We have previously
demonstrated that, though the Erwinia amylovora rcsC mutant produces more
amylovoran than the wild type strain in vitro, it is avirulent on the host plants.
In this study, we further characterized the RcsC and its homologs from
various enterobacteria. Our results showed that overexpression of Erwinia
RcsC suppressed amylovoran production in different amylovoran overproducing strains, indicating net phosphatase activity of RcsC.
Complementation studies showed that RcsC homologs from Pantoea stewartii
and Yersinia pestis, but not those from E. coli and Salmonella enterica,
partially restored virulence of the Erwinia rcsC mutant on gala apple shoots.
However, all RcsC homologs could not rescue amylovoran production
phenotype of the Erwinia rcsC mutant. In addition, a chimeric construct
containing the sensor domain of Erwinia RcsC and the output domain of E.
coli RcsC restored virulence of the Erwinia rcsC mutant, but not amylovoran
production; whereas a chimeric construct containing the sensor domain of E.
coli RcsC and the output domain of Erwinia RcsC rescued amylovoran
production phenotype of the Erwinia rcsC mutant, but not virulence. These
results suggest that the sensor domain of RcsC might be essential for
regulating bacterial virulence, whereas the output domain of RcsC might be
responsible for regulating amylovoran production.
Evaluation of foliar response of cucumber to Phytophthora capsici and
inoculation techniques
X. WANG (1), M. Hausbeck (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S133
Phytophthora blight, caused by Phytophthora capsici Leonian, has become a
limiting factor for cucumber (Cucumis sativus) growers in Michigan. Pre- and
post-emergence damping-off are very common symptoms of P. capsici
infection on cucurbit crops. Cucumber seedlings were used to investigate the
infection of P. capsici on the foliage of cucurbits under controlled laboratory
conditions. Four P. capsici isolates (OP97, SP98, 12889 and 13351) were
employed to determine the disease response on the foliage of susceptible
cucumber cultivar ‘Vlaspik’ by using three inoculation techniques including
mycelial plug, droplet and spray. Disease symptoms appeared on the
cotyledons one day after inoculation. Significant differences (P ≤ 0.05) among
AUDPC values calculated for the isolates and inoculation techniques were
observed respectively. No significant interaction was found between isolates
and inoculation techniques. Isolate 12889 was most virulent when 20 ml of
zoospore suspensions (1 × 106 per ml) were used and applied via foliar droplet
or spray. The foliar-droplet inoculation technique was quantitative, effective
and consistent in evaluating the disease response on cucumber seedlings
compared to the other two inoculation methods. Therefore, isolate 12889 and
the foliar droplet technique will be used to evaluate Phytophthora foliar blight
of cucurbits.
germplasm materials (near 84.2% among 247). The reaction type, incidence,
and severity of the three strains were lower than those of race CYR31,
CYR32, CYR33, and Su11-4 on most of varieties or germplasm materials.
The parasitic fitness of them was apparently lower than that of current
epidemic races.
Use of a strip-till cover crop system to manipulate above and below
ground organisms in cucurbit plantings
K. WANG (1), C. R. Hooks (2), S. P. Marahatta (1)
(1) University of Hawaii, Honolulu, HI, U.S.A.; (2) University of Maryland,
College Park, MD, U.S.A.
Phytopathology 100:S133
A field trial was conducted in 2008 and 2009 to evaluate the potential of using
sunn hemp (SH), Crotalaria juncea, and marigold (MG), Tagetes patula in a
strip till cover cropping (STCC) system to suppress crop pests and increase
the number of free-living soil organisms involved in nutrient cycling.
Cucumber (Cucumis sativus) and winter gourd (Benincasa hispida) were
planted as cash crops in 2008 and 2009, respectively. In 2008, SH increased
the abundance of free-living nematodes (including bacterivores, fungivores,
and omnivores) and soil microarthopods (detritivores) compared to the
monoculture bareground (BG) treatment and suppressed the total abundance
of plant-parasitic nematodes by the end of the cucumber cropping cycle.
Thrips and whitefly numbers on cucumber were reduced significantly in SH
compared to BG treatment only in 2008. However, due to more severe melon
fly (Bactrocera cucurbitae) colonization in SH plots, SH had lower cucumber
yield than the BG. Similar results were obtained in 2009, except that SH did
not reduce foliar insect pests. Winter gourd seedlings were initially infected
by Microphomina phaseolina, but more seedlings in the SH plots survived the
infection than those in the BG. Final yield of winter gourd was also
significantly higher in the SH than BG plots. In conclusion, STCC with SH
managed above and below ground beneficial organisms and pests efficiently
and resulted in increased in crop yield in the second year of the STCC system.
The striatin ortholog of Colletotrichum graminicola plays a role in
mycelial growth, conidiation, and virulence to maize
C. WANG (1), W. Shim (1), B. D. Shaw (1)
(1) Dept. Plant Pathology and Microbiology, Program for the Biology of
Filamentous Fungi, Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S133
Colletotrichum graminicola is a foliar pathogen of maize causing anthracnose
and stalk rot. In filamentous fungi, striatin orthologs have been implicated in
multiple developmental processes including sexual development and
pathogenicity, and localize to the endoplasmic reticulum and the nuclear
envelope. However, little is known about its role in C. graminicola, a
heterothallic, hemi-biotrophic pathogen of maize. We generated C.
graminicola str1 deletion mutants using split-marker recombination. Mutants
grew slower on culture media, and lost the clockwise spiral growth of wild
type on PDA. Interestingly, the mutants gained a counter-clockwise spiral
growth pattern on V8 medium. The str1 mutants produced shorter falcate
conidia and fewer, less developed acervuli on nutrient medium and autoclaved
maize leaf, resulting in reduced falcate conidium production. Oval conidium
production was low, which may reduce the secondary infection in stalk
colonization. The str1 mutants still produced appressoria and successfully
penetrated leaves, but were delayed in foliar lesion development. In stalk rot
assays, mutants produced reduced lesions at the primary infection sites. Our
study indicated that the str1 deletion mutants were defective in several
propagation-associated developmental events and were less virulent to maize.
First report of new strains of Puccinia striiformis f. sp. tritici pathogenic to
Zhong 4 (Trititrigia) in China
B. WANG (1), X. Hu (1), Q. Li (1), G. Li (1), Z. Kang (1)
(1) Plant Protection College and Shaanxi Key Laboratory of Molecular
Biology for Agriculture, Northwest A&F University, Yangling, Shaanxi, PRC
PEOPLES REP OF CHINA
Phytopathology 100:S133
Detection of Phytophthora species in retail nurseries and urban forest
environments in northern Nevada
S. WANG (1), S. Garneni (1)
(1) Nevada Department of Agriculture, Sparks, NV, U.S.A.
Phytopathology 100:S133
Trititrigia Zhong 4 has good agricultural properties and high resistances, and
has been used as an important germplasm resource in wheat breeding all over
the world. It was assigned as one of 17 differential hosts in race identification
of Puccinia striiformis f. sp. Tritici (PST) since 1983 due to its immunity to
all races. Until now, only Zhong 4 is resistant to all races of PST in China. It
has been taken as an efficient resistant material for wheat stripe rust by many
breeders. In 2007, 6 wheat leaf samples (named as T1 to T6) among 196
samples from Taibai in Shaanxi province were found to be pathogenic to
Zhong 4 with reaction type 4. The reaction type of T2, T4, and T6 on
differential hosts was very similar with race Su11-11 except for those on
Zhong 4. The severity of three isolates on Zhong 4 was 25%. The incidence
was 66.7%, 75.0%, and 60.0%, respectively. Thus, T2, T4, and T6 should be
new strains. The pathogenicity of the three strains to seeding of wheat
cultivars or germplasm materials was tested in field from 2008 to 2009.
Results showed that three strains were toxic to most of cultivating varieties or
To survey for Phytophthora ramorum, P. kirnoviae, and other Phytophthora
species in retail nurseries and urban forest environments, a total of 385
symptomatic plant samples were collected from 140 host species or varieties
in 27 nurseries and 28 urban environments. To isolate Phytophthora species,
fresh leaf tissue or phloem and xylem tissue was placed on the selective
medium PARP. Isolates of Phytophthora were then transferred to corn meal
agar and V8 juice agar for morphological identification. Molecular
identification was employed by amplifying and sequencing an rDNA region
containing partial 18S ribosomal RNA gene, ITS1, 5.8S ribosomal RNA gene,
ITS2 and 28S ribosomal RNA gene. Of the total of 12 isolates obtained, 8
were from urban maple trees showing a bleeding canker symptom, and 4 were
from plants showing leaf blight in retail nurseries. P. cactorum was
predominantly associated with maple bleeding canker (88%), whereas P.
citricola was found only from one maple tree. In nurseries, P. cactorum was
found from Fraser’s photinia, P. citricola from Red Robin cinquefoil, and P.
Vol. 100, No. 6 (Supplement), 2010
S133
citrophthora from both Canadale Gold euonymus and Vicary Golden privet.
This survey suggests that P. cactorum is the major cause of chronic decline
and death of maples in urban environments of northern Nevada and that
nursery stock carrying various Phytophthora species is a direct pathway of
introducing non-native pathogens into the urban forest environments.
Field evaluations of Simplicillium lanosoniveum as a biological control
agent for Phakopsora pachyrhizi
N. A. WARD (1), R. W. Schneider (1), C. L. Robertson (1)
(1) Louisiana State University Agricultural Center, Baton Rouge, LA, U.S.A.
Phytopathology 100:S134
Simplicillium lanosoniveum is an inhabitant of soybean rust sori where it
parasitizes urediniospores and affects sorus development. Microscopic
observations indicated that the filamentous fungus penetrated and colonized
urediniospores within 5 days of inoculation. To further evaluate the impact of
this mycoparasitic fungus, we conducted field tests in 2009 near Baton Rouge,
LA to evaluate S. lanosoniveum as a biological control agent. Treatments
included foliar applications of conidia of S. lanosoniveum at various plant
growth stages and rust severities. Leaf samples were collected weekly from
late vegetative stages through senescence and were rated for disease severity
and total number of sori. Next, we subjected total leaf genomic DNA,
including all associated microorganisms, to qPCR analyses in order to
quantify S. lanosoniveum and P. pachyrhizi. We found that S. lanosoniveum
colonized and survived on leaf surfaces for 6 weeks after the earliest
inoculation. This led to delayed disease development and reduction in
numbers of sori. We conclude that S. lanosoniveum has mycoparasitic
properties that should be exploited for biological control of soybean rust and
possibly other rust diseases. This field of investigation incorporates ecological
principles into the study of phyllosphere microbiology, which may lead to
alternative means of disease control.
Development of ELISA and qPCR for Squash vein yellowing virus detection
C. WEBSTER (1), W. Li (2), C. Kousik (3), S. Adkins (1)
(1) USDA ARS, Ft. Pierce, FL, U.S.A.; (2) UF, Lake Alfred, FL, U.S.A.; (3)
USDA-ARS, Charleston, SC, U.S.A.
Phytopathology 100:S134
Watermelon vine decline caused by Squash vein yellowing virus (SqVYV) is a
new and emerging disease that has caused severe losses to Florida watermelon
growers in recent years. First identified in 2005, SqVYV is widely distributed
in southwest and west-central Florida and has recently been found infecting
several cucurbit weeds, often without inducing symptoms. Although late stage
symptoms of the disease are basically diagnostic for the presence of SqVYV,
earlier symptoms are not as obvious and may be confused with other causes.
Thus, continued development of simple and reliable diagnostic tests for early
monitoring of SqVYV in watermelon and cucurbit weeds remains important
as accurate identification is the first step in management. After several
unsuccessful attempts to produce specific antisera from virion preparations,
peptides of the SqVYV coat protein (CP) were synthesized and used to
immunize rabbits. The resulting polyclonal antisera were tested in ELISA and
found to react with SqVYV but to none of the other cucurbit-infecting viruses
common in Florida. A real-time PCR assay is being developed that targets the
CP gene of SqVYV. Initial tests have shown the PCR assay to be sensitive
and specific for SqVYV. These newly developed ELISA and real-time PCR
methods for SqVYV detection are being compared to existing methods of
detection including: conventional RT-PCR, tissue blots and indicator hosts on
both greenhouse grown and field collected samples.
Identification of Groundnut ringspot virus in tomato in south Florida
C. WEBSTER (1), L. Horsman (2), G. Frantz (2), C. Mellinger (2), S. Adkins
(1)
(1) USDA ARS, Ft. Pierce, FL, U.S.A.; (2) GCC, Jupiter, FL, U.S.A.
Phytopathology 100:S134
Fresh market tomatoes are widely grown in Florida and are subject to
infection by several viruses. Tomato spotted wilt virus (TSWV) is common in
north Florida tomato production areas and is occasionally found in south
Florida. Tomato plants with typical tospovirus symptoms (including necrotic
flecking, ring patterns, irregular chlorotic areas, and deformation of leaves;
and necrotic lesions on the epidermis of petioles and stems) were seen in
November 2009 and February 2010 in south Florida, after being observed
sporadically for about a decade. Several thrips species were observed in these
recent symptomatic tomatoes including Frankliniella bispinosa, F.
occidentalis, F. schultzei and Thrips palmi. No TSWV was detected by
ELISA or RT-PCR tests in samples collected at either time but infection by
another tospovirus was indicated by ELISA and RT-PCR tests using broad
spectrum tospovirus antiserum and degenerate tospovirus primers,
respectively. Subsequent ELISA tests did not detect other tospoviruses known
to occur in the U.S. However, ELISA tests using an antiserum that reacts with
both Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus were
S134
PHYTOPATHOLOGY
positive. A 697 nt fragment of the nucleocapsid (N) gene sequence amplified
by RT-PCR was >95% identical to GRSV N gene sequences in Genbank
confirming the presence of GRSV. Additional characterization of the Florida
GRSV isolate from tomato is ongoing.
Rapid micro-dilution broth assay for evaluating in vitro fungicide
resistance in Botrytis cinerea
D. E. WEDGE (1), K. J. Curry (2), B. Kreiser (2), A. Curry (2), M. Abril (3),
B. J. Smith (4)
(1) USDA ARS NPURU, University, MS, U.S.A.; (2) Department of
Biological Sciences, University of Southern Mississippi, Hattiesburg, MS,
U.S.A.; (3) Department of Biological Sciences, University of Southern
Mississippi, Baton Rouge, LA, U.S.A.; (4) USDA ARS Southern
Horticultural Laboratory, Poplarville, MS, U.S.A.
Phytopathology 100:S134
Recently strawberry growers in southeastern Louisiana reported a failure of
fungicide spray programs to control Botrytis fruit rot. Since Botrytis cinerea
has become resistant to several commonly used fungicides classes we
suspected chemical insensitivity. A 96-well micro-dilution broth assay using a
3-point dose response protocol developed for fungicide discovery was used to
provide growers with a rapid assessment of the fungicide sensitivity profiles
of 13 Botrytis isolates; 12 obtained from the strawberry farms and a control
isolate obtained from blueberry. Fungicide sensitivity profiles were
established for each of 13 isolates against 11 fungicides based on mean
percent growth inhibition. We identified 3 phenotypes in the sensitivity
profiles to benzimidazole and dicarboximide fungicides: benzimidazole and
dicarboximide resistant, benzimidazole resistant and dicarboximide sensitive,
and those with benzimidazole and dicarboximide intermediate resistance.
Codon at position 198 in the -tubulin gene confirmed benomyl resistance of
10 of 11 strawberry isolates. Traditional fungicide sensitivity assays are
tedious, time consuming, and often large pathogen populations are sampled
that do not provide a rapid answer for extension personnel or growers. Our
assay provides a method to rapidly obtain resistance and sensitivity
information and allow growers to incorporate important chemical disease
management strategy during the disease cycle when they need it.
Brassica juncea seed meal amendment induces long-term suppressiveness
to Pythium abappressorium under enclosed and open soil incubation
conditions
M. WEERAKOON (1), A. Izzo (2), M. Mazzola (3)
(1) Washington State University, Wenatchee, WA, U.S.A.; (2) Elon
University, Elon, NC, U.S.A.; (3) USDA ARS, Wenatchee, WA, U.S.A.
Phytopathology 100:S134
Pythium spp. contribute to development of apple replant disease. B. juncea
seed meal (SM) soil amendment can effectively suppress Pythium via
generation of biologically active allyl isothiocyanate (AITC). AITC is
evacuated from soils within 48 h after SM application, yet preliminary
evidence indicates that long-term control of this pathogen may be attained.
Greenhouse trials were conducted to assess the capacity of B. juncea SM to
suppress P. abappressorium in AITC evacuated soils. SM was incorporated
into soil at a rate of 0.3% (wt\wt); one system was maintained under enclosed
conditions and the other open to the atmosphere for two days. Soils were then
incubated in slightly covered containers to maintain moisture conditions. Soils
were incubated for 2, 4 and 8 weeks and then infested with P. abappressorium. Regardless of the time of pathogen introduction after B. juncea SM
amendment, disease suppression was consistently observed under both
enclosed and open initial incubation conditions. However, disease suppression
was significantly greater in soils incubated under enclosed conditions for the
initial two days relative to soils incubated under open conditions during the
same period. Evacuation of AITC from these soils prior to infestation with P.
abappressorium suggests that disease suppression does not operate completely
via chemical means. Disease suppression was associated with distinct changes
in the resident fungal community prior to pathogen introduction.
Validation of the accuracy of single-kernel near-infrared technology to
sort winter wheat kernels based on scab and deoxynivalenol levels
S. WEGULO (1), K. Peiris (2), P. Baenziger (1), F. Dowell (2)
(1) University of Nebraska, Lincoln, NE, U.S.A.; (2) USDA-ARS, Manhattan,
KS, U.S.A.
Phytopathology 100:S134
Fusarium head blight (scab) of wheat caused by Fusarium graminearum
lowers yield and grain quality. F. graminearum also produces the mycotoxin
deoxynivalenol (DON) which contaminates grain. For purposes of quality
assurance, DON concentration usually is determined in grain or the products
made from it. One of the methods commonly used to measure DON is gas
chromatography (GC). Although GC is accurate, it is time consuming, costly,
and destructive since grain must be ground to flour before DON can be
measured. For purposes such as breeding for resistance to FHB, it may be
sufficient early in the breeding program to know only whether a given line
accumulates low or high DON. A rapid, inexpensive, and non-destructive
method is needed for such purposes. This study validated the accuracy of two
single-kernel near-infrared (SKNIR) systems to sort winter wheat kernels
based on scab and DON levels. Both SKNIR systems accurately discriminated
between wheat kernels with low and high DON levels.
An investigation into mixed infections by potato purple top and potato
witches’-broom phytoplasmas in tomato
W. WEI (1), W. Wu (1), I. Lee (1), R. E. Davis (1), R. A. Owens (1), D. L.
Nuss (2), Y. Zhao (1)
(1) Molecular Plant Pathology Laboratory, ARS-USDA, Beltsville, MD,
U.S.A.; (2) Center for Biosystems Research, University of Maryland
Biotechnology Institute, Rockville, MD, U.S.A.
Phytopathology 100:S135
In nature, plants are often infected by two or more pathogens simultaneously.
Mixed infections may induce “atypical” symptoms that make precise visual
diagnosis difficult. The impact of mixed infections on the host can be severe,
especially when the co-infecting agents interact synergistically. Potato purple
top (PPT) and potato witches’-broom (PWB) phytoplasmas are newly
characterized pathogens that cause serious diseases in potato and other
vegetable crops. While PWB phytoplasma is a member of subgroup 16SrVIA, several PPT-associated phytoplasma strains belonging to subgroups
16SrVI-A, 16SrXII-A, 16SrIII-M, and 16SrIII-N have been identified. The
wide distribution of their insect vectors make mixed infections inevitable. In
the present work, we developed 16S rRNA gene sequence-based molecular
markers and a sensitive diagnostic tool to study mixed infections by a 16SrVIA PPT phytoplasma strain and a PWB phytoplasma strain in tomato plants.
The distribution and relative abundance of the two co-infecting phytoplasmas
were monitored over a 60-day post-infection time course and for five passages
in plants. Our results revealed that i) the two competing phytoplasmas differ
in fitness level for tomato; ii) interactions between the two phytoplasmas
induce new symptoms unseen in infection by either phytoplasma alone; and
iii) the severity of the symptoms is correlated with the relative abundance of
the two phytoplasmas.
Grafting as a disease management tool for fusarium wilt of heirloom
tomatoes in Arkansas
K. D. WELCH (1), J. C. Correll (1), J. C. Gavin (2)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) University of
Arkansas Cooperative Extension Service, Warren, AR, U.S.A.
Phytopathology 100:S135
The cultivation of grafted vegetables began in Korea and Japan in the early
20th century and has been adopted by many countries to control soilborne
pathogens. Grafting of tomatoes was adopted in the 1960’s. Fusarium
oxysporum f. sp. lycopersici (Fol), causes Fusarium wilt of tomato, and is an
economically important disease in the commercial tomato production area of
Arkansas. There are 3 known races of Fol and all 3 have been found in
Arkansas. While there are a number of fresh-market tomato cultivars grown in
Arkansas that have resistance to all 3 races, a number of heirloom tomatoes do
not have adequate resistance. The heirloom cultivar Bradley, developed as a
pink fresh-market tomato, is commonly grown in Arkansas and is valued for
its color, flavor, and stable price. Bradley is only resistant to race 1 of Fol.
The objective of this study was to determine if Bradley could be grafted onto a
rootstock resistant to races 1, 2, and 3 and provide control of Fol. The cultivar
Crista, resistant to all 3 races, was used as the rootstock. Ungrafted Bradley,
Bradley grafted onto Bradley, and ungrafted Crista served as controls. Disease
incidence and severity was evaluated under inoculation conditions in a
greenhouse test and in commercial fields naturally infested with Fol. If
disease control and adequate yields can be obtained, grafting Bradley onto a
resistant rootstock may provide a means of growing an heirloom susceptible
tomato in fields naturally infested with Fol.
Development of a national standard for virus certification of ornamental
and fruit tree nursery stock
R. Welliver (1), M. Hansen (2), N. OSTERBAUER (3)
(1) Pennsylvania Dept. of Agriculture, Harrisburg, PA, U.S.A.; (2) Michigan
Dept. of Agriculture, St. Joseph, MI, U.S.A.; (3) Oregon Dept. of Agric,
Salem, OR, U.S.A.
Phytopathology 100:S135
Since the 1960s, U.S. nurseries have traded virus-certified Malus, Pyrus,
Prunus, Chaenomeles and Cydonia nursery stock interstate and
internationally. A hindrance towards trade has been the lack of a harmonized
national standard for virus certification of such stock. Instead of reviewing a
national standard, importers must assess several unique state regulations
designed to meet their import requirements. Adoption of RSPM No. 35 by the
North American Plant Protection Organization has also obliged movement
towards a national standard. A subcommittee of the Fruit Tree Clean Plant
Network, including members of federal and state agencies, academia, and
industry met to develop such a standard in November 2009. A draft was
developed that contained several key elements including: 1) Harmonization of
the language used in state regulations for virus certification programs; 2)
Creating a two-tiered approach towards virus certification; and, 3) Setting
minimum standards for each tier of certification. Efforts were made to
harmonize the list of viruses of concern, taking regional differences into
account. Language describing the use of a systems approach to maintain viruscertified status was included. A pilot study will be conducted in Pennsylvania,
Michigan, and Oregon to assess the efficacy of the proposed national
standard. It is anticipated the presence of the standard will facilitate trade
interstate and with other countries throughout the world.
Mutational analysis of the putative pipo of Soybean mosaic virus with
emphasis on symptom expression and virus accumulation
R. Wen (1), B. He (1), M. R. HAJIMORAD (1)
(1) University of Tennessee, Knoxville, TN, U.S.A.
Phytopathology 100:S135
The function(s) of pipo, a newly discovered open reading frame (ORF)
embedded in the P3 cistron of potyviruses, is largely unknown. The putative
pipo of Soybean mosaic virus (SMV) is 225 nucleotides long, encoding for 75
amino acids, and has a GA6 motif at its 5′-end. We recently showed that
disruption of SMV PIPO protein, without substitution in polyprotein ORF, did
not abolish virus replication, but restricted the resultant mutants to small foci
of infected cells within the inoculated leaves. Furthermore, extensive
mutagenesis of the conserved GA6 motif also generated two movementdefective pipo-mutants. We are now studying the putative pipo of SMV by
mutational analysis to find out whether it influences the expression of
symptom severity and enhancement of virus accumulation. To achieve these
goals, the differential interactions of three SMV strains with soybean cv.
Williams82 are being exploited. SMV-N induces severe symptoms and
accumulates to a high level in systemically infected leaves, while SMV-G7
and SMV-G7d accumulate at relatively lower levels and provoke mild
symptoms. Interestingly, SMV-N PIPO protein differs from those of SMV-G7
and SMV-G7d by three amino acid substitutions whereas PIPO protein of
SMV-G7 is differentiated from that of SMV-G7d by a single residue.
Analyses of reciprocal exchanges of the unique amino acid residues of PIPO
proteins of these strains, individually or in combination without substitutions
in polyprotein ORF, are underway.
Impact of Zebra Complex disease on the development of potato plants
from seed-borne infection of ‘Candidatus Liberibacter solanacearum’
A. WEN (1), X. Wang (1), J. S. Pasche (1), N. C. Gudmestad (1)
(1) North Dakota State University, Fargo, ND, U.S.A.
Phytopathology 100:S135
An emerging disease of potatoes known as ‘Zebra Chip’ or ‘Zebra
Complex’(ZC), is putatively caused by the fastidious, phloem-limited
bacterium ‘Candidatus Liberibacter solanacearum’ (Lso), vectored by potato
psyllid, Bactericera cockerelli. Although ZC is spreading, the role of seedborne ZC in dissemination of the disease is not known. Seed tubers from the
same certified seed lot, with and without ZC symptoms, were grown in the
greenhouse to assess the impact of the disease on germination, progeny tuber
production and chip discoloration. The presence of Lso was tested in seed
tubers, foliage and progeny tubers. Lso was detected in both ZC-symptomatic
and asymptomatic seed, however, Lso concentration in asymptomatic seed
was substantially lower. Significant differences in emergence between ZCsymptomatic and asymptomatic seed were observed. Plants emerged from
non-ZC seed appeared healthy with 99% emergence while plants emerged
from ZC-seed displayed typical ZC symptoms with 42% emergence. No
plants grown from asymptomatic seed were Lso positive, whereas 29/64
plants derived from ZC-seed were Lso positive. A high percentage (99%) of
progeny tubers from non-ZC seed was asymptomatic, whereas among plants
from ZC-seed, only 44% produced progeny, 34% of which displayed ZC
symptoms. None of the progeny tubers from asymptomatic seed were Lso
positive, however, 11/43 of progeny tubers from ZC-seed were positive. Chip
discoloration in progeny tubers of ZC-seed generally was more severe than
those from non-ZC seed.
Alternative fumigants for management of root-knot nematode on carrots
B. B. WESTERDAHL (1), J. S. Gerik (2)
(1) Nematology Dept., University of California, Davis, CA, U.S.A.; (2)
USDA, ARS, San Joaquin Valley Agricultural Center, Parlier, CA, U.S.A.
Phytopathology 100:S135
Two field trials were conducted to evaluate the effectiveness of iodomethane
(MI) and chloropicrin (CP) for management of root-knot nematode,
Meloidogyne javanica, on carrots. Treatments in both trials were CP at 337
kg/ha, a 50/50 formulation of MI/CP at 374 kg/ha, a 33/67 formulation of
MI/CP at 122, 187, and 288 kg/ha, and a water treated control. Treatments
Vol. 100, No. 6 (Supplement), 2010
S135
were applied pre-plant, under tarp, via drip irrigation, and each trial consisted
of 5 replications in a randomized complete block design. In the first trial,
compared to the control, all MI/CP treatments increased yield of marketable
carrots based on both number of carrots and weight of carrots (95%). MI/CP
at 187 kg/ha (95%) had lower levels of root-knot nematode present at harvest
than the control. In the second trial, compared to the control, MI/CP at 187
and 288 kg/ha increased yield of marketable carrots based on both number of
carrots and weight of carrots (95%). MI/CP at 288 kg/ha (90%) had lower
levels of root-knot nematode present at harvest than the control.
Population density development of Heterodera schachtii under
susceptible, resistant and tolerant sugar beet cultivars
A. WESTPHAL (1), M. Daub (2)
(1) Julius Kühn-Institut. Federal Research Centre for Cultivated Plants,
Toppheideweg 88, D- 48161 Münster, GERMANY; (2) Julius Kühn-Institut,
Federal Research Centre for Cutlvated Plants, Dürener Strasse 71, D-50189
Elsdorf, GERMANY
Phytopathology 100:S136
Heterodera schachtii is a severe problem in sugar beet. Cultural management
of H. schachtii may be facilitated by the use of resistant and tolerant sugar
beet cultivars. To examine effectiveness of such cultivars, we monitored
population dynamics of H. schachtii in 30 cm-diameter plots, infested with
550 H. schachtii eggs/100 g of soil at different depths: 0-60 cm, 0-30 cm, or
30-60 cm; non-inoculated layers and the control were filled with non-infested
soil. Plots were planted to a susceptible, resistant or tolerant sugar beet
cultivar. Root penetration rates of seedlings by juveniles of H. schachtii were
similar for the cultivars. Early canopy diameter was larger in non-inoculated
than in entire column-inoculated plots. Final population densities were
cultivar-specific as expected due to their host suitability and independent of
initial inoculation depth. White sugar yields were highest in non-infested soil,
next highest in the deep-infested soil, and lowest in the all-layers-infested soil.
In two trials in 1 m2 naturally infested with H. schachtii at 0-60 cm depths,
populations were suppressed with fosthiazate at 30 cm-depth layers reciprocal
to the artificially inoculated ones of the first trial. In one of these trials, yields
were highest under nematicide treatment and lowest under non-treated. In
both types of trials, deep-occurring populations of H. schachtii reached sugar
beet seedlings and caused damage. Deep-occurring populations of H.
schachtii must not be ignored in sustainable sugar beet production.
Sampling for pod rot of peanut
T. A. WHEELER (1), J. E. Woodward (2), S. A. Russell (3), M. G. Cattaneo (4)
(1) Texas AgriLife Research, Lubbock, TX, U.S.A.; (2) Texas AgriLife
Extension, Lubbock, TX, U.S.A.; (3) Texas AgriLife Extension, Brownfield,
TX, U.S.A.; (4) Texas AgriLife Extension, Seminole, TX, U.S.A.
Phytopathology 100:S136
Two peanut fields with a history of pod rot were sampled weekly at 101
randomly chosen locations. Each sample location covered 0.5 m of row. The
objective was to determine a sampling intensity which would adequately
estimate pod rot for three treatment thresholds. Low, medium, and high
treatment thresholds were chosen at ≥1%, ≥2.5%, and ≥5.5% pod rot. Pythium
pod rot was the primary disease at both sites. From the sampled locations, 5,
10, 15, 20, 25, 35, and 50 sample points were selected at random from the data
sets, with 10 simulations for each sampling intensity. In general, when a
threshold number was close to the average pod rot, there were more wrong
threshold decisions than when the average pod rot was far away from a
threshold number. There were more wrong decisions for the high threshold,
compared with the moderate and low thresholds. It was more difficult to be
accurate at the higher percentage of pod rot than the lower disease levels when
sampling up to 50 locations (of the 101 sampled points). For the low and
moderate thresholds, 15 sampling points was as likely to be accurate as higher
sampling intensity, and more likely to be accurate in terms of being over or
under threshold than with lower sampling intensity. Crop consultants must
determine how intensive to sample to identify disease problems timely, but
must also maintain profitability while scouting fields, by minimizing time
spent in a field. This project is aimed at assisting consultants.
Root susceptibility and inoculum production from roots of eastern oak
species to Phytophthora ramorum
T. L. WIDMER (1), N. Shishkoff (2), S. Dodge (2)
(1) USDA ARS FDWSRU, Frederick, MD, U.S.A.; (2) FDWSRU/
ARS/USDA, Frederick, MD, U.S.A.
Phytopathology 100:S136
Little is known about root susceptibility of eastern tree species to
Phytophthora ramorum. In this study, we examined root susceptibility and
inoculum production from roots. Roots of sprouted acorns for several eastern
oak species were exposed to zoospore suspensions of 1, 10, 100, or 1000
zoospores per ml at 20°C. After 24 h, roots were removed, rinsed in water,
planted in pots and placed in the greenhouse. After 4 weeks, the roots were
S136
PHYTOPATHOLOGY
surface sterilized and plated on PARPH+V8 medium. A root was recorded as
positive if P. ramorum was observed on the medium. Infection of oak radicles
occurred at a concentration as low as 1 zoospore per ml. Differences were
observed among the species tested. To test inoculum production, the roots of
oak seedlings were inoculated with sporangia, washed after 24 hr and
transplanted into 2 × 2 inch pots containing Turface®. Periodically, 20-25 ml
samples of runoff were collected from each pot and plated on PARPH; the
resulting colonies were counted. Counts from oaks were compared to a
positive control, Viburnum tinus, using regression analysis. Root segments
were plated to calculate percent colonization. After 16 days, inoculum
production from oak seedlings was variable and lower than V. tinus, as was
colonization of roots. After 35-days, results were similar. This study shows
that sprouted oak acorns are very susceptible to P. ramorum and may be
important epidemiologically under natural environmental conditions.
Efficacy of new fungicides for control of powdery mildew (Erysiphe
necator) and downy mildew (Plasmopara viticola) of grapes
W. F. WILCOX (1), D. G. Riegel (1)
(1) Cornell University, NY State Agr Expt Sta, Geneva, NY, U.S.A.
Phytopathology 100:S136
Materials representing two new classes of fungicides, metrafenone (220 g/ha)
and fluopyram, provided superior control of powdery mildew relative to
current products. Fluopyram was more effective at 180 vs 86 g/ha; the lower
rate mixed with 88 g/ha tebuconazole was equivalent to the higher rate solo.
Among newly-available DMI fungicides, tetraconazole (44 g/ha) and flutriafol
(73-91 g/ha) were roughly equivalent and often modestly superior to
traditional DMIs; flutriafol was significantly more effective at 225 g/ha than
at the lower rates. Difenoconazole (128 g/ha) was significantly more effective
than all other DMIs; perhaps causally, EC50 values of individual E. necator
isolates averaged 29-fold lower (i.e., more active) than those for myclobutanil.
Tank mixing difenoconazole with cyprodinil (368 g/ha) did not improve
control, and cyprodinil solo gave modest to no control, depending on pressure.
Fluopicolide, BAS 651 (ametoctradin + dimethomorph), and mandipropamid
(146 g/ha) at 14-day spray intervals all provided excellent control of downy
mildew under high disease pressure. Fluopicolide was numerically but not
statistically more efficacious at 140 vs. 105 g/ha; tank-mixing with copper
hydroxide did not improve its performance. BAS 651 was equivalent at 422 vs
537 g/ha and at 10- vs 14-day intervals. Cyazofamid (80 g/ha) solo was
slightly less effective than the above, but was equivalent when tank-mixed
with a phosphite product.
Global gene expression analysis of Pseudomonas syringae during
epiphytic and endophytic growth
J. L. WILLIAMS (1), X. Yu (2), R. Scott (3), S. P. Lund (2), A. R. Records
(4), G. A. Beattie (2), S. E. Lindow (3), D. C. Gross (1), D. Nettleton (2)
(1) Texas A&M University, College Station, TX, U.S.A.; (2) Iowa State
University, Ames, IA, U.S.A.; (3) University of California- Berkeley,
Berkeley, CA, U.S.A.; (4) University of Maryland, College Park, MD, U.S.A.
Phytopathology 100:S136
Pseudomonas syringae is an important plant pathogen with a prominent
epiphytic phase that serves as inoculum for subsequent infection. To obtain a
comprehensive understanding of the genetic networks contributing to
saprophytic and pathogenic growth, we are performing whole genome
transcriptional profiling of P. syringae pv. syringae B728a, a pathogen of
bean, using an ORF-based microarray. Gene expression of the wild-type strain
and ahlR, aefR, gacS, salA, retS, rpoE, rpoN, rpoS, and hrpL mutants are
being assessed on leaf surfaces and in the leaf apoplast as well as in culture in
a basal medium and under conditions of low iron, nitrogen and water
availability and high oxidative stress. Total RNA was collected from cells
recovered from epiphytic and endophytic sites from at least 600 and 60 leaves,
respectively, to obtain yields sufficient for analysis. Preliminary results have
indicated that the B728a cells experience distinct environments during these
distinct growth phases based on differences in gene expression. For example,
genes involved in flagellar motility and type VI secretion were expressed
more in epiphytic sites, whereas genes involved in syringomycin production,
levan synthesis, uptake of quaternary ammonium compounds, GABA
degradation and tolerance to water stress and nitrogen limitation were
expressed more in endophytic sites. The importance of the targeted regulatory
networks to the distinct growth phases of B728a is currently being evaluated.
Phenotyping the components of resistance as a bottleneck to breed rice
varieties with suitable resistance to sheath blight
L. WILLOCQUET (1), M. Noel (1), N. Magculia (1), J. Lore (2), A.
Srinivasachary (1), S. Savary (1)
(1) IRRI, Los Banos, PHILIPPINES; (2) PAU, Ludhiana, INDIA
Phytopathology 100:S136
Sheath blight (ShB) caused by Rhizoctonia solani, is a rice disease causing
important yield losses, particularly under intensive production systems. Host
plant resistance to ShB can represent a very efficient, pro-poor, and
environment-friendly way to manage this disease. No high resistance against
ShB has been deployed in Asia. Phenotyping is a key component in breeding
programs aiming at improving host plant resistance, particularly when dealing
with quantitative resistance, as in the case of ShB. We present here a
framework that allows to measure host plant resistance from physiological
resistance and from disease escape. A phenotyping method under controlled
conditions was developed to measure the components of physiological
resistance in terms of number of lesions, disease spread, and lesion expansion.
A phenotyping procedure in microfields was also developed to measure the
combined effects of physiological resistance and disease escape on disease
intensification and spread. The results obtained from a series of tests
performed on a range of rice genotypes, and using both approaches (controlled
conditions phenotyping, microfields), allow assessing the relative
contributions of physiological resistance and disease escape to the overall
resistance of rice to ShB.
Multi-state evaluation of integrated management strategies for Fusarium
head blight and deoxynivalenol in small grain
K. T. WILLYERD (1), C. Bradley (2), A. Grybauskas (3), D. Hershman
(4), L. Madden (1), M. McMullen (5), L. Osborne (6), L. Sweets (7), P. Paul
(1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.; (2) University of
Illinois, Urbana, IL, U.S.A.; (3) University of Maryland, College Park, MD,
U.S.A.; (4) University of Kentucky, Princeton, KY, U.S.A.; (5) University
of Minnesota, St. Paul, MN, U.S.A.; (6) South Dakota State University, Brookings, SD, U.S.A.; (7) University of Missouri, Columbia, MO,
U.S.A.
Phytopathology 100:S137
Field experiments were conducted in multiple states between 2006 and 2009
to determine the magnitude of Fusarium Head Blight (FHB) and
deoxynivalenol (DON) reduction achieved by integrating multiple
management strategies, relative to a single strategy. Experiments used a
randomized complete block design with a split-plot arrangement of fungicide
treatment and cultivars of wheat, barley or durum, depending on region. Some
trials incorporated previous crop for a split-split-plot arrangement. A
combination fungicide of prothioconazole and tebuconazole was applied at
anthesis to cultivars with varying levels of FHB susceptibility. Disease index
was assessed at soft dough and DON concentration was determined following
harvest. In general, fungicide, host resistance and cultivation of small grain
following a non-host crop reduced FHB and DON. However, the magnitude
and significance of individual and combined effects of these management
strategies varied among experiments. Compared to a susceptible check, the
moderately resistant cultivar reduced index by –4 to 98% and DON by 3 to
96%. Compared to untreated checks for each cultivar, fungicide alone reduced
index by 15 to 85% and DON by -157 to 74%. By combining resistance and
fungicide, index and DON were reduced by 26 to 99% and 23 to 82%,
respectively. A three-tier management approach of crop rotation, resistance
and fungicide reduced index and DON by 27 to 99% and 61 to 92%,
respectively.
Effectiveness of early-season fungicide programs for the control of
Sclerotinia homoeocarpa, the causal agent of dollar spot
C. WILSON (1), P. Koch (1), J. Kerns (1)
(1) University of Wisconsin-Madison, Madison, WI, U.S.A.
Phytopathology 100:S137
Dollar spot, caused by Sclerotinia homoeocarpa, is the most important
turfgrass disease in the United States with respect to fungicide expenditures.
Single early-season fungicide applications delay dollar spot symptom
development, but do not provide season long control of the disease. This field
study compares the efficacy of a conventional dollar spot fungicide program
to early-season programs. This study was conducted at the O.J. Noer
Turfgrass Facility and at Milwaukee C.C. in Wisconsin. Conventional
applications started June 1 and were applied on 14-day intervals using full
label rates of propiconazole and chlorothalonil. Early-season treatments were
applied May 1, followed up with applications of a tank mixture of
propiconazole and chlorothalonil at either ¾ rates every 21 days or full label
rates applied on 28-day intervals. Treatments were arranged in a randomized
complete block design with four replications with individual plots measuring
2.8 m2. Disease severity was rated visually by counting individual dollar spot
foci every two weeks. The 21-day early-season program suppressed dollar
spot development, but not to acceptable levels (<5% disease severity). The 28day early-season program provided an excellent suppression that was
comparable to the conventional program. One fungicide application could be
eliminated by using a 28-day early season program rather than a 14-day
conventional program, reducing fungicide expenditures and environmental
inputs.
Effects of temperature on growth of Sclerotinia homoeocarpa
C. WILSON (1), J. Kerns (1), D. Smith (2)
(1) University of Wisconsin-Madison, Madison, WI, U.S.A.; (2) Oklahoma
State University, Stillwater, OK, U.S.A.
Phytopathology 100:S137
Dollar spot, caused by Sclerotinia homoeocarpa, is an important disease of
most turfgrass species worldwide. S. homoeocarpa was described almost a
century ago by F.T. Bennett. However, the basic biology and epidemiology of
the pathosystem is still unclear. Four isolates of S. homoeocarpa from WI and
6 isolates from OK were grown on native soils and sand. WI isolates were
grown with and without creeping bentgrass (Agrostis stolonifera) debris and
incubated at temperatures of 11, 14, 17, 20, 23, 26, 29, 31 and 34°C. OK
isolates were grown with creeping bentgrass debris only at temperatures of 15,
20, 25, 30, and 35°C. Radial growth of mycelia was recorded at 24, 48, 72,
and 96 hours. Growth for all isolates was most rapid between 17 and 26°C.
WI isolates grew best on native silt loam with bentgrass debris. Growth was
significantly reduced and highly variable at temperatures below 15°C. These
data suggest that S. homoeocarpa is strongly saprophytic, and that higher
temperatures (17–26°C) are conducive to growth. To assess pathogen
aggressiveness, 3 WI isolates and 6 OK isolates were inoculated on live
creeping bentgrass incubated at 14, 20, 26 or 34°C. Disease severity was
assessed every 24 hours. Four days post-inoculation, disease was most severe
at 14 and 20°C for all isolates, with average severity as high as 25%. These
initial data suggest that S. homoeocarpa infects creeping bentgrass between 14
and 26°C.
Exploring the role of nitroalkane dioxygenases in Magnaporthe oryzae
morphogenesis and infection
R. WILSON (1)
(1) University of Nebraska-Lincoln, Lincoln, NE, U.S.A.
Phytopathology 100:S137
Rice blast, mediated by Magnaporthe oryzae, is the most destructive disease
of rice. Foliar infection is mediated by a specialized infection structure called
the appressorium. Generation of cellular turgor in the mature appresorium
forces a penetration peg through the rice leaf cuticle and allows the fungus
access to the plant interior. Once inside the host cell, the fungus forms an
intimate association with the plant cell and is able to suppress or neutralize the
host defense response and grow unimpeded for the first 72 hrs of infection.
We are interested in understanding the molecular and cellular processes that
underlie this important interaction. Nitroalkanes are produced by some plant
species in response to pathogen attack, and can also result from ROS damage
to cellular alkanes such as fatty acids. We have identified five genes encoding
nitroalkane dioxygenases that could neutralize the harmful affects of
nitroalkanes produced during plant infection. Homologous gene replacement
of two of these genes affects sporulation and growth of the fungus. Moreover,
susceptibility to nitroalkanes is increased in one of these mutant strains during
growth on cysteine, compared to wild type. Together, this work suggests that
nitroalkane dioxygenases are important for the normal development of the
fungus and might protect M. oryzae against harmful products of the plant
defense system. Characterization of the remaining nitroalkane dioxygenases,
and their role in virulence, will be discussed.
Interactions of the endophyte Acremonium zeae and Aspergillus flavus in
maize hybrids in the field
G. L. WINDHAM (1), W. P. Williams (1)
(1) USDA ARS, Mississippi State, MS, U.S.A.
Phytopathology 100:S137
The maize endophyte Acremonium zeae has recently been shown to produce
pyrrocidines which are toxic to a number of fungi commonly found in maize
kernels including Aspergillus flavus. Field studies were conducted to
determine the effect of A. zeae on aflatoxin accumulation and A. flavus kernel
infection of two maize hybrids. Acremonium inoculation methods included
injecting conidia under the husks of ears, spraying conidia on silks, and
inoculating stalks just below developing ears with infested toothpicks.
Aspergillus flavus inoculation methods included the side-needle and spray
techniques. In 2007 in three experiments where A. zeae was inoculated using
three different methods, aflatoxin accumulation was significantly higher in A.
flavus-resistant hybrid plants inoculated with both A. zeae and A. flavus than
in plants inoculated with A. flavus alone. Aspergillus flavus kernel infection
was also significantly higher in plants inoculated with both fungi using the
side-needle technique compared to plants inoculated with A. flavus alone. In
2008, aflatoxin accumulation was significantly higher in the resistant hybrid
inoculated with both fungi using the side-needle technique compared to plants
inoculated with A. flavus alone. Although A. zeae has been reported as a
protective endophyte, we demonstrated it can act synergistically with A. flavus
to produce higher levels of A. flavus kernel infection and aflatoxin
accumulation in a resistant maize hybrid.
Vol. 100, No. 6 (Supplement), 2010
S137
Emergence and establishment of Cucurbit yellow stunting disorder virus in
California and Arizona poses a threat to desert melon production
W. M. WINTERMANTEL (1), R. L. Gilbertson (2), E. T. Natwick (3), J. K.
Brown (4)
(1) USDA ARS, Salinas, CA, U.S.A.; (2) Department of Plant Pathology,
University of California, Davis, CA, U.S.A.; (3) University of California
Desert Research and Extension Center, Holtville, CA, U.S.A.; (4) Department
of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S138
Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus, family
Closteroviridae) was identified in the large melon production region of the
American Desert Southwest (CA, AZ, SON) in fall 2006, and affected most
fall melon crops in the region. CYSDV is transmitted efficiently by the sweet
potato whitefly (Bemisia tabaci biotype B). Whitefly populations accumulate
gradually during the spring melon season, but reach high levels during the fall
melon season. Analysis of weeds and crops in and adjacent to infected fields,
along with subsequent laboratory studies, demonstrated that the host range of
CYSDV included not only cucurbits as previously believed, but also several
crop and weed plants native to the region. Many of these hosts do not exhibit
symptoms, but can be sources for virus transmission to crops. Over a period of
3 years, all fields in Imperial County, CA and fields in central Arizona were
monitored for CYSDV during both spring and fall production seasons, and
whiteflies from fields were tested for CYSDV. During this period, CYSDV
incidence in the fall crop was nearly 100%, resulting in a dramatic reduction
in fall production and yields. In contrast, incidence in spring melons was
initially low and limited to a small number of fields in 2007, but increased to
63% of fields by spring 2009, indicating establishment in native vegetation
and an increasing threat to the spring crop. Southwest production accounts for
80% of the U.S. cantaloupe crop and 96% of the U.S. honeydew melon crop.
quality (r = –0.77; P = 0.01). Under low disease pressure, however, cone
yield, alpha acid content, and quality were similar if fungicide applications
were made through 27 July. Cone color was negatively affected in treatments
that ended before this date. Further characterization of ontogenic resistance in
cones may enable control measures to be targeted to critical periods of cone
susceptibility and potentially reduce unnecessary fungicide applications.
Effects of irrigation and crop rotation on Verticillium wilt of cotton in
Texas
J. E. WOODWARD (1), T. A. Wheeler (2), J. P. Bordovsky (2)
(1) Texas AgriLife Extension Service, Lubbock, TX, U.S.A.; (2) Texas
AgriLife Research, Lubbock, TX, U.S.A.
Phytopathology 100:S138
Verticillium wilt, caused by the soilborne fungus Vertcillium dahliae, is an
increasingly important disease of cotton (Gossypium hirsutum L.) on the
Southern High Plains of Texas. Disease incidence is correlated with soil
populations of the fungus and management options are currently limited to the
use of partially resistant cultivars. The impact of other cultural practices on
disease development, or V. dahliae is unknown. Field trials were conducted to
determine the influence of three irrigation levels (Base, Base + 50% and Base
– 50%) on disease development under four rotation schemes (continuous
cotton, and three rotations containing sorghum (Sorghum bicolor L.)). Disease
incidence increased from 0.9 to 14.5% and 4.0 to 19.3% for the low and high
ET treatments in 2008 and 2009, respectfully. Populations of V. dahliae
increased from 2.7 to 50.7 cfu/cc soil over a three year period where cotton
was planted, whereas, populations remained similar where sorghum was
planted. These studies indicate that lower irrigation levels and rotation with a
non-host can negatively impact V. dahliae and result in lower disease
incidence. Additional studies are required to optimize the use of such practices
to ensure sustainable cotton production in fields infested with V. dahliae.
Response by Aspergillus flavus to a sublethal atmosphere of ozone
C. P. WOLOSHUK (1), L. Zhang (1), B. N. Reese (1), G. A. Payne (2)
(1) Purdue University, Department of Botany and Plant Pathology, West
Lafayette, IN, U.S.A.; (2) North Carolina State University, Raleigh, NC,
U.S.A.
Phytopathology 100:S138
Evaluation of the edge factor in epidemiology of zebra chip disease in
potato fields
F. WORKNEH (1), D. C. Henne (1), A. C. Childers (1), C. M. Rush (1)
(1) Texas AgriLife Research, Bushland, TX, U.S.A.
Phytopathology 100:S138
Ozone is a powerful oxidant with numerous beneficial applications. When
fungi are exposed to a sublethal atmosphere of ozone, surface growth and
development are inhibited. To better understand the molecular responses to
ozone, liquid cultures of Aspergillus flavus were grown for 3 days in an ozone
atmosphere. Fungal growth below the surface of the medium was not affected,
but aerial growth was suppressed. In contrast, cultures exposed to air
produced abundant aerial mycelia and conidia. When the ozone-treated
cultures were shifted to an air environment, aerial hyphae were visible after 4
h and conidia were visible after 24 h. Total RNA was isolated from cultures 0,
4, 12 and 24 h after removal from ozone and from cultures grown in an air
environment. The RNA was hybridized to microarrays that contain probes
representing 14,163 A. flavus genes. Expression profiles indicated that
transcription of hydrophobins and conidiation genes were significantly
reduced in the ozone-treated cultures. Among the few genes significantly upregulated in ozone-treated cultures was CAT5, one of five putative catalase
genes in A. flavus. Within 4 h after shifting the ozone-treated cultures to an air
environment, CAT5 expression decreased to control levels. Furthermore, when
cultures grown in air were shifted to the ozone environment, CAT5 expression
increased 3-fold after 4 h. Based on these results, we hypothesize that CAT5
has a role in protection against ozone.
Zebra chip (ZC), caused by the fastidious bacterium ‘Candidatus Liberbacter
solanacearum’, is an emerging disease of potatoes that affects all market
classes. Initially observed in Mexico in the mid 1990s, the disease now has
been identified in several potato producing regions of the U.S. including
Texas. The pathogen is vectored by the potato psyllid, Bactericera cockerelli,
and affected plants exhibit a variety of foliar symptoms, and unacceptable
discoloration of fried potato products (chips and french fries). In 2009, studies
were conducted in three fields in the Texas Panhandle to determine whether
there was a greater incidence of ZC on the edges of fields than within fields.
Plots of 20 m x 10 m were established around the edges of the fields (50 ha
each), approximately160 m apart (n = 15 to 18). The same number of plots
also were established 100 m inward (corresponding to individual plots on the
edges), and symptomatic plants in all plots were counted. In all three fields,
ZC incidence was significantly greater (P < 0.05) on the edges than in the
infield plots, and some directional effects were evident. In a separate study,
temporal progression of ZC on the edges was monitored in two fields in which
new symptoms were recorded weekly. In both fields, ZC incidence peaked 2
weeks after the first detection and declined steeply thereafter. The findings
suggest that greater emphasis in psyllid management should be directed
towards the edges of the fields for better results.
Ontogenic resistance to powdery mildew in hop cones: Implications for
disease management
J. L. WOODS (1), M. E. Nelson (2), G. G. Grove (2), D. H. Gent (3)
(1) USDA ARS, Corvallis, OR, U.S.A.; (2) Washington State University,
Prosser, WA, U.S.A.; (3) USDA-ARS/Oregon State University, Corvallis,
OR, U.S.A.
Phytopathology 100:S138
Evaluation of southern highbush blueberry cultivar and propagation
methods for stem blight mortality during the first year of growth in
Florida
A. F. WRIGHT (1), P. F. Harmon (1)
(1) University of Florida, Gainesville, FL, U.S.A.
Phytopathology 100:S138
Podosphaera macularis, causal agent of hop powdery mildew, can cause
substantial losses in crop yield and quality. Ontogenic resistance has been
described in leaves, although this resistance has not been examined in detail in
cones. Greenhouse produced cone tissues were inoculated on a time course to
assess their susceptibility to powdery mildew in different developmental
stages. Field-based fungicide programs also were evaluated to determine the
impact of omitting late-season fungicide applications on cone yield, bittering
acids, and quality factors. In greenhouse assays, flowers were highly
susceptible to powdery mildew, but susceptibility of bracts and bracteoles
decreased linearly with increasing cone maturity. In fungicide trials conducted
under high disease pressure, there was a tendency for later season fungicide
applications (up to 24 August) to improve cone yield, alpha acid content, and
quality. The incidence of diseased cones was correlated with alpha acid
content (r = –0.62; P = 0.04), cone color (r = –0.62; P = 0.01), and aroma
S138
PHYTOPATHOLOGY
Stem blight is caused by fungi in Botryosphaeriaceae; infection results in
substantial southern highbush blueberry (SHB) mortality during the first two
years of growth. Cultivar and propagation method effects on Botryosphaeria
(Bot) disease incidence and severity were evaluated. Three commercial sites
were selected and are located in Alachua, DeSoto, and Polk, Co. FL. Plot
installation was a RCBD and consisted of four cultivars each propagated from
softwood cuttings (sw) and tissue culture (tc). The cultivars Emerald,
Premadonna, and Snowchaser were planted at all locations. Star was planted
at Alachua, and Jewel was planted at the DeSoto and Polk, Co. sites. Plots
were sampled periodically for stem blight, and data were analyzed with
ANOVA. For the Alachua Co. site very few plants died and the model was not
significant p > 0.45. At the Desoto and Polk Co. sites, cultivar and
propagation had a significant effect on plant mortality p < 0.05. Plants
propagated from tc survived more frequently and had less stem blight than
plants propagated from sw. Cultivar differences in Bot susceptibility observed
in this study are similar to reports of relative cultivar susceptibility in
production fields. New cultivar development presents a long-term potential
management option for stem blight. Additionally, industry-wide propagation
practices need to be reviewed and improved with the input of the industry and
results of further research.
AV2 protein encoded by Tomato yellow leaf curl China virus is a RNA
silencing suppressor
J. Wu (1), J. Zhang (1), X. ZHOU (1)
(1) Hangzhou, PRC PEOPLES REP OF CHINA
Phytopathology 100:S139
GFP green fluorescence in the leaves of 16c transgenic N. benthamiana plants
co-infiltrated with the A. tumefaciens harboring GFP gene and the A.
tumefaciens harboring Tomato yellow leaf curl China virus (TYLCCNV)
isolate Y10 AV2 gene could be observed and a narrow red ring around the
edge of infiltrated patch could also be found at 6 days post inoculation (dpi),
which indicated that TYLCCNV Y10 AV2 protein can suppress local RNA
silencing of GFP gene, but can not interfere with the spread of RNA silencing
signal of GFP gene by cell-to-cell. In addition, GFP green fluorescence could
be observed in the systemic leaves of 16c transgenic N. benthamiana plants
co-infiltrated with GFP and AV2 genes at 30 dpi, which suggested that
TYLCCNV Y10 AV2 protein can inhibit systemic RNA silencing of GFP
gene. Moreover, TYLCCNV Y10 AV2 protein could interfere with the spread
of systemic RNA silencing signal of GFP gene by agrobacterium-mediated
infiltration assay. The above results indicated TYLCCNV Y10 AV2 protein is
a RNA silencing suppressor. TYLCCNV Y10 AV2 protein is a pathogenicity
determinant in the PVX heterogenous system. Northern blot analysis indicated
that the concentration of PVX RNA was higher in the N. benthamiana
infiltrated with PVX-AV2 than that in the N. benthamiana infiltrated with
PVX. It could be speculated that the higher concentration of PVX in the N.
benthamiana infiltrated with PVX-AV2 was due to the AV2 protein
suppressing the antivirus RNA silencing of the plants.
Effects of sampling methods on the assessment of populations of
Xanthomonas hortorum pv. carotae on carrot plants and on harvested
carrot seeds
B. M. WU (1)
(1) Oregon State University, Madras, OR, U.S.A.
Phytopathology 100:S139
To optimize sampling techniques for assessing population levels of
Xanthomonas hortorum pv. carotae (Xhc) in carrot seed crops, populations of
Xhc on carrot plant parts and on harvested carrot seeds were estimated by
using different sampling methods. The influence of sample methods,
including the number of samples, sample size (the dry weight and the number
of plants per tissue sample, or the number of seeds per seed sample), and
number of subsamples, on the estimation of mean bacterial population size
(Log CFU) per gram dry tissue or dry seed and on the variability among
sampling units was investigated. In general, bacterial populations were highly
variable among sampling units. The variation in population size of Xhc was
greater (0.53 to 3.59 Log CFU) among plants than among subsamples (0.007
to 0.48 Log CFU) suggesting that a good sample method should collect
samples from as many plants as possible, and increasing number of
subsamples or the number of medium plates for enumeration cannot improve
the estimate of population size as much as increasing the number of
plants sampled. The sample size (the number of seeds per sample) is
important in estimation of the bacterial population on harvested seeds, and a
large sample size is especially important to accurately estimate the bacterial
population for seed lots with low infestation levels. The population sizes
among sample units sometimes, but not consistently, followed a lognormal
distribution.
Determining the development of practical fungicide resistance in cucurbit
powdery mildew of pumpkin
C. A. WYENANDT (1)
(1) Rutgers University, Bridgeton, NJ, U.S.A.
Phytopathology 100:S139
In 2006 and 2007, nine fungicides were evaluated to determine if practical
fungicide resistance could be identified in cucurbit powdery mildew
(Podosphaera xanthii) following five different season-long fungicide
programs using different modes-of-action and rotations. The nine fungicides
evaluated included: sulfur (FRAC code M2), chlorothalonil + myclobutanil
(M5 + 3), famoxadone + cymoxanil (11 + 27), myclobutanil (3), azoxystrobin
(11), pyraclostrobin + boscalid (11 + 7), pyraclostrobin (11), quinoxyfen (13),
chlorothalonil (M5) and a control (water only). Based on percentage of leaf
surface with symptoms of powdery mildew, a FRAC code 11 resistance
cucurbit powdery mildew population developed where a FRAC code 11
fungicide had not be applied directly season-long, or where a FRAC code 11
fungicide had been applied weekly or in rotation with another fungicide
chemistry. Resistance did not develop where a FRAC code 3 fungicide had
been applied season-long, or in rotation, or where no FRAC code 3 fungicide
had been applied season-long. Control was improved with a FRAC code 7 +
11 fungicide, suggesting that control of the cucurbit powdery mildew
population in this study was achieved with the FRAC code 7 fungicide. In
both years, of the nine fungicides evaluated during this study, end of season
cucurbit powdery mildew control was best with a FRAC code 3, 11 + 7, or 13
fungicide.
Sensitive and cost-effective immunocapture RT-PCR for routinely viral
detection in large number of plant samples
J. Q. XIA (1), K. Ling (2)
(1) AC Diagnostics, Inc., Fayetteville, AR, U.S.A.; (2) USDA-ARS, U.S.
Vegetable Laboratory, Charleston, SC, U.S.A.
Phytopathology 100:S139
It has long been a challenging task to develop timely and accurate diagnostic
tests for diseases caused by viruses or virus-like agents in plants in agriculture
production. Current ELISA detection methods may not provide the sensitivity
needed for samples with low viral concentrations. Conventional RT-PCR,
which requires relatively pure nucleic acid samples, is costly and timeconsuming. By combining two widely used virus detection methods, ELISA
and RT-PCR, Immunocapture RT-PCR was developed for practical detection
of plant viruses. The immunocapture sample preparation allows RT-PCR to be
performed in a 96-well format equivalent to that of a regular ELISA test.
The entire RT-PCR assay, viral immunocapturing from samples, captured
viral RNA amplification and amplicon detection, can be conducted in a
single PCR reaction tube within a relatively short time. This novel technology makes it possible to routinely detect plant viruses in large number
of samples by RT-PCR. This sensitive and cost-effective immunocapture RT-PCR assay has huge potential applications in plant viral disease
diagnosis.
Development of commercial products based on immunocapture RT-PCR
technology
J. XIA (1), C. Feng (2), K. Ling (3)
(1) AC Diagnostics, Inc., Fayetteville, AR, U.S.A.; (2) Dept. of Plant
Pathology, University of Arkansas, Fayetteville, AR, U.S.A.; (3) USDA-ARS,
U.S. Vegetable Laboratory, Charleston, SC, U.S.A.
Phytopathology 100:S139
A sensitive, reliable and user-friendly commercial product has been developed
based on immunocapture RT-PCR technology. This diagnostic kit includes all
assay components and is ready to use. Pre-coated PCR tubes for capturing
virus particles which can be directly amplified by RT-PCR allows entire assay
to be performed in a single PCR tube. Sample is simply ground in a PCR
sample extract buffer and added to PCR plate/tubes pre-coated with antibody.
After incubation and washing, the captured target viral RNA is ready to be
amplified by RT-PCR. Robust and consistent result can be obtained every
time running a RT-PCR by using this product because all PCR inhibitors are
eliminated through viral immunocapture. This immunocapture RT-PCR
product provide one of the most sensitive diagnostic tools for an effective
detection of plant viruses in seeds, stock materials or other plant tissues of
economically important vegetables, fruits, ornamentals and field crops.
Cuticle plays an important role in basal as well as induced defense
against bacterial and fungal pathogens
Y. XIA (1), Q. Gao (1), K. Yu (1), A. Kachroo (1), P. Kachroo (1)
(1) University of Kentucky, Lexington, KY, U.S.A.
Phytopathology 100:S139
Systemic acquired resistance (SAR) is a phenomenon in plants that confers
protective immunity in the distal tissues towards secondary infections by
related or unrelated pathogens. SAR involves the generation of mobile signal
(s) at the site of primary infection, which then translocates to, and activates
defense responses in the distal tissues. Although several signals have been
implicated to play a role in SAR, the signaling events leading to activation of
SAR still remains unclear. Recently, we showed an intact cuticle is required
for decoding of the mobile signal in the distal tissues. Genetic mutations
leading to abnormal cuticle or physical damage of cuticle on the distal leaves
compromised SAR. The requirement for intact cuticle was only relevant
within the time frame of mobile signal generation and translocation to the
distal tissues. Since most mutations affecting cuticle development also impair
fatty acid (FA) and/or lipid biosynthesis, we studied a role for these in SAR.
Our results show impaired biosynthesis of FAs or lipids do not contribute to
SAR. we have uncovered several mutations that specifically alter cuticle
without influencing FA or lipid biosynthesis and they were impaired in SAR.
Besides SAR, most mutants with abnormal cuticle showed enhanced
susceptibility to necrotrophic fungal pathogens and this phenotype did not
correlate with cuticular permeability. The studies demonstrate an important
role for cuticle in induced as well as basal defense responses.
Vol. 100, No. 6 (Supplement), 2010
S139
Gene regulation during asexual development in the oomycete
Phytophthora infestans
Q. XIANG (1), H. Judelson (1)
(1) Dept. of Plant Pathology and Microbiology, University of California,
Riverside, CA, U.S.A.
Phytopathology 100:S140
Phytophthora infestans, the causal agent of potato and tomato late blight,
undergoes a serial of distinct morphological differentiations during asexual
reproduction. Our study focuses on identifying the genes involved in
differentiation, and the transcription factors and cognate promoter binding
sites that are responsible for the expression of these genes. Microarray studies
revealed that thousands of genes were up-regulated in different stages of
asexual reproduction. These genes encode proteins involved in flagellar
function, vesicle transport, protein posttranslational modification, signaling
and other activities that may be important in asexual development. Dissections
of the promoters of several genes identified transcription factor binding sites
required for their developmental regulation. One of the binding sites is
CCGTTG, which is significantly enriched in promoters specifically active in
sporulation. As CCGTTG is known to bind MYB transcription factors from
animals and plants, we characterized the MYB transcription factor families
from four sequenced oomycete genomes. Several groups of MYB proteins
were predicted, with some resembling those found in plants and animals and
others having oomycete-specific configurations of their DNA binding
domains. Many of these myb genes are differentially expressed during asexual
development based on RT-PCR. Gene silencing and over-expression
experiments are being conducted to determine how MYB proteins regulate the
spore cycle.
Characterization of Sclerotiium rolfsii isolates affecting vegetables and
row crops in the southern U.S.
C. XIE (1), G. Vallad (1)
(1) University of Florida - Gulf Coast Res & Ed Ctr, Wimauma, FL, U.S.A.
Phytopathology 100:S140
Southern blight (caused by Sclerotium rolfsii Sacc.) is a serious fungal disease
affecting diverse crops grown around the world especially in tropical and
subtropical regions. While mostly a problem in the production of peanut in the
southern U.S., the disease is becoming more problematic in vegetable
production with the phase out of methyl bromide and the adoption of organic
and other low-input production strategies. An initial 38 isolates from peanut,
pepper, tomato, pumpkin, cantaloupe, watermelon, and a Ruellia sp. were
partially characterized for cultural morphology and mycelial compatibility. An
initial characterization of fourteen isolates found variation in the number and
size of sclerotia produced on PDA. Most peanut isolates produced less than
250 sclerotia per plate, while most isolates from other hosts produced more.
Sclerotia size ranged from from 0.60mm to 0.80mm in diameter for four
isolates from pepper, cantaloupe, and watermelon; whereas, 7 of 9 peanut
isolates ranged from 1.14mm to 1.49mm, with the remaining two isolates
measuring 0.76mm and 0.93mm in diameter. The 38 isolates were assigned to
14 MCGs. No common MCGs were observed among peanut and vegetable
isolates, with the exception of a single peanut isolate. Initial results suggest
vegetable isolates are distinct from peanut isolates, but further characterization
of additional isolates is in progress. Isolates will also be tested for differences
in virulence on peanut, pepper, and tomato.
Visualization of Clavibacter michiganensis subsp. michiganensis infection
of tomato seedlings using a bioluminescent strain
X. XU (1), G. Rajashekara (1), S. A. Miller (1)
(1) Ohio State University, OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S140
Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive
bacterium that causes severe economic losses in commercial tomato
production worldwide. The disease is transmitted from infected seed to
seedlings, and mechanically from plant to plant. To study Cmm movement
from tomato seed and seedlings, healthy tomato seeds were inoculated with a
virulent, bioluminescent Cmm strain (BL-Cmm17) by vacuum infiltration.
The transmission and localization of BL-Cmm17 from seed to seedling was
monitored under controlled conditions using an in vivo imaging system (IVIS)
and culturing. Our results indicated that Cmm aggregated on hypocotyls and
cotyledons at the early stage of germination. Cmm transmission was also
investigated in tomato plants inoculated by cotyledon clipping with BLCmm17. The bacterial infection process was visualized every 5 days. The
bacteria multiplied quickly in the petiole remaining after clipping the
cotyledon and reached a titre of 108 cfu/g fresh tissue 5 days post inoculation.
Translocation of bacteria to stem tissue was also observed with a titre of 106
to 107 cfu/g. The first true leaf was infected without symptoms 10 days post
inoculation. Leaf wilting was observed 15–20 days post inoculation and the
bacterial titre in stem reached 109 cfu/g.
S140
PHYTOPATHOLOGY
A screening strategy of fungal biocontrol agents towards Verticillium wilt
of cotton
L. Xu (1), J. GUO (1)
(1) Nanjing, PRC PEOPLES REP OF CHINA
Phytopathology 100:S140
Verticillium wilt caused by Verticillium dahliae Kleb. is one of the most
destructive diseases in cotton. A screening strategy was developed to assess
the potential biocontrol agents (BCAs) of this disease. 373 fungal isolates
were obtained from the endorhiza, rhizosphere, and bulk soil of cotton plants.
105 of them produced obvious inhibition zones against V. dahliae were
selected as the antagonists towards this pathogen. An assessment system was
established to evaluate these 105 antagonists for their biocontrol potential and
plant growth-promoting potential. Their biocontrol potential was assessed
according to their in vitro antagonistic activity against V. dahliae and
activities of plant cell wall degrading enzymes including protease, cellulase,
and chitinase. Their plant growth-promoting potential was assessed according
to their in vitro activities of solubilizing phosphate and fixing nitrogen. 33
antagonists received at least 3 points of the total assessed value of biocontrol
potential and plant growth-promoting potential; they were tested for
biocontrol efficacy and growth-promoting effect on cotton in greenhouse. 12
of them achieved positive biocontrol efficacy of 8.58–69.78% in greenhouse.
Their biocontrol efficacy is positively correlated with their assessed biocontrol
potential, the correlation coefficient is 0.926.
Biological characterization and complete genomic sequence of Apium
virus Y infecting celery
D. Xu (1), H. LIU (2), S. T. Koike (3), F. Li (1), R. Li (1)
(1) USDA-ARS, Beltsville, MD, U.S.A.; (2) USDA ARS, Salinas, CA,
U.S.A.; (3) University of California Cooperative Extension, Salinas, CA,
U.S.A.
Phytopathology 100:S140
Apium virus Y (ApVY) isolated from celery plants with ring spot and line
pattern symptoms from a commercial field in California was characterized in
this study. The experimental host range of the virus included 14 plant species
in the families Apiaceae, Chenopododiaceae and Solanaceae, and almost all
infected plant species showed foliar chlorosis and distortion or severe stunting
and systemic chlorosis. ApVY was transmitted to all 10 host species in the
Apiaceae by green peach aphids (Myzus persicae) and induced symptoms on
the inoculated plants. The virus reacted with the potyvirus group antibody and
Celery mosaic virus (CeMV) antiserum. The complete genomic sequence of
ApVY was determined to be 9917 nucleotides in length, excluding the 3’
poly(A) tail, and it comprises a large open reading frame encoding a
polyprotein of 3184 amino acid residues. Its genomic organization is typical
of potyviruses, and contains conserved motifs found in the genus Potyvirus.
Comparisons with available sequences of other potyviruses indicate that
ApVY shares 26.1–52.9% identities with species of the existing genera and
unassigned viruses in the Potyviridae at the polyprotein sequence level.
Extensive phylogenetic analysis based on 3’-partial sequences containing NIb
and CP genes confirms that ApVY is more closely related to CeMV and is a
distinct species of the genus Potyvirus.
Multiple infection of Sugarcane streak mosaic virus in a single sugarcane
plant and complete genomic sequences of two SCSMV genotypes
D. XU (1), G. Zhou (2), F. Li (1), R. Li (1)
(1) USDA ARS, Beltsville, MD, U.S.A.; (2) South China Agricultural
University, Guangzhou, PRC PEOPLES REP OF CHINA
Phytopathology 100:S140
At least four genetic variants of Sugarcane streak mosaic virus (SCSMV)
were identified from a single sugarcane accession (Co 6304) in the germplasm
collections of South China Agricultural University at Guangdong, China.
Complete genomic sequences of two Co6304 variants were determined to be
9782 nucleotides (nt). Comparison of the full-length sequences among these
variants and a Pakistan isolate shows that they share identities of only 87.5–
88.2% at the nt sequence level, but 97.9–98.2% at the polyprotein sequence
level. Most mutations are point and silent, resulting in highly conserved
polyprotein sequences. Similar results are also observed from all individual
genes except the coat protein (CP) gene. Comparison of the CP gene
sequences of four Co6304 variants and 62 isolates available in GenBank
reveals a wide range of divergences not only at the nt sequence level (0–
18.1%) but also at the amino acid sequence level (0–14.3%). Phylogenetic
analysis based the CP gene sequences shows that these isolates are grouped
into four distinct clusters. The two completely sequenced Co6304 variants
were equally dominant in the original host. However, only one of them was
detected after transmission to sorghum, indicating a strong genetic drift and
differentiation between host plants. These results provide evidence of
extensive variation and complicated population structures among SCSMV
genomes, and the data will be useful for the viral genotyping and control
strategy development.
A novel root-knot nematode secretory protein interacts with a Golgiassociated host plant protein
B. XUE (1), G. Huang (2), T. Baum (3), R. Hussey (2), E. Davis (1)
(1) North Carolina State University, Raleigh, NC, U.S.A.; (2) University of
Georgia, Athens, GA, U.S.A.; (3) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S141
Secreted parasitism proteins of the southern root-knot nematode (RKN),
Meloidogyne incognita, play crucial roles in modulating successful host root
infection and the formation of elaborate feeding cells called giant-cells. The
4D03 parasitism gene is expressed exclusively within the single dorsal
esophageal gland secretory cell of RKN parasitic life stages and encodes a
novel 191 amino acid secretory protein. The 4D03 genomic clone has three
introns of 50bp, 107bp, and 47bp within the predicted ORF and this gene also
exists in the genome of other RKN species. No phenotypic changes have been
observed when 4D03 was expressed in Arabidopsis thaliana. Plant hostderived RNA interference targeted to the 4D03 transcript in transgenic
Arabidopsis thaliana plants significantly reduced root galling induced by M.
incognita. A positive interaction of the 4D03 protein with a Golgi-associated
protein of tomato was detected in yeast two-hybrid screens and confirmed in
subsequent co-transformation experiments. The interacting Golgi-associated
protein has a predicted domain of the RGP (Reversibly Glycosylated
Polypeptide) superfamily that is putatively involved in polysaccharide
biosynthesis. The observed protein-protein interaction suggests that secreted
RKN 4D03 protein may play a functional role in feeding site establishment by
modulating wall synthesis in giant-cells.
Inoculum sources and spore survival in field soil of the sour rot pathogen,
Geotrichum candidum
M. A. YAGHMOUR (1), R. M. Bostock (1), J. E. Adaskaveg (2), T. J.
Michailides (3)
(1) UC Davis, Davis, CA, U.S.A.; (2) UC Riverside, Riverside, CA, U.S.A.;
(3) University of California, Parlier, CA, U.S.A.
Phytopathology 100:S141
Post-harvest decay of peaches and nectarines complicates storage and
shipment of fruits and has serious economic consequences. Geotrichum
candidum causes sour rot of fruit both in the field and after harvest. In some
packing houses, inferior but otherwise sound fruit treated with fungicide are
typically culled and returned to orchards where they are discarded between
rows in stone fruit fields. Sour rot developed significantly more on fruits
culled than on fruit collected from the same orchards without any treatment.
Depending on the variety, cull fruit with sour rot ranged from 3 to 26%. Since
cull fruits are a source of inoculum in the field and we have shown that it is a
source of propiconazole-insensitive isolates, a study on spore survival at two
different soil depths was undertaken to assess the possibility of burying cull
fruits in orchards as a measure to manage the disease. Over a one-year period,
spore populations of G. candidum at 20 cm depth were significantly less than
at 10 cm until March. At both depths, the population remained low after
March, declining to less than 2% of the initial populations until the last
sampling point in August. The results suggest that survival of spores from
decaying fruits in the orchard is minimal if those fruits are crushed and
plowed under, thus providing a safe way to dispose of culled fruit.
Incidence of Agrobacterium tumefaciens on walnut seeds used for
rootstock production: Implications for crown gall management strategies
L. E. YAKABE (1), S. R. Parker (1), D. A. Kluepfel (1)
(1) USDA ARS Crops Pathology/Genetics Research Unit, Davis, CA, U.S.A.
Phytopathology 100:S141
Crown gall of walnut, caused by Agrobacterium tumefaciens, is traditionally
managed by pre-plant soil fumigation since soil-borne, A. tumefaciens
populations in newly planted orchards and nurseries are thought to be the
primary inoculum source. Despite these practices, crown gall persists. The
distribution of disease development in walnut nursery plots suggests A.
tumefaciens may be seed-borne. Extensive surveys of two production nursery
seed sources revealed A. tumefaciens is not present in or on seeds collected
directly from the mother trees. However, if seeds contact the orchard floor, as
is common in traditional harvesting practices, A. tumefaciens could be
detected on seeds. The bacterium is limited to the husk material surrounding
the walnut, is not found inside the hull, and prevalence of husk infestation
increased with time of floor exposure. Artificial inoculation studies
corroborate field survey findings. In order to develop management strategies
to control seed-borne populations of A. tumefaciens, heat treatments (50, 55,
and 60°C for 30, 45, and 60 min) are being evaluated. Disease incidence and
germination rates resulting from these heat treated seeds will be discussed. In
conjunction with our previous research demonstrating increased colonization
of fumigated soils by A. tumefaciens, planting clean walnut seeds may be key
when managing crown gall in walnut production.
The ColS/ColR two-component system is involved in virulence of citrus
canker pathogen Xanthomonas axonopodis pv. citri 306
Q. YAN (1), N. Wang (1)
(1) Lake Alfred, FL, U.S.A.
Phytopathology 100:S141
Bacterial citrus canker disease, which is caused by Xanthomonas axonopodis
pv. citri, is one of the most devastating diseases on citrus. To investigate the
virulence mechanism of this pathogen, a X. axonopodis pv. citri mutant library
was constructed by randomly mutagenesis using EZ::Tn5Tm. Multiple
mutants were identified by screening the mutants using plant assay on
grapefruit. Among the mutants with reduced virulence, four of them, named
256A10, 421E7, 386C6 and 417E10, are mutants of two-component system
ColS/ColR (colS::Tn5 in mutants 256A10 and 421E7; colR::Tn5 in mutants
386C6 and 417E10). The pathogenicity of the mutants could be
complemented using wild-type colS or colR, respectively. Collectively, our
data demonstrated that the two-component system ColS/ColR is involved in
the virulence of X. axonopodis pv. citri. How ColS/ColR system is involved in
virulence of X. axonopodis pv. citri 306 is under further investigation.
Population dynamics of Dactylella oviparasitica in a Heterodera schachtii
suppressive soil
J. YANG (1), J. O. Becker (2), J. Borneman (1)
(1) Department of Plant Pathology and Microbiology, University of
California, Riverside, Riverside, CA, U.S.A.; (2) Department of Nematology,
University of California, Riverside, Riverside, CA, U.S.A.
Phytopathology 100:S141
The initial objective of this study was to identify and isolate different strains
of the fungus, Dactylella oviparasitica, from a Heterodera schachtiisuppressive soil. Toward this end, we endeavored to enumerate D.
oviparasitica populations in H. schachtii cysts extracted from 16 regions of
the field containing the suppressive soil. Using a sequence-selective
quantitative PCR assay, D. oviparasitica was detected in only one of the 16
locations. D. oviparasitica was also not detected in the soil or purslane weed
seeds (Portulaca oleracea) collected from this field. These results led to the
development of a new hypothesis, which was that D. oviparasitica parasitizes
newly formed females and eggs within the cysts, and that its population
densities decrease after this food source (females and eggs in the cysts) has
been utilized. To test this hypothesis, we performed root box experiments to
collect freshly developed H. schachtii females and cysts from roots of sugar
beets grown in the suppressive soil. When these samples were examined using
the sequence-selective qPCR assay, high levels (as high as 109 copies per
cyst) of D. oviparasitica were detected in most samples, providing evidence to
support our hypothesis. Further examination of these samples by analysis of
the rRNA intergenic transcribed spacer region identified two D. oviparasitica
phylotypes. Future experiments will examine the relative abilities of the two
strains to reduce H. schachtii populations.
A screening strategy of bacterial biocontrol agents towards Ralstonia wilt
of ginger
W. Yang (1), J. GUO (1)
(1) Nanjing, PRC PEOPLES REP OF CHINA
Phytopathology 100:S141
Bacterial wilt caused by Ralstonia solanacearum (Smith) has become a severe
problem on ginger in China, there are no effective measures to control this
disease up to now. In order to develop a new method to control it, four
hundred and twenty strains were isolated from different habitats of ginger
including soil, stem and leaves, eighty-five of them were selected for BOXand ARDRA-PCR based on the results of in-vitro antagonistic activity against
R. solanacearum. An assessment of 19 antagonists that from different BOX
clusters was established based on the activities of enzymes including protease,
chitinase, cellulose, glucanase, and production of siderophores. Also they
were chosen for greenhouse experiment on ginger. Their biocontrol efficacies
were 26–69%. Efficacies more than 50% were achieved by Bacillus subtilis
1JN2, Myroides odoratimimus 3YW8, Bacillus amyloliquefaciens 5YN8 and
Stenotrophomonas maltophilia 2JW6. This is the first report to use Myroides
sp. and Stenotrophomonas sp. as biocontrol agents against ginger wilt caused
by R. solanacearum, and these strains can be good candidates for further
exploration to develop a commercial biocontrol agent.
Biological control of take-all of wheat by fluorescent Pseudomonas spp.
from China
M. YANG (1), J. Guo (2), L. S. Thomashow (3), D. M. Weller (3)
(1) Nanjing Agriculture University and Washington State University,
Pullman, WA, U.S.A.; (2) Nanjing Agricultural University, Nanjing, PRC
PEOPLES REP OF CHINA; (3) USDA-Agricultural Research Service,
Pullman, WA, U.S.A.
Phytopathology 100:S141
Vol. 100, No. 6 (Supplement), 2010
S141
Take-all disease of wheat caused by the soilborne fungus Gaeumannomyces
graminis var. tritici (Ggt) is one of the most important root diseases of wheat
worldwide. In order to find biocontrol agents, bacteria were isolated from
wheat at different stages of development in Hebei and Jiangsu provinces in
China. Samples from rhizosphere soil, roots, stems and leaves were plated
onto King’s B agar. All of 105 isolates that inhibited Ggt in vitro were
identified as Pseudomonas spp. by ARDRA analysis. Twenty-seven isolates,
which represented 24 BOX-PCR groups, were selected for further study. Of
these, 14 suppressed take-all in biocontrol assays under greenhouse
conditions. The antibiotics phenazine-1-carboxylic acid (PCA) and 2,4diacetylphloroglucinol (DAPG) are major determinants of biocontrol of
soilborne plant pathogens by fluorescent pseudomonads. Using PCR and
primers specific for sequences within the biosynthetic operons responsible for
production of these antibiotics, 4 of the 14 strains were found to produce PCA
but none produced DAPG. High-pressure liquid chromatography (HPLC)
analysis of 2-day-old cultures of these four strains confirmed the production
of PCA. DNA sequence analysis within the phzF gene indicated that three
strains were similar to the well-described PCA producer P. fluorescens 2-79.
This is the first report of a 2-79-like strain isolated from outside of
Washington State.
TMV inclusion bodies: Their formation and relationship to virus
accumulation
X. YANG (1), R. S. Nelson (1), S. Bhat (1)
(1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.
Phytopathology 100:S142
Many viruses produce intercellular inclusions during infection. Their
relationship with the accumulation and intra- and inter-cellular movement of
these viruses and to disease is not fully understood. Previous studies with
TMV have identified a positive correlation between the size of an inclusion,
referred to as the virus replication complex (VRC), induced in infected cells
and the intensity of disease symptoms displayed by the infected host, N.
benthamiana. In addition, there was a similar positive correlation between the
size of these VRCs and the inclusions produced by ectopic expression of the
126 kDa protein of the virus fused with GFP (Liu et al. Plant Physiology 2005
138:1853-1865). We have since determined that the size of the VRCs can be
modified by silencing specific host factors. For example, silencing rubisco
activase expression results in greater accumulation of virus (up to 7 fold) and
smaller, but much more numerous VRCs. In addition, using chimeric
constructs of 126 kDa protein and a homolog from TVCV, the 125 kDa
protein, which poorly forms inclusions, we determined that particular domains
within the 126 kDa protein control formation of inclusions. In addition, the
formation of inclusions is a temporal process involving multiple domains.
TVCV induces milder symptoms than TMV in N. benthamiana and the
relationship of symptoms and inclusion body formation will be discussed
based on these results.
Effect of limestone on development of Verticillium wilt of spinach
W. YANG (1), A. Iglesia-Garcia (1), L. du Toit (2), B. Bluhm (1), J. Correll
(1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) Mount Vernon, WA,
U.S.A.
Phytopathology 100:S142
Verticillium wilt, caused by Verticillium dahliae, is an important vascular wilt
disease of many agricultural crops and, more recently, has become
problematic for spinach seed production. Development of Verticillium wilt is
influenced by soil conditions such as pH, moisture, and temperature. In an
effort to optimize greenhouse pathogenicity and virulence tests, the effect of
limestone (5 g/L CaCO3) on the development, incidence, and severity of
Verticillium wilt symptoms on spinach was evaluated. In addition, the
infection process was monitored using wild-type isolates and genetically
marked strains in the form of nitrate non-utilizing (nit) mutants. Limestone
and no limestone treatments were evaluated and compared to a non-limed
control treatment. Treatments consisted of amending potting mix with
different rates of limestone application, or drenching the potting medium with
a limestone solution. Potting mix pH was recorded before and after each
experiment. Plants were inoculated by adding V. dahliae spores to the root
plug and monitoring disease development. Both wild-type isolates and nit
mutants caused symptoms of Verticillium wilt, and both types of isolates were
recovered from inoculated plants. Limestone treatments caused a significant
increase in disease severity of Verticillium wilt of spinach.
Use of formulation and adjuvant approaches to identify potential
limitations in field performance of an experimental fungicide relative to
benchmarks
C. YAO (1), J. Owen (1)
(1) Dow AgroSciences, Indianapolis, IN, U.S.A.
Phytopathology 100:S142
S142
PHYTOPATHOLOGY
Compound A has shown a promising activity profile against a spectrum of
fungal pathogens. In preliminary greenhouse bio-assays using technical
material and a high-volume spray format, it was 4-fold more potent as a
protectant treatment, based on ED50 values, than azoxystrobin and
epoxiconazole vs. wheat brown rust (Puccinia recondita, PUCCRT), while its
curative potency was 3-fold weaker than azoxystrobin, and 10-fold weaker
than epoxiconazole. Rust activity of Compound A formulated as a base SC
was further compared to the commercial formulations of azoxystrobin
(Amistar®) and epoxiconazole (Opus®) in low-volume spray applications.
Compound A showed better protectant rust activity than both commercial
products, while curative activity was better than Amistar® but weaker than
Opus®. However, PUCCRT efficacy of Compound A was significantly
weaker than Amistar® and Opus® in field trials. This discrepancy between
greenhouse and field efficacies was partially attributed to the presence of a
constant rate (0.1%) of an alcohol ethoxylate adjuvant in the spray solutions
of Compound A. When it was compared to similar base SC of azoxystrobin
and epoxiconazole and the same adjuvant added to 0.1% in all spray solutions,
curative activity of Compound A vs. PUCCRT was at least 26-fold less
than the two commercial fungicides, even though the protectant activities
of all three fungicides were comparable. Data generated using the latter
approach may improve predictions of field performance of experimental
fungicides.
Soil amendments with Brassica cover crops for control of Phytophthora
blight on squash
J. YIN (1), D. Koné (1), M. Purvis (1), K. L. Jackson (1), A. S. Csinos (1), P.
Ji (1)
(1) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.
Phytopathology 100:S142
Phytophthora blight caused by Phytophthora capsici has become an
increasing concern in vegetable production in Georgia and several other states
in the U.S. Cover crops that produce general biocides, such as mustard,
collards, canola, and other Brassica species, were studied under greenhouse
and field conditions to determine host status and evaluate the efficacy to
suppress P. capsici. In greenhouse studies, disease incidence on squash was
significantly reduced by soil amendment with mustard leaves or roots. Soil
amendments with leaves of canola or carrot also reduced disease incidence
significantly. In field studies, cover crops were grown in winter and
incorporated into soil in spring and squash seedlings were transplanted after
soil amendment. Mustard and canola provided the greatest disease reduction
while radish and collards reduced disease to a lesser extent. None of the cover
crops showed symptoms when leaves or roots of the crops were inoculated
with P. capsici and most of the cover crops did not appear to be a host of the
pathogen. The results indicated that some Brassica cover crops had the
potential to inhibit P. capsici and suppress disease development on squash
plants when used as soil amendments.
Comparison of bacterial communities from inside and outside of
Rhizoctonia bare patches in wheat
C. Yin (1), S. H. Hulbert (1), K. L. Schroeder (2), O. Mavrodi (1), D. Mavrodi
(1), A. Dhingra (3), T. C. PAULITZ (2)
(1) Dept. of Plant Pathology, Washington State University, Pullman, WA,
U.S.A.; (2) USDA-ARS, Root Disease and Biological Control Research Unit,
Pulllman, WA, U.S.A.; (3) Dept. of Horiculture and Landscape Architecture,
Washington State University, Pullman, WA, U.S.A.
Phytopathology 100:S142
Rhizoctonia solani AG-8 causes distinct patches of stunted wheat in the field.
Bacterial communities from bulk soil and rhizospheres of wheat were
analyzed with pyrosequencing. Replicated samples were taken from inside
and outside of patches; and from patches that had recovered the previous 1–2
years. Pyrosequencing was performed on amplified products from primers
designed to the V3 hyper-variable region of bacterial 16S rDNA. Between
2070 and 5746 sequences of >150 bp were obtained from each sample, and
these were assembled into 345 OTUs, with 151 identified to the genus level.
Community diversity was higher and abundance was lower in the bulk soil
compared to the rhizosphere soil. In the bulk soil, abundance was highest
inside the patches. Chitinophaga and Acidobacteria GP 3 were the most
abundant genera, followed by Enterobacteriaceae, Pseudomonas, Pedobacter,
Variovorax, Sphingomonas, Solirubrobacter, Nitriliruptor and TM7.
Flavobacterium and a single OTU in the family Enterobacteriaceae and the
order Sphingobacteriales were more frequent in the rhizospheres of plants
inside the patch compared to outside or recovered patches, and this was
confirmed by real-time quantitative PCR. Dyella and Acidobacteria GP 7
were more frequent in recovered patches. These results show that the
rhizosphere community on wheat plants in diseased patches is distinct from
healthy plants, and this technique may be useful for finding organisms
associated with disease suppression.
Molecular characterization of boscalid resistance in field isolates of
Botrytis cinerea from apple in Washington State
Y. YIN (1), Y. Kim (1), C. Xiao (1)
(1) Washington State University, TFREC, Wenatchee, WA, U.S.A.
Phytopathology 100:S143
To monitor boscalid resistance in the field, Botrytis cinerea isolates obtained
from 5 commercial apple orchards were screened for resistance to boscalid
using a conidial germination assay at the discriminatory concentration of 5
µg/ml. Of the 220 isolates tested, 42 were resistant to boscalid. There was
cross resistance between boscalid and either carboxin or penthiopyrad.
Analysis of partial sequences of the iron-sulphur subunit of succinate
dehydrogenase gene (BcSdhB) from 13 boscalid-resistant and 9 -sensitive
isolates showed that point mutations in the BcSdhB gene leading to amino acid
substitutions at the codon position 272 from histidine to either arginine
(H272R) or tyrosine (H272Y) were associated with boscalid resistance.
Allele-specific PCR analysis of the 66 boscalid-resistant isolates (including 24
additional isolates obtained from decayed apples) showed that 46 had the
point mutation H272R and 19 exhibited the point mutation H272Y, but one
resistant isolate gave no amplification product. Analysis of BcSdhB sequence
of this isolate revealed a different point mutation at the codon position 225,
resulting in a substitution of proline (P) by phenylalanine (F) (P225F).
A multiplex allele-specific PCR assay was developed to detect point
mutations at the position 272 in BcSdhB conferring resistance to boscalid in a
single PCR amplification. There was no correlation between types of pointmutation in the BcSdhB gene and levels of boscalid resistance in the resistant
isolates.
Characterization of pyraclostrobin resistance and detection of the Bcbi143/144 intron in the cytochrome b gene in Botrytis cinerea isolates from
apple
Y. YIN (1), Y. Kim (1), C. Xiao (1)
(1) Washington State University, TFREC, Wenatchee, WA, U.S.A.
Phytopathology 100:S143
To monitor resistance to the QoI fungicide pyraclostrobin (PYR) in the
orchard, Botrytis cinerea isolates obtained from 5 orchards where PYR had
been used for 4 consecutive years were screened for resistance to PYR in a
mycelial growth assay on potato dextrose agar amended with PYR at the
discriminatory concentration of 5 µg/ml and salicylhydroxamic acid at 100
µg/ml, which was used to inhibit the alternative oxidase respiration. Of the
220 isolates tested, 43 (19.5%) were resistant to PYR. All resistant isolates
were highly resistant to PYR with resistance factors >1000. Cross-resistance
was observed between PYR and two other QoIs azoxystrobin and
trifloxystrobin. Analysis of partial sequence of the cytochrome b gene (cytb)
and molecular diagnosis based on an allele-specific PCR showed that all
PYR-resistant (PYR-R) isolates had the same mutation leading to the
substitution of glycine by alanine at codon position 143 in cytb (G143A).
Structural analysis of cytb indicated that all PYR-R isolates did not have the
Bcbi-143/144 intron. Of the 123 PYR-sensitive (PYR-S) isolates collected
from 4 major apple producing regions in Washington State, only 17 (13.8%)
had the Bcbi-143/144 intron in cytb, indicating a high inherent-risk for
development of QoI resistance among the PYR-S isolates as the presence of
the Bcbi-143/144 intron in cytb is believed to prevent the occurrence of
G143A mutation-mediated QoI resistance.
The source of polypeptone in culture medium affects lipopeptide
production by Bacillus subtilis
K. YOKOTA (1), E. Miwa (1), K. Higuchi (1)
(1) Tokyo University of Agriculture, Tokyo, JAPAN
Phytopathology 100:S143
Iturin and surfactin are antimicrobial lipopeptides thought to play a key role in
antimicrobial activity of biological control strains of Bacillus spp., including
B. subtilis. We report here that the source of polypeptone used in submerged
culture medium influences production of iturin and surfactin. These
lipopeptides were produced if the polypeptone source was either crude
soybean cake or potato, but not if the source was purified soybean protein,
milk casein, or fish meat. The data suggest that the lipopeptide-inducing factor
in polypeptone from crude soybean is lost after protein purification, and that it
is not present in polypeptone from certain animal sources.
Effect of solar radiation on disease severity of soybean rust
H. M. YOUNG (1), D. F. Narvaez (2), J. J. Marois (3), D. L. Wright (3)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) Monsanto, St. Louis,
MO, U.S.A.; (3) University of Florida, Quincy, FL, U.S.A.
Phytopathology 100:S143
Soybean rust (SBR), caused by Phakopsora pachyrhizi, is the most damaging
fungal disease of soybean. While it is known that solar radiation can reduce
SBR spore survival, increase canopy temperature and reduce humidity,
limited information is available on how solar radiation affects SBR progress
within the soybean canopy. Such information can aid in accurate SBR
prediction models. To manipulate light penetration into soybean canopies
shade structures (using 30, 40, and 60% shade cloth) were constructed over
soybeans and weekly evaluations of severity of lower, middle, and upper
canopies were recorded, as well as, daily environmental conditions. Detached
leaf assays assessed cuticular wax amount and the susceptibility of leaves in
the lower, middle, and upper canopies. The experiment was conducted over 2
years, with 2 plantings each year, and 3 replications each planting.
Temperature and relative humidity were affected by shade cloth, regardless of
thickness. A trend of decreasing cuticular wax from upper to lower canopy
level was observed, for all treatments. Greater disease severity occurred in 40
and 60% shaded field plots and in detached leaf assays of middle and upper
canopy leaves from 40 and 60% shaded plots. These results provide an
understanding of the effect solar radiation has on the progression of SBR
within soybean canopy.
Selection of single chain variable fragments (scFv) against Xylella
fastidiosa subsp. pauca by phage display
Q. YUAN (1), R. Jordan (1), R. H. Brlansky (2), O. Minenkova (3), J.
Hartung (1)
(1) USDA ARS MPPL, Beltsville, MD, U.S.A.; (2) University of
Florida, Lake Alfred, FL, U.S.A.; (3) Sigma-tau Pharmaceuticals, Rome,
ITALY
Phytopathology 100:S143
Xylella fastidiosa is a gram-negative member of the gamma proteobacteria.
Xylella fastidiosa subsp. pauca causes citrus variegated chlorosis in Brazil and
enjoys ‘select agent’ status in the United States. Antibody based detection
assays are commercially available for Xylella fastidiosa, and are effective at
the species, but not at the subspecies level. We have made a library of scFv
antibody fragments directed against Xylella fastidiosa subsp. pauca strain
9a5c (citrus) by using phage display technology. BALB/c mice were
immunized with 9a5c bacteria at a concentration of 108 cfu/100 ul buffer.
mRNA from the spleens of the immunized mice was purified and converted
into cDNA. Antibody gene repertoires were PCR-amplified using 23 primers
for the heavy chain variable region (VH) and 21 primers for the light chain
variable region (VL). The VH and VL were joined by overlap extension PCR,
and then the genes of the scFv library were ligated into the phage vector
pKM19. The library contained 1.2 × 107 independent clones with full-length
scFv inserts. In each of 3 cycles of affinity-selection with 9a5c, about 1.0 ×
1012 phage were used for panning with 4.1 × 106, 7.1 × 106, 2.1 × 107
phage recovered after the first, second and third cycles respectively. 66%
of clones from the final library bound Xylella fastidiosa 9a5c. Some of
these phage expressing scFv antibodies recognized strain 9a5c but did
not recognize Xylella fastidiosa strains that cause Pierce’s disease of
grapevine.
Development of conventional monoclonal antibody and recombinant
antibody (scFv) against Candidatus Liberibacter asiaticus
Q. YUAN (1), R. Jordan (1), R. C. Bohannon (2), R. H. Brlansky (3), O.
Minenkova (4), J. S. Hartung (1)
(1) USDA ARS MPPL, Beltsville, MD, U.S.A.; (2) Agdia, Elkhardt, IN,
U.S.A.; (3) University of Florida, Lake Alfred, FL, U.S.A.; (4) Sigma-tau
Pharmaceuticals, Rome, ITALY
Phytopathology 100:S143
A non-culturable member of the alpha-proteobacteria, ‘Ca. Liberibacter
asiaticus’, is consistently associated with huanglongbing (HLB) disease. This
bacterium is transmitted by citrus psyllids and grows systemically in infected
citrus phloem tissues. Control of the disease requires effective, convenient and
inexpensive methods for detection of the pathogen in infected plants and
insects. Antibodies are the most widely used tool to detect pathogens, and they
are also uniquely useful as experimental reagents. Therefore antibodies
against the HLB pathogens would greatly aide detection of the pathogen and
eventual control of this disease. Extracts of psyllids fed on HLB infected
citrus in Florida were assayed individually for ‘Ca. Liberibacter asiaticus’ by
q-PCR. Extracts with more than 108 ‘Ca. Liberibacter asiaticus’/100 ul were
used to immunize BALB/C mice. Monoclonal antibodies were made by
spleen/myeloma fusion and screened against both plant extracts containing
‘Ca. Liberibacter asiaticus’ and against outer membrane protein (OMP)
purified from Escherichia coli cells expressing the cloned gene. A
recombinant library of scFv antibodies also was made using the phage vector
pKM19 to clone cDNAs prepared from the mRNA isolated from the mouse
spleens. This scFv library in recombinant phage is currently being screened
against extracts of plants infected with high concentrations of ‘Ca.
Liberibacter asiaticus’ as well as against purified outer membrane protein
from ‘Ca. Liberibacter asiaticus’.
Vol. 100, No. 6 (Supplement), 2010
S143
Foliar symptoms expression and early infection of soybean sudden death
syndrome
M. L. ZACCARON (1), X. Yang (1), S. S. Navi (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S144
Soybean sudden death syndrome (SDS), caused by Fusarium virguliforme, is
an important root disease that can express foliar symptoms leading to
defoliation and yield reduction. Our earlier report showed that root cap is one
of the penetration sites of infection for F. virguliforme, which will be followed
by foliar symptoms in soybean. There is not much information about infection
process of SDS and how environmental conditions influence the process and
subsequent foliar symptoms development. The objective of this study was to
investigate infection sites on soybean roots leading to foliar symptoms
expression. Seeds were germinated using paper towel method at room
temperatures; radicles of 72-h were inoculated with 20 µl of conidial
suspension F. virguliforme using inoculation loop and micropipette methods
under sterile conditions. Three sites of infection on different locations were
examined. After inoculation germinating seeds were transplanted to cones
filled with sterile potting mixture. Rhizosphere temperature was controlled
with water bath at (20°C). Plants were evaluated three weeks after
transplanting. Our preliminary results indicate that plants inoculated at root tip
had higher incidence than plants inoculated at other sites. The incidence was
even higher when the inoculation loop was used, suggesting mechanical
wounding early at soybean planting may be critical to foliar symptom
expression.
Transposon mutagenesis of Pantoea ananatis: Isolation and
characterization of a Tn5-induced mutant with reduced virulence to
onion
A. M. Zaid (1), J. M. Bonasera (1), S. V. BEER (1)
(1) Department of Plant Pathology and Plant-Microbe Biology, Cornell
University, Ithaca, NY, U.S.A.
Phytopathology 100:S144
Center rot of onion, caused by Pantoea ananatis, is a disease of increasing
concern to onion growers in New York State. We recently isolated both
pathogenic and non-pathogenic strains of P. ananatis from New York onions.
The strains are indistinguishable from each other based on numerous
biochemical assays. OC5a, a highly virulent New York strain was mutated
using transposon Tn5. Two-thousand mutants were screened for virulence in
small onion bulbs (sets). Southern blot analysis of 27 mutants revealed that
Tn5 has a strong insertion-site preference in the P. ananatis genome. Thus,
not surprisingly, only a single less virulent mutant was found; it contained two
additional insertions of Tn5. These insertion sites will be characterized as to
the genes they may have inactivated and the potential role of the disrupted
genes in virulence to onion. We seek to identify genes of P. ananatis that are
involved in virulence to advance our understanding of the genetic basis for
pathogenesis and to provide molecular markers for pathogenic strains that
would likely be useful for future diagnostic and epidemiological studies.
Effect of temperature on latent period of Stagonospora nodorum blotch
in winter wheat under field conditions
A. D. ZEARFOSS (1), C. Cowger (2), P. S. Ojiambo (1)
(1) Department of Plant Pathology, North Carolina State University, Raleigh,
NC, U.S.A.; (2) USDA-ARS, Department of Plant Pathology, North Carolina
State University, Raleigh, NC, U.S.A.
Phytopathology 100:S144
Stagonospora nodorum blotch (SNB) is caused by Stagonospora nodorum
(teleomorph Phaeosphaeria nodorum) and yield losses from severe disease
epidemics can be as high as 50%. To establish a model for SNB development
based on the effects of temperature on pathogen latent period relative to the
host, batches of two winter wheat cultivars (AGS 2000 and USG 3209) were
inoculated with pycnidiospores of S. nodorum at weekly intervals from
February 2009 to June 2009. After an incubation period of 72 h, plants were
exposed to field conditions where temperatures ranged from –6.6°C to 35.8°C
with a mean batch temperature of 9.7°C to 23.7°C. Latent period expressed as
the time from inoculation until the first visible symptoms, ranged from 8 to 34
days. A shifted cumulative gamma distribution model with a base temperature
of 0.5°C best described the relationship between number of lesions with
pycnidia and accumulated thermal time. When defined as time to 50% of the
maximal lesions with pycnidia, latent period was estimated as 297 and 313
degree-days above the base temperature of 0.5°C for USG 3209 and AGS
2000, respectively. The relationship between the inverse of latent period,
defined as time to 50% maximal lesions with pycnidia, and the mean batch
temperature was best described using a linear model (r2 = 0.93, P < 0.001).
This study provides data that link wheat growth with SNB progress and will
facilitate the construction of disease development models for use in timing of
fungicide application.
S144
PHYTOPATHOLOGY
Survey of Stagonospora nodorum toxins and wheat sensitivity genes in the
southeastern U.S.
A. D. ZEARFOSS (1), T. L. Friesen (2), P. S. Ojiambo (1), C. Cowger (3)
(1) Department of Plant Pathology, North Carolina State University, Raleigh,
NC, U.S.A.; (2) USDA-ARS, Department of Plant Pathology, North Dakota
State University, Fargo, ND, U.S.A.; (3) USDA-ARS, Department of Plant
Pathology, North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology 100:S144
Stagonospora nodorum blotch (SNB), caused by the necrotrophic fungal
pathogen Stagonospora nodorum (teleomorph: Phaeosphaeria nodorum), is
among the most ubiquitous diseases of winter wheat in the U.S. The disease is
favored by warm, moist weather conditions prevalent in the southeastern U.S.,
and can cause substantial reductions in yield and test weight. Host resistance
is the most effective and economically viable option for SNB control. Recent
discovery of several host-selective toxins (HSTs) produced by S. nodorum,
and their corresponding host sensitivity genes, has offered new resources for
SNB resistance breeding. Knowledge of HSTs and toxin-sensitivity genes in
regionally adapted wheat breeding materials can aid small grains breeding
programs in the southeast. Fifty-four isolates of S. nodorum collected from
wheat debris from nine states in the southeastern U.S. were used to obtain
culture filtrates for host infiltration. Twenty-four advanced soft red winter
wheat (SRWW) lines with varying levels of resistance to SNB were chosen
from nine southeastern states. Three toxin controls, three fungal isolate
controls and six differential wheat lines were also used. Each cultivar × isolate
reaction was assessed 3, 5 and 7 days after inoculation and scored as positive,
intermediate or negative. We will report HSTs found in S. nodorum isolates
and postulate sensitivity genes in elite SRWW lines and released varieties
commonly grown in the southeastern U.S.
The Irony of Silicon: Accumulation in a non-accumulator induced by
TRSV
W. L. ZELLNER (1), J. Frantz (2), S. M. Leisner (1)
(1) University of Toledo, Toledo, OH, U.S.A.; (2) U.S. Department of
Agriculture-Agricultural Research Service, Toledo, OH, U.S.A.
Phytopathology 100:S144
While silicon protects a variety of plants against fungal and bacterial
pathogens, silicon-aided resistance to viral pathogens is as unclear as the
amount of silicon required to aid in plant defenses. Therefore, the effects of
and accumulation of silicon on Tobacco ringspot virus (TRSV) were
investigated in the “low-accumulator”, N. tabacum. Plants were grown
hydroponically and prior to inoculation the levels of soluble silicon were
increased. TRSV symptoms on N. tabacum treated with increased silicon
typically took two days longer to appear and covered less leaf area then plants
grown under control conditions. Furthermore, preliminary studies using TMV
show no change in the onset or distribution of symptoms in N. tabacum,
suggesting that the beneficial effects of silicon are pathogen-specific. ICP
analysis of leaf and root tissue indicated a significant accumulation of silicon
in leaves of TRSV-infected plants grown under increased silicon compared to
leaves of mock-inoculated plants. However, root silicon levels were elevated
for all plants supplemented with silicon, independent of TRSV infection. This
suggests that N. tabacum accumulates silicon in root tissue which is then
released during TRSV infection, permitting acquisition by leaves. Salicylic
acid (SA) treatment of N. tabacum also resulted in an increase in silicon
accumulation in upper leaves compared to control plants, suggesting that SA
may play a role in the signaling pathway.
Regulation of Dickeya dadantii type III secretion system by polynucleotide
phosphorylase
Q. ZENG (1), A. Ibekwe (2), C. Yang (1)
(1) University of Wisconsin-Milwaukee, Milwaukee, WI, U.S.A.; (2) USDAARS, US Salinity Laboratory, Riverside, CA, U.S.A.
Phytopathology 100:S144
The type III secretion system (T3SS) is an essential virulence factor of the
phytopathogenic bacterium Dickeya dadantii. In D. dadantii, the transcription
of T3SS structural and effector genes is positively regulated by the master
regulator, HrpL. Expression of hrpL is regulated at the transcriptional level by
RpoN and post-transcriptionally by RsmB, a regulatory small RNA, which
enhances hrpL mRNA stability. Polynucleotide phosphorylase (PNPase) is
one of the major exoribonucleases in bacteria and plays important roles in
general mRNA degradation, tRNA processing, and sRNA turnover. In this
study, we showed that PNPase down-regulates the transcription of T3SS
genes. This negative regulation of T3SS by PNPase occurs through the
repression of hrpL expression. By decreasing rpoN mRNA stability, PNPase
down-regulates the transcription of hrpL, which leads to a reduction in T3SS
gene expression. Moreover, we have found that PNPase down-regulates T3SS
by reducing hrpL mRNA stability. Our results suggest that PNPase decreases
the amount of functional RsmB transcripts, which could result in the reduced
hrpL mRNA stability.
Genotypic and phenotypic diversity of Pyricularia oryzae in the
contemporary rice blast pathogen population in Arkansas
L. ZHAI (1), C. Feng (1), R. Cartwright (1), J. Correll (1)
(1) University of Arkansas, Department of Plant Pathology, Fayetteville, AR,
U.S.A.
Phytopathology 100:S145
Rice blast, caused by Pyricularia oryzae, is one of the most economically
important diseases of rice worldwide. It is also one of the most important rice
diseases in Arkansas. The objective of this study was to examine the
genotypic and phenotypic diversity of P. oryzae in the contemporary
population in Arkansas. The weather during the 2009 rice season was
particularly conducive for disease development. Over 500 isolates were
recovered from symptomatic rice cultivars in Arkansas during the 2009 rice
season. Theses isolates were evaluated for vegetative compatibility, MGR586
(Magnaporthe grisea repeat sequence) DNA fingerprint diversity, and
virulence on a set of 30 commercial cultivars or advanced breeding lines.
Although four VCG groups (US-01 to US-04) have been identified among
contemporary and archived isolates of P. oryzae in Arkansas, only three VCG
groups (US-01, US-02 and US-04) were identified in our 2009 collection.
VCG US-01 remains predominant and over 50% of these isolates recovered
from 2009 belonged to VCG US-01. Pathogenicity test of a subset isolates on
the commercial cultivars and advanced breeding lines indicated that there was
considerable virulence diversity present both between and among isolates in a
VCG or MGR fingerprint group. The genetic population analysis will provide
us a better understanding of host coevolultion in plant pathosystems and
provide us direction for effectively managing rice blast.
Bactericidal activities of antimicrobial molecules against huanglongbingassociated ‘Candidatus Liberibacter asiaticus’ in the diseased periwinkle
M. Zhang (1), Y. Duan (2), C. A. POWELL (3)
(1) University of Florida, Ft. Pierce, FL, U.S.A.; (2) United States Department
of Agriculture-ARS-USHRL, Fort Pierce, FL, U.S.A.; (3) IRREC-IFAS
University of Florida, Fort Pierce, FL, U.S.A.
Phytopathology 100:S145
Candidatus Liberibacter is a phloem-limited, gram-negative, fastidious
bacterium that is associated with the citrus Huanglongbing (HLB) disease.
Periwinkle was used as a model plant to test molecules for bactericidal
activity against Liberibacter. Liberibacter-infected, 4-cm periwinkle cuttings
were soaked in solutions containing one of several molecules including a
biocide (DBNPA), two peptides (D2A21 and D4E1), a fungicide (Zineb) and
SAR substances (SA, antiguard and ortho-phenylphenol). Cuttings prior to
treatment and their regenerated plants at 2 and 3 months post treatment were
analyzed for Liberibacter by quantitative real-time (q)PCR with primer set
HLBas/HLBr/HLBp. The peptides consistently reduced the bacteria titers with
an average Ct values for both peptides of 34.87 ± 4.45 and 37.00 ± 1.77 by
qPCR in the regenerated plants at 2 and 3 months post treatment respectively,
compared to the water-control (22.59 ± 4.96 and 24.89 ± 1.39). Biocide
(DBNPA) could also suppress the Las bacteria. The fungicide zineb and three
SARs (SA, antiguard and ortho-phenylphenol) were not effective in
controlling Liberibacter bacteria. Whether treated with zineb or not, the
Liberibacter bacteria can keep reproducing. The Ct value was lower in the
zineb-treated, regenerated plants than those treated with peptides. Because
peptides were very effective but expensive in eliminating the bacterium in the
Liberibacter-infected citrus in the field, they can be expressed in the
transgenic citrus to control citrus HLB disease.
Baseline sensitivity of Cercospora sojina to azoxystrobin, trifloxystrobin,
and pyraclostrobin
G. ZHANG (1), D. V. Phillips (2), C. A. Bradley (1)
(1) University of Illinois, Urbana, IL, U.S.A.; (2) University of Georgia,
Griffin, GA, U.S.A.
Phytopathology 100:S145
Frogeye leaf spot (FLS) is a disease of soybean caused by Cercospora sojina.
Quinone outside inhibitor (QoI) fungicides such as azoxystrobin,
trifloxystrobin, and pyraclostrobin are applied to manage FLS. A total of 58
“baseline” isolates collected from 9 states prior to QoI fungicide registration
in the U.S. was tested with an in-vitro spore germination assay to determine
the effective fungicide concentration at which 50% of conidial germination
was inhibited (EC50). The effect of salicylhydroxamic acid (SHAM) on
conidial germination also was tested to determine if C. sojina can use the
alternative respiration pathway to bypass the effect of the fungicides.
Significantly greater (P 0.05) EC50 values were observed when media was
amended with azoxystrobin alone compared with media amended with both
azoxystrobin and SHAM. This indicates that C. sojina may use alternative
respiration to overcome the effect of QoI fungicides in-vitro, and that the
inclusion of SHAM is necessary when testing the sensitivity of C. sojina
isolates to QoI fungicides. Baseline isolates had EC50 values for
pyraclostrobin, trifloxystrobin, and pyraclostrobin that ranged from 0.0029 to
0.0323, 0.00018 to 0.00311, and 0.00014 to 0.00076 ug/ml, with means of
0.0127, 0.0012 and 0.00027 ug/ml, respectively. The establishment of these
baseline QoI sensitivity values is an important first step in developing a
fungicide resistance monitoring program for C. sojina.
Evaluation of biologically-based products for managing bacterial spot
disease of tomato
S. ZHANG (1), T. L. White (1), Y. Fu (1)
(1) TREC - University of Florida, Homestead, FL, U.S.A.
Phytopathology 100:S145
Bacterial spot, caused by Xanthomonas spp., is a devastating disease of
tomato in Florida and worldwide. Despite the tremendous efforts towards
managing this disease, it still remains a major challenge in the Southeastern
states. Greenhouse experiments were conducted to evaluate efficacy of six
biologically-based products alone or in combination with Actigard®, an
inducer of systemic acquired resistance (SAR) registered for tomato.
Beginning 1 week after transplanting into 4-inch plastic pots, plants were
sprayed four times either with suspensions of biological products alone or in
combination with Actigard® at weekly intervals. Kocide 3000 plus Manzate
(K+M) was used as a standard chemical control, and water sprays were served
as the nontreated control. Tomato plants were spray-inoculated with
suspensions of X. perforans (2 × 107 CFU/ml) 3 days after the last treatment
and placed on a greenhouse bench for 10 days when the disease was rated on a
0–5 scale. The products HMO 736 and Companion each alone or in
combination significantly (P < 0.05) reduced the disease severity compared to
the nontreated control, and was as effective as K+ M. Other products in
combination with Actigard® resulted in significantly lower disease than each
of these biologicals alone. Combined treatments numerically reduced the
disease compared to Actigard® alone. Results indicate that these biologicallybased products could potentially be incorporated into integrated management
programs for control of bacterial spot on tomato.
Management of powdery mildew on squash with biologically-based
products
S. ZHANG (1), T. L. White (1)
(1) TREC - University of Florida, Homestead, FL, U.S.A.
Phytopathology 100:S145
Powdery mildew, caused by Sphaerotheca xanthii, is a serious disease of
cucurbits worldwide. Many fungicides have been registered for this disease
control. However, management of fungicide resistance in the pathogen is a
challenge. Efficacy of six biologically-based products, alone or in alternation
with a conventional fungicide, against powdery mildew was evaluated on
squash under greenhouse and field conditions. In greenhouse experiments,
products Regalia and BU EXP 1216 S each significantly (P < 0.05) reduced
the disease severity compared to the nontreated control, and they were as
effective as Procure, the standard fungicide treatment. The products HMO 736
and BU EXP 1216 S, each in alternation with Procure, consistently resulted in
significantly lower disease than the nontreated control. Similarly, Regalia,
Actinovate, Companion, and BU EXP 1216 C each when alternated with
Procure provided significant protection in two out of three experiments. More
interestingly, Regalia and HMO 736 had a synergistic effect when alternated
with Procure in one of the experiments. In the field trial, the biological
products applied individually or in alternation with Procure significantly
reduced powdery mildew severity at the early stage of disease development
and significantly improved the control efficacy of the biologicals and Procure
at the late stage. These data suggest that these biologically-based products
alternated with Procure could be used for managing powdery mildew of
squash in Florida.
Organization and structure of two stable Ca. Liberibacter asiaticus
prophage lysogens that become lytic in plant infections
S. ZHANG (1)
(1) University of Florida, Gainesville, FL, U.S.A.
Phytopathology 100:S145
Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus
(Las), is a psyllid transmitted, lethal disease of citrus now found widespread in
Florida citrus growing regions. The recently published Las strain psy62
genome, obtained from a single psyllid (GenBank NC_012985.2), revealed
prophage DNA integrated in the genome. Phage have not been previously
associated with HLB. A fosmid DNA library from curated Las strain UF506,
isolated from an infected Florida citrus tree, seemed surprisingly biased
towards phage DNA inserts. Two highly related circular phage genomes (SC1
and SC2) were assembled and annotated from the UF506 library, including 5
additional genes not previously identified in psy62. Extensive Southern blot
and PCR analyses were used to: 1) confirm the presence of lytic cycle SC1
and SC2 phage in Las infected citrus and periwinkle but not in Las infected
psyllids; 2) confirm the SC1 and SC2 circular phage genomic assemblies; 3)
map the cos sites of both phage, and 4) determine the genomic DNA
Vol. 100, No. 6 (Supplement), 2010
S145
integration sites and gene order of both SC1 and SC2 prophage. Semiquantitative RT-PCR revealed that the copy number of (lytic cycle) SC-1 in
infected citrus and periwinkle averaged 10X and 20X higher, respectively,
than in (lysogenic cycle) infected psyllids. The SC-2 phage DNA appeared at
a level 2-3X higher in planta than in psyllids. Phage particles associated with
Las were found in the pholem of infected periwinkles by transmission EM.
Antagonistic role of ethylene and abscisic acid in mediating rice sheath
blight resistance
J. Zhang (1), D. Park (2), Y. YANG (1)
(1) Department of Plant Pathology and Huck Institute of Life Sciences,
Pennsylvania State University, University Park, PA, U.S.A.; (2) National
Yeongnam Agricultural Research Institute, Rural Development
Administration, Milyang, KOREA
Phytopathology 100:S146
Sheath blight, caused by Rhizoctonia solani, is one of the most important rice
diseases in the U.S. and around the world. However, little is known about the
molecular mechanism and defense signaling pathways that mediate basal and
partial resistance against this necrotrophic pathogen. In this study, we attempt
to determine the role of ethylene (ET) and abscisic acid (ABA) in sheath
blight resistance using transgenic rice lines defective in hormone pathways as
well as exogenous treatments of hormones or their chemical inhibitors.
Treatments of Nipponbare cultivar with 0.2 mM ethephon, which releases ET,
significantly reduced lesion length and disease severity. By contrast,
treatments with 0.1 mM ABA greatly increased lesion length on both
Nipponbare and resistant cultivar Jasmine 85. Application of fluridone (an
ABA biosynthesis inhibitor) on Nipponbare rice markedly reduced lesion
length, whereas treatments with aminooxyacetic acid and 1-MCP (ET
biosynthesis and signaling inhibitors, respectively) increased lesion length on
Nipponbare and Jasmine 85. Furthermore, transgenic rice lines defective in
ET signaling exhibited increased susceptibility to Rhizoctonia solani
infection, whereas transgenic lines with an increased ET level showed
enhanced sheath blight resistance. Together, these results suggest that basal
and partial resistance to rice sheath blight is positively regulated by ET
biosynthesis and signaling, but negatively modulated by ABA pathway.
Biological control of banana wilt
Y. Zheng (1), J. GUO (1)
(1) Nanjing, PRC PEOPLES REP OF CHINA
Phytopathology 100:S146
Banana wilt is one of the most destructive diseases on banana production
worldwide, caused by Fusarium oxysporum f. sp. cubense (Foc). No effective
control measure for Fusarium wilt has been found other than the use of
resistant cultivars. In our study, 539 bacterial strains were isolated from soil,
roots, pseudostem, corn and leave of banana plants. According to their in vitro
antagonistic activities against Foc race 1, race 4 and their enzyme activities
including protease, chitinase, and cellulase, 177 strains with obvious activities
were selected for diversity study using amplified ribosomal DNA restriction
analysis (ARDRA) and BOX-PCR. Through analysis of the fingerprints, 22
strains from different groups were selected for greenhouse experiments on
banana. These 22 antagonists belonged to genus of Bacillus, Serratia, Pantoea,
Enterobacter, Burkholderia, Stenotrophomonas and Lysinibacillus based on
the 16S rRNA gene sequences, were confirmed in greenhouse study with the
biocontrol efficacy of 38.64–84.09%, and increased biomass by 15.92–
64.73%. Finally, 5 potential biological control agents appearanced in field
experiment in Guangdong Province, and achieved biocontrol efficacy of
35.96–69.65%.
Complete genome sequence of Capsicum chlorosis virus from
Phalaenopsis orchid and prediction of the unexplored genetic information
of tospoviruses
Y. Zheng (1), C. Chen (1), F. JAN (1)
(1) National Chung Hsing University, Taichung, TAIWAN
Phytopathology 100:S146
Phalaenopsis orchids are popular ornamentals all over the world. A
tospovirus, Capsicum chlorosis virus (CaCV-Ph) had been identified to cause
the chlorotic ringspots on leaves of Phalaenopsis orchids in Taiwan. The
tripartite genome of CaCV-Ph was found to contain 3608, 4848 and 8916 nts
of S, M and L RNAs, respectively. The phylogenetic relationship of the
nucleocapsid (N) protein indicated that CaCV-Ph was a member of
Watermelon silver mottle virus (WSMoV) serogroup in the genus Tospovirus.
Based on the relations among the nonstructural protein (NSs), glycoprotein
(GnGc), thrips genera, host and geographical distribution, they could be
classified into two major types: WSMoV-Thrips-Asian and Tomato spotted
wilt virus (TSWV)-Frankliniella-EuroAmerican. The proline (P459) of all
tospoviral Gn proteins was indispensable and the RGD motif maintained by
partial tospoviruses may not involve in the viral transmission. A RdRp
catalytic domain found in the conserved region of L protein may recognize the
S146
PHYTOPATHOLOGY
typically conserved sequences on the 5′ and 3′ terminal regions (5′
AGAGCAAU 3′). In this study, the genetic information of CaCV-Ph was
investigated by bioinformatic analyses for further investigation of CaCV-Ph
properties.
Occurrence of a rice dwarf disease in China caused by Southern rice
black-streaked dwarf virus, a new species in genus Fijivirus
G. ZHOU (1), Q. Wang (1), J. Wen (1), D. Xu (1)
(1) Laboratory of Plant Virology, South China Agricultural University,
Guangzhou, PRC PEOPLES REP OF CHINA
Phytopathology 100:S146
Southern rice black-streaked dwarf virus (SRBSDV) is a newly proposed
species in the genus Fijivirus, family Reoviridae. It was discovered on rice for
the first time in 2001, in Yangxi county, Guangdong Province, China, and
recorded previously as a new strain of Rice black-streaked dwarf virus
(RBSDV) or RBSDV-2. During the past several years, it spread rapidly
throughout southern China and northern Vietnam, and became one of the most
important rice pathogens in those regions. In the 2009 crop season, 300 000 ha
and 15 000 ha of rice was affectted in China and Vietnam, respectively, and
more than 6 500 ha of crop failure was estimated. This virus is naturally
transmitted by white-backed plnathopper (Sogatella furcifera), and infects
rice, maize, barnyard grass (Echinochloa crusgalli), chinese sorghum (Coix
lacryma-jobi), flaccid grass (Pennisetum flaccidum) and Juncellus serotinus.
In artificial tests, rice plants at any growth stage could be infected and, the
earlier they got infected, the more severe symptoms they developed.
Symptoms include severe stunting, dark green leaf, small galls on stem, and
rootlets and tillers on the upper nodes. A field survey at rice earing stage in
October 2009 in Guangzhou, China, revealed that approximate 15% of plants
were pronounced dwarf with all above symptoms and 11% showed slight
dwaf with small galls on the stem and, moreover, 20% (10/50) symptomless
plants were SRBSDV positive in RT-PCR detection.
Mechanism of suppression of a no-till hairy vetch cover crop on the
spread of anthracnose on watermelon
X. ZHOU (1), K. L. Everts (2), C. Zhou (3)
(1) AgriLife Research, Texas A&M University System, Beaumont, TX,
U.S.A.; (2) University of Maryland and University of Delaware, Salisbury,
MD, U.S.A.; (3) University of Maryland, Salisbury, MD, U.S.A.
Phytopathology 100:S146
Anthracnose caused by Colletotrichum orbiculare is one of the most common
and destructive diseases of watermelon worldwide. Previous experiments
indicated that production of watermelon on a no-till hairy vetch (Vicia villosa)
cover crop was effective in reducing anthracnose. The objective of this study
was to examine the spread of anthracnose on watermelons when grown on
vetch and other ground coverings to better understand the mechanism of
action of disease suppression. Spread of watermelon anthracnose was
evaluated in field plots over three years by assessing disease severity at a
range of distances from an introduced point source of infection. Ground
covers were bare soil, black plastic, or a cover crop of vetch that was grown
on raised beds in the fall, killed in the spring and left on the soil surface. At all
assessment times, there was a significant reduction in disease severity with
increasing distance from the infection source. Severity was significantly lower
at most of the distances evaluated in plots with vetch compared with bare soil
or plastic. Percentage of diseased and sunburned fruit was 47% lower in plots
with vetch than with bare soil or plastic. Soil splash dispersal also was
reduced in plots with vetch or plastic versus bare soil. These results indicate
that the use of no-till vetch cover crop can reduce the dispersal of spores by
rain splash and thus have a significant suppressive impact on the spread of
anthracnose on watermelon.
Screening of bacterial antagonists for suppression of sheath blight in rice
X. ZHOU (1), K. Kumar (2), M. Reddy (2), J. Kloepper (2), S. Zhang (3)
(1) AgriLife Research, Texas A&M University System, Beaumont, TX,
U.S.A.; (2) Dept. Entomology and Plant Pathology, Auburn University,
Auburn, AL, U.S.A.; (3) Tropical REC, University of Florida, Homestead,
FL, U.S.A.
Phytopathology 100:S146
Sheath blight caused by Rhizoctonia solani is the most important disease of
rice in the southern United States. Since none of the leading high-yielding
cultivars have acceptable levels of resistance, sheath blight management has
been largely depended on fungicides. However, the use of fungicides is costly
and unsustainable. Biological control has been recently considered as a
promising option. In vitro and greenhouse assays were conducted to screen
biocontrol agents for suppression of sheath blight in rice. Nineteen bacterial
strains that were previously demonstrated growth promotion in other plants
and antibiosis against other plant pathogens were examined for their
antifungal activity on mycelial growth of R. solani, germination of sclerotia
and hyphal growth of germinated sclerotia. They were also examined for their
ability to inhibit lesion development on leaf blades and sheaths of seedlings in
the greenhouse. Ten out of 70 strains showed significant inhibition of mycelia
growth and the hyphal growth of germinated sclerotia. Four of these 10 strains
also significantly inhibited the germination of sclerotia. When tested in the
greenhouse, 10 strains significantly reduced the lesions on leaf blades and
sheaths. The performance of these strains, most of which belong to Bacillus
subtilis, under field conditions will be further evaluated.
A cucumber mosaic virus mutant that induces resistance to its aphid
vector in tobacco
H. ZIEBELL (1), A. Murphy (2), M. G. Lewsey (2), J. H. Westwood (2), K.
L. Perry (1), M. Stevens (3), J. P. Carr (2)
(1) Cornell University, Ithaca, NY, U.S.A.; (2) Cambridge University,
Cambridge, UNITED KINGDOM; (3) Broom’s Barn Applied Crop Sciences,
Bury St Edmunds, UNITED KINGDOM
Phytopathology 100:S147
Cucumber mosaic virus (CMV) is transmitted by aphids in a non-persistent
manner. The CMV coat protein is the only known factor required for aphid
transmission. We have found that the CMV 2b protein plays an indirect but
important role in transmission by protecting the vector against the induction of
anti-insect defenses. The 2b protein is a counter-defense factor that suppresses
the initiation of RNA-silencing, amongst others. While investigating the
potential of CMVΔ2b (a CMV mutant in which the gene for the 2b protein is
deleted) as a cross-protection agent, we noted that aphid (Myzus persicae)
infestation was inhibited on CMVΔ2b-infected plants of tobacco (Nicotiana
tabacum). We found statistically significant decrease in aphid survival and an
increases in aphid mortality on CMVΔ2b-infected plants compared to mockinoculated plants or plants infected with wild-type CMV. The data indicates
that in CMVΔ2b-infected plants a viral gene product other than 2b (or the
stress of viral infection) induces resistance to aphids. However, in plants
infected with wild-type CMV we suspect that this effect is neutralized by the
2b protein. The results suggest that an RNA-silencing suppressor may also
target anti-aphid defenses as well as anti-viral responses in plants. Gene
expression analysis of jasmonic acid-regulated genes in tobacco support this
hypothesis. Furthermore, aphid transmission of CMVΔ2b is drastically
reduced compared to wild-type CMV. Work funded by grants from BBSRC
and Leverhulme Trust.
Sphingoid bases and their 1-phosphates, but not fumonisins, are
translocated from roots to aerial tissues of maize seedlings watered with
fumonisins
N. C. ZITOMER (1), S. Jones (2), C. Bacon (1), A. E. Glenn (1), T. Baldwin
(1), R. T. Riley (1)
(1) USDA – ARS, Toxicology and Mycotoxin Research Unit, Athens, GA,
U.S.A.; (2) South Carolina State University, Department of Biological
Sciences, Orangeburg, SC, U.S.A.
Phytopathology 100:S147
In an earlier study using maize seedlings grown from kernels inoculated with
Fusarium verticillioides, fumonisin B1 (FB1) was preferentially accumulated
in leaf tissue compared to FB2 and FB3. The present study tested whether
maize seedlings preferentially translocate FB1 when plants are watered with
FB1 and/or FB2, without the fungus present. The results show that neither FB1
nor FB2 was translocated when administered in the watering solution and
while both FB1 and FB2 were taken up by the roots the accumulation of FB2 in
roots was significantly less than predicted indicating that FB1 was
preferentially accumulated. In addition there was clear evidence of ceramide
synthase inhibition in the roots and sphingoid base and sphingoid base 1phosphates accumulated in leaf tissue presumably due to translocation from
the roots. These findings suggest that the fungal/plant interaction is necessary
for FB1 translocation in maize seedlings infected with F. verticillioides.
Combination of genetic resistance, reduced-risk fungicides and Tom-Cast
for tomato disease control
T. A. ZITTER (1), J. L. Drennan (1), M. A. Mutschler (1)
(1) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S147
Late blight (LB), early blight (EB), and Septoria leaf spot (SLS) are the major
foliar tomato diseases in temperate regions. Growers currently rely upon
fungicides applied on a weekly basis for control. Hybrid tomato varieties with
EB tolerance and/or LB resistance are becoming available commercially, and
the addition of SLS resistance is progressing. The objectives of this research
were: to confirm the need for homozygous resistance for EB, to determine the
efficacy of reduced-risk fungicides (azoxystrobin + difenoconazole and
boscalid) following Tom-Cast, compared with weekly application of
chlorothalonil or an organic practice (Bacillus subtilis + cupric hydroxide) for
EB and SLS, and to reduce the environmental impact quotient (EIQ) for the
fungicides chosen. EB homozygous tolerant genotypes performed
significantly better than heterozygous genotypes or susceptible controls over a
3 year period. Genotypes with LBR conferred by Ph3 plus Ph2 genes were
immune to LB-US22 in 2009. All fungicide treatments provided control of
both EB and SLS compared with the unsprayed control. Chlorothalonil when
applied weekly developed tolerance to EB, but provided season-long control
of SLS. The reduced-risk treatments provided superior control of both
diseases with 4 fewer sprays for EB and 3 fewer sprays for SLS control. The
EIQ values of these fungicides were 80% lower than those for the
conventional or organic treatments.
Silencing of Cysteine protease, acidic chitinase or PR1-a individually,
does not hamper BTH mediated resistance to P. infestans in tomato
A. ZULUAGA (1)
(1) Cornell, Ithaca, NY, U.S.A.
Phytopathology 100:S147
Induced resistance by chemicals such as benzothiadiazole BTH (Syngenta
Inc) mimics the biological activation of Systemic Acquired Resistance (SAR)
by necrogenic pathogens. BTH takes the place of salicylic acid (SA) in the
SAR signal pathway, inducing the same molecular markers and range of
resistance. Previous work in our laboratory found that BTH activates
resistance against late blight caused by P. infestans, on petunias and tomatoes
while it did not activate resistance against the same pathogen on potatoes,
suggesting that the spectra of resistances activated by BTH are crop and
pathogen specific. The goal of our work was to understand the molecular
mechanism by which BTH mimics the SAR response and further
understanding why BTH works in some plants and not others. To address this
question we used microarray technology to identify the genes expressed in
response to BTH in tomatoes. Of these we selected three candidate genes
(cysteine protease, acidic chitinase and PR1-a) to characterize further by
silencing using Virus Induced Gene Silencing (VIGS). Our hypothesis was
that silencing of these genes will reduce the resistance response observed in
plants after BTH treatment. However, silencing of cysteine protease, PR1-a or
acidic chitinase II individually did not reduce the effect of BTH on plants. The
lack of phenotype after silencing PR1-a supports previous conclusions from
our lab that partial resistance to P. infestans in tomato is not dependent of the
SA pathway.
Sensitivity of Fusarium graminearum isolates to pyraclostrobin
M. ZWINGMAN (1), J. Hernandez Nopsa (1), K. Eskridge (1), S. Wegulo (1)
(1) University of Nebraska, Lincoln, NE, U.S.A.
Phytopathology 100:S147
Fusarium graminearum causes Fusarium head blight (FHB) of wheat as well
as ear and stalk rots of corn. Pyraclostrobin is one of the most frequently
applied fungicides for control of foliar diseases of wheat and corn in
Nebraska. The objective of this study was to determine if Nebraska isolates of
F. graminearum differed in sensitivity to pyraclostrobin. The isolates were
collected from wheat fields and elevators in 2007 following severe epidemics
of FHB in the south central and eastern parts of the state. Potato dextrose agar
was amended with salicylhydroxamic acid (SHAM, dissolved in methanol) at
100 µg/ml of PDA, then with technical grade pyraclostrobin (95 percent)
dissolved in acetone at 0, 0.001, 0.01, 0.1, 1.0, and 10.0 µg/ml. A 5-mmdiameter PDA mycelial plug from an actively growing edge of each of 15 F.
graminearum isolates was placed, mycelial face down, at the center of the
amended PDA plates which were then incubated at 25°C in 12 hr light and 12
hr dark. An alpha lattice randomized design with 3 replications was
used. EC50 values calculated from mycelial area measured after 10 days
ranged from 0.063 µg/ml to 1.585 µg/ml for 12 isolates. However,
isolates NE90, NE101, and NE91 had EC50 values of 15.85, 100.0, and
398.1 µg/ml, respectively. These preliminary results indicate the development
of resistance to pyraclostrobin in some Nebraska populations of F.
graminearum.
Vol. 100, No. 6 (Supplement), 2010
S147
2010 APS Annual Meeting
Abstracts of Special Session Presentations
Biology of Plant Pathogens
10th I. E. Melhus Graduate Student Symposium:
Seed Pathology—Epidemiology, Management,
and Phytosanitary Concerns
Quorum sensing affects virulence and seed-to-seedling transmission of
Acidovorax avenae subsp. citrulli, the causal agent of bacterial fruit blotch
K. JOHNSON (1)
(1) University of Georgia, Athens, GA, U.S.A.
Phytopathology 100:S148
Watermelon fruit and seed production can be significantly limited by bacterial
fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli (AAC). BFB
can cause up to 100% loss in fields and infested seed is the most important
source of inoculum. During AAC colonization of seed there is a switch from
non-pathogenic to pathogenic growth that could be regulated by quorum
sensing (QS). QS is the ability of bacteria to communicate with each other and
respond collectively to environmental cues. AAC has the QS homologs, luxI
and luxR, based on genomic sequence analysis. The role of QS in seed
colonization and seed-to-seedling transmission was investigated using luxR
and luxI mutants of AAC strain 00-1. The luxR and luxI mutants were able to
colonize germinating watermelon seed to levels similar to wildtype. The QS
mutants were efficiently transmitted from seed-to-seedling when high levels
of initial inoculum, (106 CFU), were used. Seed infiltrated with AAC00-1, the
luxI or luxR mutant had 97.5, 93 and 95% seed-to-seedling transmission,
respectively. However, the luxI mutant was significantly reduced in its ability
to be transmitted from seed-to-seedlings compared to wildtype when low
levels of initial inoculum, (103 CFU), were used. Seed infiltrated with
AAC00-1 or the luxI mutant had 76 and 33% seed-to-seedling transmission,
respectively. These results suggest that QS plays a role in seed-to-seedling
transmission of the BFB pathogen.
Effect of the mechanism of infestation on the localization of A. avenae
subsp. citrulli in naturally infested watermelon seeds
B. DUTTA (1)
(1) University of Georgia, Athens, GA, U.S.A.
Phytopathology 100:S148
Previously, it was determined that watermelon seeds could become infested
by Acidovorax avenae subsp. citrulli (Aac) via two mechanisms: 1)
penetration of the pericarp of the ovary that results in fruits with bacterial fruit
blotch (BFB) symptoms and 2) invasion of the pistil that results in infested
seeds within symptomless fruits. In this study, we investigated the effect of
the mechanism of seed infestation on localization of Aac in seeds.
Watermelon seeds from symptomatic fruit (pericarp invasion) and
asymptomatic fruit (pistil invasion) were tested for Aac by PCR and plating
on semi-selective media. Samples (n = 50 seeds) from each type of seedlot
were dissected into sections including seed coat (testa), perisperm-endosperm
layer (a thin suberized envelope that encloses the cotyledon), and endosperm.
The abstracts are published as submitted. They were formatted but not edited
at the APS headquarters office.
S148
PHYTOPATHOLOGY
The mean proportions of Aac-positive PE layer sections by real-time PCR
were not significantly different for pistil (83%) and pericarp-invaded (99%)
seedlots. In contrast, for the same seedlots, the mean proportions of Aacpositive endosperm tissue samples were significantly higher for pistil-invaded
(98%) than for pericarp-invaded (11%) seedlots. Additionally, less than 8% of
the testa samples were Aac-positive for both seedlot types. These results
indicate that for seeds infested by pericarp penetration, Aac becomes localized
on or outside the PE layer. In contrast, when seeds become invaded by pistil
penetration, Aac becomes localized in the endosperm under the PE layer.
Characterization of genes in Fusarium verticillioides regulating
colonization of maize kernels
R. L. HIRSCH (1), B. H. Bluhm (1)
(1) University of Arkansas, Fayetteville, Fayetteville, AR, U.S.A.
Phytopathology 100:S148
Fusarium verticillioides is a common seedborne pathogen of maize and
produces fumonisin mycotoxins during kernel colonization. Identifying genes
underlying seed infection is crucial to elucidate pathogenesis at the molecular
level. The objective of this research was to characterize genes in F.
verticillioides that regulate kernel colonization. The overall approach was to
identify genes through forward genetics and determine their function through
targeted disruption. First, a collection of >3000 random insertional mutants
was generated via Restriction Enzyme Mediated Integration (REMI) with a
novel promoter-trapping cassette. A high throughput in vitro screen was
developed to quantify the hydrolysis of starch, the predominant carbohydrate
in maize kernels. Nine mutants with altered starch hydrolysis were analyzed
for their ability to colonize maize kernels; of these, one mutant was
significantly impaired in kernel colonization and fumonisin production. In this
mutant, the REMI cassette disrupted a gene encoding a putative ubiquitin
ligase (designated UBL1). Targeted disruption of UBL1 in the wild-type strain
confirmed the phenotype of the REMI mutant. The discovery of a novel regulatory gene underlying seed colonization and fumonisin biosynthesis significantly expands the working model of kernel pathogenesis in F. verticillioides.
Interactions between viruses and Phomopsis infection in soybean, and
effects of integrated management strategies
J. SOTO-ARIAS (1), G. Munkvold (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S148
Bean pod mottle virus, Soybean mosaic virus, bean leaf beetles, soybean
aphids and Phomopsis spp. all affect soybean seed quality in addition to
causing yield losses. However, interactions among these pests and pathogens,
and the effects of combined management practices, are not well understood.
To understand these interactions, greenhouse studies were established to
determine the effects of virus infection on susceptibility of soybean plants to
infection by Phomopsis longicolla at different growth stages. Virus
inoculation with either SMV or BPMV, significantly increased seed infection
by P. longicolla, compared with the control or plants only inoculated with P.
longicolla. To evaluate the effects of management strategies, four experiments
were established in 6 locations in Iowa during 2008 and 2009. Applications of
Headline at R5, or Stratego Pro by itself or in combination with insecticide at
R3, significantly reduced Phomopsis spp. infection of stems and seeds.
Insecticide applications alone reduced aphid and bean leaf beetle populations.
In some experiments insecticide applications also reduced Phomopsis spp.,
SMV and BPMV infection of seeds, but this effect was not consistent. Virus
incidence and beetle populations were very low in both years, and seed
mottling was not observed. Phomopsis spp. infection affected seed
germination in some experiments. Few treatments aimed at insect and disease
control had an effect on yield.
Advances in Plant Virus Evolution
set of genes specifically responding for phylogenetically related viruses
represents interactions that acquired during the evolutionary diversification of
a viral family. Those interactions shared by phylogenetically unrelated viruses
represent anon-specific responses.
The evolution of plant virus evolution: A historical overview
W. L. SCHNEIDER (1)
(1) USDA-ARS FDWSRU, Fort Detrick, MD, U.S.A.
Phytopathology 100:S149
Plant viruses face challenges at every phase of their life cycle. There is strong
selection pressure to interact with the host during uncoating, translation,
replication, cell to cell movement and long distance movement. At the same
time there is a constant battle to avoid the host defenses and RNA silencing.
In addition, plant viruses face selection pressures during horizontal
transmission. From the early days of plant virus research virologists noted
the flexible traits of their subjects. Not surprisingly, viruses, with high
mutation frequencies and large populations are adept at evolving to deal with
new selection pressures and challenges. However, recent research has
provided valuable insight into just how these variable populations of
RNA viruses, DNA viruses and viroids contribute to plant virus evolution.
This symposium covers some of the latest work on plant virus evolution,
including population processes, the factors affecting viral emergence, viral
breakdown of host resistance, the evolution of plant DNA viruses, and viroid
evolution.
Population processes and plant virus evolution
R. FRENCH (1)
(1) USDA, ARS, University of Nebraska, Lincoln, NE, U.S.A.
Phytopathology 100:S149
The number of studies detailing levels of sequence diversity within plant virus
populations are growing at a rapid pace. At the same time, recent work has
provided empirical estimates of parameters important in the life cycle of plant
viruses, which in turn can help in understanding observed patterns of
polymorphism. Despite the fact that plant viruses are prolific replicators,
producing upwards of millions of virions per cell, they are subjected to severe
genetic bottlenecks at virtually all stages of growth, including cell to cell
movement, systemic infection, and horizontal transmission to new hosts.
Thus, the effective population size (Ne) of plant viruses is many orders of
magnitude smaller than their census numbers. Ne is of crucial importance in
determining both the rate of genetic drift in a population (drift is faster in
smaller populations), and the efficacy of selection relative to drift. Evidence
also suggests that intracellular replication of RNA viruses (as well as DNA
viruses replicating by a rolling circle mechanism) is a nearly linear ‘stamping
machine’ process. This profoundly reduces the number of mutant genomes
that are produced in a viral population. A ‘stamping machine’ mode of
replication also increases the variation in offspring number among potential
parental genomes and reduces Ne. Nevertheless, strong selection can still
effect changes even in small populations, with better adapted genotypes
replacing those with deleterious mutations.
Evolutionary and systems biology of plant RNA virus emergence
S. F. ELENA (1)
(1) Instituto de Biología Molecular y Celular de Plantas, Valencia, SPAIN
Phytopathology 100:S149
Understanding the underlying mechanisms by which viruses are able to
overcome the host’s defenses and proliferate has been a challenging problem
because the large number of cellular factors involved and the complexity of
interactions established during infection. The classic approach has been the
identification of one or few host genes involved in the interaction. The
generalization of the “omic” techniques is opening the possibility of taking a
whole picture of the interaction. Here, I present results from an evolution
experiment in which Tobacco etch virus has been adapted to Arabidopsis
thaliana. I show that adaptation has profound effects in the way virus and
plant interact. Next, I present a comparative study of the lists of over/underexpressed genes from infection experiments with the potyviruses Tobacco
etch virus, Turnip mosaic virus and Plum pox virus, and the phylogenetically
unrelated Turnip crinkle virus. We analyze lists in terms of biological
functions. Then, taking advantage of the recently inferred regulatory networks
of A. thaliana, we dissect the viral mode of action showing a directed
mechanism by altering the expression of key genes on the interactome. The
Evolution of natural populations of BNYVV to overcome host resistance
C. M. Rush (1), R. ACOSTA-LEAL (1)
(1) Texas AgriLife Research, Amarillo, TX, U.S.A.
Phytopathology 100:S149
Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae,
causes rhizomania, a devastating root disease of sugar beet. Genetic resistance
against BNYVV, conferred by the single dominant gene Rz1, is commercially
available in regionally adapted cultivars in all production regions of the
U.S.A. In the last few years, cultivars with a second resistance gene, Rz2, and
cultivars with a combination of Rz1 and Rz2 have been released. Based on
results of variety trials, it appears that resistance in cultivars with Rz1 + Rz2>
Rz2> Rz1. BNYVV can be isolated from all of these resistant cultivars but the
virus is typically low titer and disease symptoms are absent or minimal.
However, in 2002, sugar beets with Rz1 resistance from the Imperial Valley of
California, displayed severe symptoms of rhizomania. Although isolates of
BNYVV typically are highly conserved, most isolates from symptomatic Rz1
plants exhibited an unique aa motif in the hyper variable region of p25 on
RNA3. Furthermore, isolates of BNYVV from infected, but asymptomatic,
Rz1 plants were also different from wild type BNYVV. Resistance breaking
isolates from other regions of the U.S.A. are similar, but not identical, to
California RB isolates. Greenhouse experiments revealed that strength of
genetic resistance in the host significantly affects virus mutation frequency.
How do Geminiviruses evolve as quickly as RNA viruses?
S. DUFFY (1)
(1) Rutgers, The State University of New Jersey, New Brunswick, NJ, U.S.A.
Phytopathology 100:S149
Geminiviruses are significant emerging pathogens of crops worldwide. Their
ability to emerge and adapt to novel hosts has long been attributed to
their frequent recombination. However, evidence is mounting that they
evolve quickly in the absence of recombination, and have similar nucleotide
substitution rates as RNA viruses of plants and animals. The mechanisms
by which geminiviruses could accumulate mutations as quickly as RNA
viruses are evaluated: high mutation rates, short generation times and
recurrent selective sweeps. Mutation frequencies and mutational spectra
suggest that geminiviruses have mutation rates much higher than previously
assumed.
Advances in the understanding of viroid evolution
R. W. HAMMOND (1)
(1) USDA, ARS, PSI, MPPL, Beltsville, MD, U.S.A.
Phytopathology 100:S149
Viroids are the smallest known pathogenic agents of plants and cause diseases
of considerable economic importance. Their genomes are composed of a
single-stranded, covalently-closed, circular, highly-structured RNA molecule
of 246 – 401 nt. They are classified into two families—those that replicate in
the nucleus (pospiviroids) and those that replicate in the chloroplast
(avsunviroids). Viroids lack the capacity to code for proteins, are not
encapsidated, and are replicated by host-encoded polymerases. As such,
viroids interact with their host through specific structural/sequence motifs for
replication, movement, and pathogenesis. Viroid infections are typically
characterized by the presence of a population of sequence variants that
conform to a quasispecies model and where predominant forms accumulate
during infection. Point mutations and RNA recombination contribute to the
sequence diversity of viroids, and the requirements to maintain conserved
structures, the host response to infection, and environmental selective
pressures all contribute to influence the population of variants. Analysis of
accumulated sequence data from natural and experimental populations of
pospi- and avsunviroids has led to the proposal of several intriguing models of
viroid RNA evolution. In addition to a discussion of these models, the
implications of viroid evolution to agriculture will be discussed.
Vol. 100, No. 6 (Supplement), 2010
S149
Refining Systematics (Taxonomy, Nomenclature,
Phylogenetics) for Better Resolution in the
Population Biology and Evolution of the Oomycetes
Pythium, Pythiogeton and prov. name Phytopythium: The current status
for the species in the genera
A. W. DE COCK (1), G. P. Robideau (2), K. Bala (2), M. D. Coffey (3), Z. G.
Abad (4), C. A. Lévesque (2)
(1) CBS-KNAW Fungal Biodiversity Centre, Utrecht, NETHERLANDS; (2)
Agriculture and Agri-Food Canada, Ottawa, CANADA; (3) Department of
Plant Pathology and Microbiology, University of California, Riverside, CA,
U.S.A.; (4) USDA-APHIS-PPQ-PHP-RIPPS-Molecular Diagnostics Laboratory, Beltsville, MD, U.S.A.
Phytopathology 100:S150
Traditionally, genera and species in Oomycetes have been distinguished and
defined based on morphological characteristics. The genus Pythium is
characterized by its well developed hyaline mycelial thallus and the way
zoospores are developed and discharged: the sporangium forms a discharge
tube through which the contents move out and form a vesicle at the tip with an
undifferentiated mass of protoplasm. This mass then differentiates into
biflagellate zoospores. Although this way of zoospore discharge is shared by
all Pythium species, the genus is heterogeneous with regard to morphological
characters like e.g. the sporangium shape. The genera Pythiogeton and
Lagenidium display a way of zoospore discharge similar to that in Pythium,
though they are considered different genera based on other characteristics.
DNA sequence analyses allow an evaluation of the morphological
classification over a phylogenetic framework. Analysis of ribosomal DNA
regions and the mitochondrial COI showed that a clade within Pythium is
actually more closely related to Phytophthora than to Pythium; this clade is
provisionally named Phytopythium. Moreover, molecular analyses revealed
the close relationship of Pythiogeton and some Lagenidium species to Pythium.
The phylogenetic results will be discussed with regard to morphology.
How to avoid misidentifying your isolates: The value of the
Morphological / Phylogenetic Key of Phytophthora extypes and neotypes
Z. ABAD (1)
(1) USDA-APHIS-Molecular Diagnostics Laboratory, Beltsville, MD, U.S.A.
Phytopathology 100:S150
Phytophthora with 105 species is a major genus of plant pathogens. Although
there have been considerable advances in its molecular taxonomy, there is still
confusion in recognizing new Phytophthora species and in identifying
described species. This confusion is due in part to the great number of
misidentified or incorrectly annotated sequences submitted to the GenBank.
Such errors in identification make it difficult to recognize many of the clusters
of the “sensu stricto”. The “Holotype” (= Type) is the single isolate that
defines the species and the “Ex-holotype” is the isolate originated from the
“Holotype”. Interestingly, taxonomic manuscripts published after the
description of P. infestans in 1876 (until present) rarely contain information
on the codes of the types. This information, which has rarely been presented,
is vital for refining the systematics of the Genus. We are reviewing the
original manuscripts to compile information of the Primary Types, assigning
Lectotypes and selecting potential Neotypes. Our goal is to establish a
database of sequences of the types and to use selected cultures to develop a
Morphological/Phylogenetic Phytophthora Key, and to publish a manuscript
to update the Taxonomy of the Genus. The USDA-APHIS-MDL is
collaborating with the World Phytophthora Collection and the Phytophthora
Database on this important initiative. We expect that the database and the key
will be useful tools to avoid misidentifying isolates of this important genus.
The Phytophthora Database: Current status and future directions
S. KANG (1)
(1) Department of Plant Pathology, Penn State, University Park, PA, U.S.A.
Phytopathology 100:S150
The Phytophthora Database (http://www.phytophthoradb.org) supports rapid
identification of Phytophthora species via comparison of sequences at one or
more marker loci. Besides archiving marker sequences from most of the
known and newly discovered species to support identification and new species
description, the database provides a comprehensive overview of Phytophthora
molecular diagnostics, morphological and pathological characteristics of many
of the archived species, and references. Data search and analysis tools in the
database include BLAST, Phyloviewer (a program for building phylogenetic
trees using selected sequences), Virtual RFLP (a program for generating
expected restriction patterns for given sequences), GIS tool (a program for
visualizing the geographic origins of species and isolates through a global
map), and Cart & Folder (a customized means of storing and sharing data via
the database). The current status and future directions of the database will be
presented.
S150
PHYTOPATHOLOGY
The Oomycetes Database: The initiative for an international web-based
informatics platform
F. N. MARTIN (1)
(1) USDA-ARS, Salinas, CA, U.S.A.
Phytopathology 100:S150
Over the years the taxonomic classification of Oomycetes based on
morphological features has been in a state of flux and the application of
molecular techniques has not always provided the clarity that is desired. In
part this has been due to a lack of consistency of the loci that have been
sequenced as well as a historical under representation of some groups of
organisms among the different studies. The objective of this project is to
establish a collaborative initiative among researchers working this diverse
group of organisms to facilitate a broader scale analysis of the kingdom using
the same set of nuclear and mitochondrially encoded loci. This data will be
presented on a web-based informatics platform patterned on what has been
developed for Phytophthora (www.phytophthoradb.org). In addition to
providing sequence data and a comprehensive multigene phylogenetic
analysis, there will also be an overview on the biology and ecology of the
orders, genus and species descriptions and their morphological features, tools
supporting species identification based on molecular and morphological
criteria and visualizing the geospatial, environmental, and/or temporal
contexts of archived species and isolates. Efforts will initially focus on
analysis of the Peronosporomycetidae (sensu Dick) and will be expanded at a
later time to include members of the Saprolegneomycetidae.
Mitochondrial genomics of Oomycetes, tools for phylogenetics and
development of molecular markers
F. N. MARTIN (1)
(1) USDA-ARS, Salinas, CA, U.S.A.
Phytopathology 100:S150
Due to its comparatively small size, similar number of genes and rate of
evolutionary divergence the mitochondrial genome can be a valuable resource
for elucidating phylogenetic relationships and development of molecular
makers. In an effort to facilitate the use of this region for these purposes the
mitochondrial genomes of 15 Pythium and 20 Phytophthora spp. have been
sequenced. Comparative genomics has been useful for identification of genes
useful for estimating evolutionary relationships and development of conserved
primer sequences for their amplification. A mitochondrial multigene
phylogeny for the genus Phytophthora was recently completed and efforts are
underway to include Pythium and other Oomycetes in the analysis using the
same regions. Comparison of genomic sequences among Phytophthora spp.
has identified the types of polymorphisms associated with intraspecific
compared to interspecific genome evolution. This has facilitated the
identification of regions more prone to evolutionary divergence that are useful
for classification of mitochondrial haplotypes. Conserved mitochondrial gene
order differences among Phytophthora compared to Pythium and plants have
also been useful for development of a systematic approach for development of
multiplexed TaqMan real time PCR diagnostic marker system for
identification of Phytophthora at a genus as well as species specific level. A
similar approach is under investigation for Pythium as well.
Population genetic insights into emergence of oomycete pathogens
N. J. GRUNWALD (1)
(1) USDA ARS, Corvallis, OR, U.S.A.
Phytopathology 100:S150
Oomycetes include notable pathogens that have repeatedly emerged as
significant threats to plant biosecurity. Among these are for example the
sudden oak death pathogen Phytophthora ramorum and the potato late blight
pathogen P. infestans. Population genetic tools, whether based on molecular
markers such as microsatellites or nucleic acid sequences, can provide unique
insights into the evolutionary dynamics underlying invasion or emergence of
Oomycete plant pathogens. Select examples of population genetic approaches
used to understand the emergence of Oomycete pathogens will be presented
and explored.
Aquatic habitats—A reservoir for population diversity in the genus
Phytophthora
J. HWANG (1), S. N. Jeffers (1), S. W. Oak (2)
(1) Clemson University, Clemson, SC, U.S.A.; (2) USDA Forest Service,
Southern Region-FHP, Asheville, NC, U.S.A.
Phytopathology 100:S150
Occurrences of oak decline and sudden oak death in forests of Europe and the
west coast of the U.S.A., respectively, have focused attention on the species of
Phytophthora present in natural ecosystems. We have been investigating the
diversity of species of Phytophthora present in forest streams in the eastern
U.S.A. Phytophthora spp. are well adapted to aquatic environments and can
be recovered from stream water by baiting and filtration. Extensive surveys in
multiple states revealed that a diversity of species occurs naturally in forest
streams. In one study, five forest streams in western North Carolina were
monitored monthly for a year. Seven species—P. cambivora, P. cinnamomi,
P. citricola, P. citrophthora, P. gonapodyides, P. heveae, and P. pseudosyringae—and seven morphologically and genetically distinct groups of
isolates were detected. Samples of stream-side soils and plants with symptoms
also were collected, but only three species were detected: P. cinnamomi and
P. heveae in soils and P. citricola and P. heveae on plants. Species of
Phytophthora consistently were detected in streams during winter months
when air temperatures were near or below freezing, which are not conducive
to lesion development and sporulation. These results suggest that the native
population of Phytophthora spp. in stream water is different from those in
terrestrial habitats. The species of Phytophthora present in streams may
occupy a unique niche—i.e., they appear to be aquatic inhabitants and not
transient visitors.
Ecological adaptations in Phytophthora. Understanding their role in
forest ecosystems
Y. BALCI (1)
(1) University of Maryland, College Park, MD, U.S.A.
Phytopathology 100:S151
The Sophistication of Host-Pathogen Interactions
Involving Necrotrophic Fungi
Live and let die: The smart lifestyle of Botrytis cinerea
J. A. VAN KAN (1)
(1) Wageningen University, Wageningen, NETHERLANDS
Phytopathology 100:S151
It becomes increasingly apparent that interactions between plants and
necrotrophic fungi are surprisingly subtle and complex, and host plants in fact
play a much more active role in disease than previously anticipated. Just
causing ‘death’ isn’t good enough, the execution of programmed cell death by
a host plant in response to a pathogen is crucial for many necrotrophs to be
successful. Botrytis cinerea is a ubiquitous pre- and post-harvest pathogen
infecting a wide range of host plants and tissues. I will present an overview of
current knowledge on pathogenicity factors of B. cinerea, with emphasis on
phytotoxic metabolites and proteins that can cause (programmed?) plant cell
death. I will subsequently discuss processes occurring in the host plant during
the interaction, with emphasis on the formation of Reactive Oxygen Species
and nitric oxide, as well as on cell death pathways. Examples will be presented of host defense responses during B. cinerea infection, that contribute to
(partial) resistance. The capacity of B. cinerea to counteract the growth inhibitory activity of defence compounds, by a combination of enzymatic detoxification and secretion mechanisms, also contributes to its successful lifestyle.
Necrotrophy in Sclerotinia sclerotiorum: To oxalate and beyond
J. ROLLINS (1)
(1) University of Florida, Gainesville, FL, U.S.A.
Phytopathology 100:S151
Oxalate production is positively associated with the broad host range
macerating symptoms of Sclerotinia sclerotiorum diseases. Yet, many nonpathogens produce oxalate; many pathogens manifesting different symptoms
produce oxalate; and other Sclerotinia species produce oxalate but have
unique or restricted host ranges. What attributes of oxalate regulation and
what other factors account for the host range and symptomatology of S.
sclerotiorum disease? Our studies have revealed that the ambient pH
environment, via Pac1 molecular regulation, plays a key role in controlling
oxalate accumulation. This regulation appears to act directly on at least one
structural gene in the oxalate biosynthetic pathway. This gene, oah1; encoding
oxaloacetate acetylhydrolase, is down-regulated in the low-oxalate pac1 lossof-function mutant. Two independently isolated oah1 deletion mutants fail to
accumulate oxalate in culture or in planta. Despite the lack of oxalate
production, these mutants infect a range of host plants albeit, with greatly
attenuated symptoms. In addition, their culture filtrates retain necrosisinducing activity when infiltrated into host leaves. These findings indicate that
factors other than oxalate may be responsible for establishing basic
compatibility. Current investigations are aimed at distinguishing between
direct toxic roles and more subtle host-modulating activities of oxalate and in
identifying other factors that condition host-pathogen compatibility.
Systematic characterization of the kinome of the wheat scab fungus
Fusarium graminearum
J. XU (1)
(1) Purdue University, West Lafayette, IN, U.S.A.
Phytopathology 100:S151
We have examined two species of Phytophthora for their role in plant health
in Appalachian oak ecosystems; one from soil and the other from streams; P.
cinnamomi and P. appalachiensis, respectively. P. cinnamomi was found to be
the most common and widespread species in eastern U.S. oak forests during a
multi State survey. P. appalachiensis has been identified as a new species
from a stream in West Virginia. It could only be isolated during June-October
and no other species was isolated from the same stream. It also was found
infecting fallen leaves in the stream and live foliage if shoots of
rhododendrons were dipped into the stream. During leaf inoculations, it was
pathogenic, but significantly more when wounded. P. cinnamomi was the
most common Phytophthora species below the N 40° latitude range. Its
occurrence in the eastern U.S. oak forests most likely is restricted by the low
minimum temperature extremes as reflected by the overlapping incidences
with plant hardiness zone maps. In infested sites, multiple woody plants
harbored the pathogen. When examined with the oak decline incidences in
Ohio, we found significant root mortalities on infested white oaks (Q. alba)
and greater inoculum levels in lower moist bottomlands. This pathogen
appears to be mainly affecting tree health by killing fine roots particularly
when site conditions are favorable, whereas, P. appalachiensis seems to be
opportunistic in behavior.
Wheat scab caused by Fusarium graminearum is one of the most important
diseases of wheat. Beside yield losses, infested wheat kernels are often
contaminated with mycotoxins. Like in many other eukaryotes, protein
kinases play major regulatory roles in filamentous fungi. In F. graminearum,
there are 126 predicted protein kinases that belong to different protein kinase
groups and families. To determine their functions, we have undertaken a
systematic approach to generate gene replacement mutants. For a number of
protein kinase genes, we were able to isolate mutant although their
orthologues are essential in the budding yeast. All the mutants have been
assayed for their defects in wheat head infection, DON production,
conidiogenesis, sexual reproduction, responses to various stresses, and hyphal
growth. Several protein kinases were found to be important for pathogenesis
and conidiogenesis. The interaction among these protein kinases and their
association with other F. graminearum proteins were predicted based on their
yeast orthologues. For a few predicted pathways or networks that are
important for plant infection, affinity purification and co-immunoprecipitation
assays will be used to determine their interactions in vivo.
Pathogen hijacking of disease resistance mechanisms in wheat
J. D. FARIS (1), Z. Zhang (2), S. Lu (2), H. Lu (3), J. Fellers (4), S. Cloutier
(5), S. Xu (2), R. Oliver (6), J. Rasmussen (2), S. Meinhardt (2), T. Friesen (2)
(1) Northern Crop Science Laboratory, USDA-ARS, Fargo, ND, U.S.A.; (2)
Fargo, ND, U.S.A.; (3) Amarillo, TX, U.S.A.; (4) Manhattan, KS, U.S.A.; (5)
Winnipeg, Manitoba, CANADA; (6) Murdoch, AUSTRALIA
Phytopathology 100:S151
Plant disease resistance is often conferred by genes with NBS-LRR or protein
kinase (PK) domains. Much less is known about mechanisms of susceptibility,
particularly to necrotrophic fungal pathogens. The pathogens that cause the
diseases tan spot and Stagonopora nodorum blotch on wheat produce effectors
(host-selective toxins) that induce susceptibility in wheat lines harboring
corresponding toxin sensitivity genes. The effector ToxA is produced by both
pathogens, and sensitivity to ToxA is governed by the Tsn1 gene in wheat. We
cloned Tsn1 and found that it contains features of disease resistance genes,
including PK and NBS-LRR domains. Mutagenesis revealed that all three
domains are required for ToxA sensitivity, and hence disease susceptibility.
Tsn1 alleles are unique to ToxA-sensitive genotypes and insensitive genotypes
are null. Sequencing and phylogenetic analysis indicated that Tsn1 arose in the
B-genome diploid progenitor of polyploid wheat through a genome shuffling
event that gave rise to its unique structure. Functional analysis indicated that
the Tsn1 protein does not interact directly with ToxA. Tsn1 transcription is
tightly regulated by the circadian clock and light, providing further evidence
that Tsn1-ToxA interactions are associated with photosynthesis pathways.
This work suggests that these necrotrophic pathogens thrive by subverting the
resistance mechanisms acquired by plants to combat other pathogens.
Dissection of effector-induced host susceptibility pathways
Stagonospora nodorum blotch of wheat
S. LU (1), T. L. Friesen (1), J. D. Faris (1)
(1) USDA-ARS Northern Crop Science Laboratory, Fargo, ND, U.S.A.
Phytopathology 100:S151
in
The necrotrophic Stagonospora nodorum-wheat interaction is characterized
by several pathogen-derived proteinaceous host-selective toxins (SnToxA,
SnTox1, SnTox2, SnTox3 and SnTox4) that induce diseases in the host
carrying a corresponding dominant susceptibility gene (Tsn1, Snn1, Snn2,
Vol. 100, No. 6 (Supplement), 2010
S151
Snn3 and Snn4, respectively). The major susceptibility gene Tsn1 has been
found to encode a novel protein kinase-NBS-LRR disease resistance-like
protein (Faris et al, unpublished) that does not directly interact with SnToxA.
To dissect the pathways associated with these toxin-susceptibility gene
interactions, we have undertaken yeast two-hybrid studies in conjunction with
co-immunoprecipitation to identify wheat proteins that are directly targeted by
the toxin or interact with the host susceptibility gene product. Several new
ToxA-interacting proteins were identified including members of the
pathogenesis-related protein (PR) families. Preliminary cDNA library
screening also revealed that Tsn1 may interact with a protein potentially
involved in the transfer of lipid receptors to the plasma membrane and two
chloroplast proteins known to be involved in photosynthesis. These raise the
possibility that Tsn1 may have a dual function and likely act as a key mediator
for ToxA internalization. Results from further characterization of these
candidate Tsn1-interacting proteins will be presented. Hypotheses on how
Tsn1 governs ToxA-induced susceptibility in wheat will be discussed.
Diseases of Plants
Biology and Management of Rhizoctonia Diseases
in Turfgrass Systems
Thanatephorous and Ceratobasidium species is critical for their effective
management on turfgrass.
New Rhizoctonia-like pathogens associated with diseases of warm-season
turfgrasses
P. F. HARMON (1), S. Kammerer (1)
(1) University of Florida, Gainesville, FL, U.S.A.
Phytopathology 100:S152
Management of leaf and sheath spot of ultradwarf bermudagrasses
B. MARTIN (1)
(1) Pee Dee Res & Ed Center, Clemson University, Florence, SC, U.S.A.
Phytopathology 100:S152
Fungi with Rhizoctonia-like biology recently have been associated with a
range of disease symptoms on warm-season turfgrasses in the Southeast.
These fungi lack fruiting bodies, have hyphae of regular diameter with rightangle branching, and occasionally form sclerotia of various size and color on
agar media. Disease symptoms range from discrete patches or rings of
blighted turfgrass to diffuse canopy thinning and foliar necrosis. Timing of
symptoms typically occurs during the hottest months of a year, but symptoms
may become more noticeable in fall or periods of semi-dormancy. Recovery
has been observed to take an extended period of time despite extensive
fungicide application and even after environmental factors begin to favor
turfgrass growth and recovery. Turfgrass hosts associated with these
symptoms include all warm-season species suitable for amenity uses such as
cultivars of bermudagrass and seashore paspalum. Isolates are not always
easily obtained from symptomatic turfgrass, especially after the initial
symptom development, but plating on a medium amended with a
benzimidazole fungicide increases odds of isolation. Phylogenetic analysis of
informative sequences indicates isolates form distinct clades related to the zea,
oryzae, and circinata varieties of Waitea circinata. Additional work is needed
to determine if these fungi constitute new varieties of W. circinata or if the
anamorphic diversity and lack of known teleomorphs warrant designation of
new Chrysorhiza spp.
The biology of brown ring patch disease on cool-season turfgrasses
F. P. WONG (1), C. Chen (1), K. A. de la Cerda (1), G. W. Douhan (1), L.
Stowell (2)
(1) University of California-Riverside, Riverside, CA, U.S.A.; (2) PACE Turf
LLC, San Diego, CA, U.S.A.
Phytopathology 100:S152
Brown ring patch is an emergent disease of cool season turfgrass in the U.S.
caused by a variety of Waitea circinata [proposed as var. circinata (Wcc)].
The pathogen was described as causing ‘brown ring patch’ on creeping
bentgrass (Agrostis stolonifera) in Japan in 2005. Following outbreaks of
yellow rings associated with a Rhizoctonia-like pathogen on annual bluegrass
(Poa annua) in the western U.S. in 2003, Wcc was indentified as the causal
agent of the disease. The disease was subsequently diagnosed throughout the
U.S. on annual bluegrass, on roughstalk bluegrass (P. trivialis) in the
southeastern and southwestern U.S., and a few locations in the western U.S.
on creeping bentgrass. Characterization of a diverse collection of Wcc using
the ribosomal intergenic spacer and beta tubulin sequences differentiate it
from other W. circinata anamorphs (Rhizoctonia oryzae and R. zeae) and from
Thanatephorous and Ceratobasidium species. Experiments have demonstrated
that Wcc is genotypically diverse, insensitive to benzimidazole fungicides, but
can be controlled by other Rhizoctonia-active fungicides, especially flutolanil,
polyoxin-D and certain demethylation inhibitors (DMIs). Unlike some
Rhizoctonia diseases, brown ring patch is less severe on putting greens with
higher nitrogen fertility. Understanding these differences between Waitea,
Cryptic Foes: Gathering the Latest Advances
on Pythium
Ecology and biology of Pythium spp. and their impact on crop production
F. N. MARTIN (1)
(1) USDA-ARS, Salinas, CA, U.S.A.
Phytopathology 100:S152
S152
PHYTOPATHOLOGY
Leaf and sheath spot, caused by Rhizoctonia zeae and Rhizoctonia oryzae,
have been well documented as pathogens of cool season grasses during warm
to hot (28–38 C) and humid weather. Although studies are fewer, isolates of
these fungi have also been proven pathogenic to warm-season grasses,
including bermudagrass (Cynodon spp.), centipedegrass (Eremochloa
ophiuroides), St. Augustinegrass (Stenotaphrum secundatum) and seashore
paspalum (Paspalum vaginatum). Concurrent with the adoption of
‘ultradwarf’ bermudagrasses, leaf and sheath spot has increased in locations
utilizing these grasses. Identification has been based on cultural characteristics
and sequencing of the ITS1 and ITS2 regions of the rDNA. Rhizoctonia zeae
has been most commonly identified, but R. oryzae and R. circinata var.
circinata have also been isolated. Symptoms include rings and patches of a
few centimeters up to a meter. Symptoms in transition zone environments may
persist for months and fungicides have frequently been unsuccessful in
alleviating symptoms as the turf slows in growth. Turfgrass culture in sandy
root zones with low fertility has been associated with increased severity, as
well as practices that injure the turf during periods of low recovery potential.
Management is based on alleviating nutrient stress, avoiding injurious cultural
practices, and use of effective fungicides. Preventive applications of
azoyxystrobin, pyraclostrobin, fludioxanil, flutolanil, and propiconazole have
been shown to be efficacious.
Rhizoctonia species causing turfgrass disease in the transition zone:
Identification, host resistance and management
B. J. HORVATH (1), D. S. McCall (2), B. S. Amaradasa (3), M. A. Cutulle
(4), V. R. Sykes (1)
(1) University of Tennessee, Knoxville, TN, U.S.A.; (2) Virginia Tech,
Blacksburg, VA, U.S.A.; (3) Virginia Tech, Blacksburg, TN, U.S.A.; (4)
Virginia Tech, Ellicott City, MD, U.S.A.
Phytopathology 100:S152
Rhizoctonia species cause some of the most destructive diseases on turfgrasses
grown in the transition climatic zone. There are currently at least five
Rhizoctonia-like pathogens that infect cool season turfgrasses in the transition
zone. My lab has focused on a three-fold approach to better understanding the
pathogen(s) involved in causing disease. First, correct identification of the
pathogen is critical for the development of host resistance and disease control.
Current work is using various molecular techniques (ITS sequencing, UPPCR, AFLP, SCAR) to attempt to better understand the relationships within
and among the Rhizoctonia species causing disease. Second, digital imaging
techniques have been developed to accurately assess the potential for host
resistance in tall fescue (Festuca arundinacea) germplasm accessions. These
techniques are also useful to quantitatively assess disease that is present on
turfgrass plants. Third, cultural management tools such as, mowing height and
N fertility, and chemical management tools such as; nozzle types and granular
fungicide applications are being examined to provide new solutions for
disease control in the transition zone while attempting to minimize the impact
of blanket preventative fungicide applications.
Pythium spp. occupy a diverse ecological habitat ranging from terrestrial
ecosystems to aquatic habitats. The genus has a world-wide distribution with
over 120 species described, all but a few of which are homothallic. Although
some species are not important as pathogens of economic crop plants (some
have shown promise as biocontrol agents), a large number of them are
responsible for causing diseases ranging from pre- and post emergence
damping-off to reduced vigor and yield of mature plants due to root pruning.
Some pathogenic species have a broad host range and are capable of attacking
a range of plants while others have a more limited host range or may be
restricted more to graminaceous hosts. Differential levels of virulence may
also be observed with some species having a significant impact on a particular
host while others may be capable of root colonization but cause limited
disease. Management of these diseases has relied primarily on fungicides and
cultural practices as host resistance is not widely encountered. Other resident
microflora can have a profound effect on disease incidence, which can be
useful in the development of biological approaches for disease management.
Recent efforts to clarify the taxonomic boundaries of species in the genus and
evaluate the population biology of some species using molecular tools will
facilitate future research on this important genus of plant pathogens.
Sampling and processing of samples for Pythium
G. W. MOORMAN (1)
(1) Pennsylvania State University, University Park, PA, U.S.A.
Phytopathology 100:S153
Pythium species are readily isolated from soil, sediment, water, or infected
mycelium, roots, stems, or fruits by plating directly on media or media
amended with antibiotics. Semi-selective media do not work equally well for
all species or against all contaminants. Pythium can grow into solid medium
that is nutrient-poor and other organisms eliminated by transferring hyphal
tips to fresh media. Alternatively, parts of a host plant or other bait can be
employed. Each bait is semi-selective in that certain species of Pythium
readily colonize it while others do not. A protocol that fails to eliminate
Pythium in one study may be a good one for Pythium work. The rapid
colonization of the cut edges of rhododendron leaf disks by Pythium reported
by Phytophthora researchers leads them to use whole leaves but indicates that
Pythium researchers should use leaf disks. That old seeds produce seedlings
highly susceptible to Pythium indicates that old seeds or seedlings grown from
old seeds could be effective baits or trap plants for Pythium. Once in pure
culture, there are simple methods of storing Pythium without frequent subculturing. Blocks of colonized water agar suspended in sterile tap water at
room temperature works well as does inoculating sterile hemp seeds
suspended in sterile tap water stored at room temperature. Bacteria greatly
shorten the duration of Pythium viability in storage. Water agar amended with
rose bengal (50–100 µg/ml) is often sufficient to eliminate bacteria.
Assessment of Pythium diversity in forest nurseries
J. WEILAND (1)
(1) USDA ARS, Corvallis, OR, U.S.A.
Phytopathology 100:S153
Pythium species are one of the most important and common damping off
pathogens affecting conifer seedling production in the Pacific Northwest.
Seedling losses can approach 100% when soil moisture is abundant. Despite
their prevalence and importance, relatively little is known about the species of
Pythium found in nursery soils. A limited number of studies report that P.
irregulare, P. mamillatum, and P. ultimum are the predominant species in the
PNW, but most studies do not report Pythium species identity. In an attempt to
further characterize Pythium species associated with conifer seedling production, a field survey was conducted at three forest nurseries (2 in OR, 1 in WA)
in 2008. Pythium species were isolated by plating soil onto PARP and by
baiting with Rhododendron leaf disks and split Douglasfir needles. One
hundred isolates were randomly selected from each method and nursery and
identified on the basis of ITS sequence. A total of 19 Pythium species were identified from the survey. Species richness and abundance were strongly influenced by both nursery and method. Each nursery was associated with a different predominate Pythium species (P. dissotocum, P. irregulare, and “P. vipa”).
Role of Pythium spp. in the seedling disease complex on cotton; results
from the National Cottonseed Treatment Trials
S. A. Winters (1), C. S. ROTHROCK (1), E. E. Gbur (1), L. L. Verhalen (2),
T. S. Isakeit (3), W. E. Batson (4), F. M. Bourland (5), P. D. Colyer (6), H. W.
Kaufman (7), T. A. Wheeler (8), G. L. Sciumbato (9), K. S. Lawrence (10), A.
Y. Chambers (11), P. M. Thaxton (12), W. S. Gazaway (10), T. L. Kirkpatrick
(13), P. M. Phipps (14), D. R. Sumner (15), L. J. Littlefield (2), G. B. Padgett
(16), F. M. Shokes (14), R. B. Hutmacher (17), R. M. Davis (18), K. W.
Seebold (19), J. D. Mueller (20), J. D. Barham (13), M. A. Newman (11), R.
H. Garber (21)
(1) University of Arkansas, Fayetteville, AR, U.S.A.; (2) Oklahoma State
University, Stillwater, OK, U.S.A.; (3) Texas AgriLife Research, College
Station, TX, U.S.A.; (4) Mississippi State University, Mississippi State, MS,
U.S.A.; (5) University of Arkansas, Keiser, AR, U.S.A.; (6) LSU AgCenter,
Bossier City, LA, U.S.A.; (7) Texas AgriLife Extension, Lubbock, TX,
U.S.A.; (8) Texas AgriLife Research, Lubbock, TX, U.S.A.; (9) Mississippi
State University, Stoneville, MS, U.S.A.; (10) Auburn University, Auburn,
AL, U.S.A.; (11) University of Tennessee, Jackson, TN, U.S.A.; (12)
Mississippi State University, Cypress, TX, U.S.A.; (13) University of
Arkansas, Hope, AR, U.S.A.; (14) Virginia Tech University, Suffolk, VA,
U.S.A.; (15) University of Georgia, Tifton, GA, U.S.A.; (16) LSU AgCenter,
Winnsboro, LA, U.S.A.; (17) University of California, Davis, Five Points,
CA, U.S.A.; (18) University of California, Davis, Davis, CA, U.S.A.; (19)
University of Kentucky, Lexington, KY, U.S.A.; (20) Clemson University,
Edisto, SC, U.S.A.; (21) USDA, Shafter, CA, U.S.A.
Phytopathology 100:S153
Fungicides are universally sold on cottonseed to control a number of seedborne and soilborne pathogens that affect the germination of seed and
emergence, survival and vigor of seedlings. The National Cottonseed
Treatment Program evaluates seed treatment combinations in 15 to 20 trials
annually. The importance of Pythium species in the seedling disease complex
on cotton in these trials was examined with the fungicide treatment metalaxyl,
disease ratings, and pathogen isolations from seedlings and soil populations
from the non-treated control plots at each location. Fungicides improved stand
over the non-treated control in 120 of the 214 trials conducted by cooperators,
56% of the trials. For the 120 trials with a fungicide response, metalaxyl alone
gave a significant response in 40 trials indicating the importance of Pythium
spp. in these trials. Based on seedling stand response for the metalaxyl
treatment compared to seed not treated, responses were found more frequently
and stand responses were greater as minimal soil temperatures at planting
decreased from 20 to 12 C and total rainfall increased the first three days after
planting. Isolation frequency from seedlings and soil populations of Pythium
spp. were poorly correlated with disease symptoms and stand response
suggesting these data have a limited role in characterizing the importance of
Pythium spp. in the seedling disease complex on cotton.
Pythium species associated to plants: The aggressive vs. the moderately,
low and non aggressive
Z. ABAD (1)
(1) USDA-APHIS-Molecular Diagnostics Laboratory, Beltsville, MD, U.S.A.
Phytopathology 100:S153
The genus Pythium, with over 150 known species is an exceptional group of
organisms not only pathologically, but also ecologically and physiologically.
Pythium species occupy a high level of niche diversity in aquatic and
terrestrial environments world wide that is not observed in other like-fungi or
fungi. Plant pathogenic species present levels of pathogenicity including:
high, moderate, low and non-pathogenic. High and moderate pathogenic
species comprise only the 25% of the reported species, but they can have
devastating impact on crops of economic importance around the world.
Although numerous species are known to be major plant pathogens, they are
frequently omitted in disease diagnosis or considered to be secondary
pathogens. Identification of Pythium isolates often stop at the Genus level.
Some pathogenic species have wide host ranges and are widely distributed
(i.e. P. aphanidermatum), others are host and locality specific (i.e. P. solare).
The wide ranges for highly and moderately pathogenic species is very ample
but turfgrasses and other related hosts (corn, wheat, oats, barley, rice, and
sugarcane) are highly susceptible to Pythium and a great number of species
have been found associated to these hosts. Factors that influence in the
pathogenicity of species will be evaluated as well as the correct morphological
and molecular characterization of species which is important in order to apply
the adequate measures for the control of the Pythium diseases.
DNA barcode, genomics and phylogenetics of Pythium species
C. LEVESQUE (1), G. P. Robideau (2), A. W. de Cock (3), K. Bala (1), Q. A.
Eggertson (2), T. L. Rintoul (1), J. Hamilton (4), N. Tisserat (5), C. Buell (4)
(1) Agriculture and Agri-Food Canada, Ottawa, ON, CANADA; (2)
Department of Biology, Carleton University, Ottawa, ON, CANADA; (3)
CBS KNAW Fungal Biodiversity Centre, Utrecht, NETHERLANDS; (4)
Department of Plant Biology, Michigan State University, East Lansing, MI,
U.S.A.; (5) Colorado State University, Ft. Collins, CO, U.S.A.
Phytopathology 100:S153
High throughput Sanger sequencing has changed the way we do taxonomy
and next generation sequencing is changing the way we do genomics. The
goal of DNA barcoding is to generate an identification reference database by
sequencing one or a few genes for all species. Some significant advances were
made in Pythium by the sequencing of both ITS and cytochrome oxidase I
(COI) for approximately 1000 strains covering all known Pythium species.
Both of these markers provide an appropriate amount of resolution and show
good complementarity. Several species that were conspecific with ITS
remained as such with COI but P. graminicola, P. periilum, and P.
tardicrescens were among the notable exceptions of significance to plant
pathology. The sequence of the genome of P. ultimum var. ultimum was
recently completed, showing unique features of this plant pathogen compared
to Phytophthora. A second strain of P. ultimum was sequenced and
comparative analyses were performed to find highly variable genes. The P.
ultimum complex with the two different varieties was analysed using these
highly variable genes to test the phylogenetic species concept within this
group. Additional genomes are being sequenced in other Pythium clades using
Vol. 100, No. 6 (Supplement), 2010
S153
next generation sequencing. This will reveal more on the pathogenicity
mechanisms of the genus and give better tools to resolve the species
complexes within Pythium.
Population genetics and inter-species boundaries within the Pythium
irregulare complex
C. D. GARZON (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S154
The genus Pythium Pringsh. includes more than a hundred species.
Historically described on the basis of few morphological characters, the
validity of many species and the systematics of the genus have been
questioned. With the advent of molecular phylogenetic tools, several attempts
have been made to revise the systematics of Pythium. Recent phylogenetic and
population genetics studies of several morphologically defined Pythium
Virus Fishing with Chips: Plant Virus Microarrays
and Next Generation Sequencing
Universal plant virus microarray development and validation
B. Bagewadi (1), K. Fischer (2), D. C. Henderson (3), R. L. Jordan (3), D.
Wang (4), K. L. Perry (5), U. Melcher (6), J. Hammond (3), C. M. FAUQUET
(7)
(1) ILTAB/ Donald Danforth Plant Science Center, St. Louis, MO, U.S.A.; (2)
University of Utah, Salt Lake City, UT, U.S.A.; (3) USDA-ARS, MPPL,
Beltsville, MD, U.S.A.; (4) Washington University, St. Louis, MO, U.S.A.;
(5) Cornell University, Ithaca, NY, U.S.A.; (6) Oklahoma State University,
Stillwater, OK, U.S.A.; (7) Donald Danforth Plant Science Center, St. Louis,
MO, U.S.A.
Phytopathology 100:S154
The number of plant viruses known is increasing exponentially, and new
methods such as deep sequencing will increase further their number.
However, with the drastic decline in the number of expert plant virologists,
virus identification becomes more challenging. DNA microarray methods
based on oligonucleotide probes offer cheap, rapid, reliable, and parallel
detection of plant viruses. Our preliminary data with a prototype broad range
plant virus chip containing 70 mer oligonuleotide probes specific to viruses at
the species or genera levels suggests the possibility to build a universal plant
virus microarray (UPVM) with probes for every taxon of the plant virus
kingdom. The design of 60 mer oligonucleotide probes based on taxonomic
principles greatly helps not only detection/identification but also classification
of known or new viruses. Here we report the first design of taxonomy-based
universal plant virus microarray probes for every taxon/node of the taxonomic
tree for all plant viruses available in GeneBank. A robust computational
strategy will be used for objectively interpreting the resulting hybridization
pattern data in an automated fashion. Preliminary in silico evaluation of the
designed oligos will be reported to demonstrate the feasibility of using them
on a chip.
High throughput sequencing - next wave diagnostics
N. BOONHAM (1), I. Adams (1), R. Glover (1), W. Monger (1), T. Hodges
(2), P. Ashton (2)
(1) Food and Environment Research Agency, York, UNITED KINGDOM; (2)
University of York, York, UNITED KINGDOM
Phytopathology 100:S154
Taken as a 30-year average, one new plant virus is found each year in the UK
alone; these are either known viruses new to the region, variants within a
known genus or new, previously un-described viruses. Detecting these new
viruses is notoriously difficult, usually involving traditional investigational
techniques and molecular methods such as PCR using degenerate
family/genus primers. Whilst micro array techniques promised much in the
area of viral diagnostics they have not yet become established as a routine
tool. Microarrays have proven most useful for screening for viruses where the
sequences are known and the diversity understood. Adoption is still likely if a
high throughput platform can be exploited to deliver cost savings over running
multiple parallel PCR tests. Development of virus discovery arrays based on
probes designed at a higher taxonomic level has been disappointingly slow
and results frequently need significant follow up. Next generation sequencing
however offers enormous potential to simplify this work. Completely de-novo,
generic work flows can be developed, and the volume of sequence generated
(GS-FLX = 1 billion nt per run) means even low titre infections can be seen
amongst the sea of host (and other pathogen) sequence. Short term the
challenge is developing effective bioinformatics pipelines to ‘sift’ the data;
longer term it is ascribing the causal agent of disease to one of the multitude
of candidates discovered.
S154
PHYTOPATHOLOGY
species have revealed the existence of multiple cryptic and closely related
species within species complexes. Pythium irregulare sensu lato is a
morphologically diverse group of species within the F clade of Pythium that
also has highly polymorphic molecular markers. Previous studies based on
ribosomal DNA sequences have identified four highly divergent lineages
within P. irregulare, including some newly described species. Analyses of
multiple genomic and mitochondrial DNA sequences suggest more species
should be described. Resolving closely related species in this complex is
challenging since most are impossible to distinguish morphologically and
their gene sequences can be highly similar. The reported research uses
multigene phylogenies to define inter-species boundaries within the P.
irregulare complex complemented with population genetic analyses to
determine the levels of genetic exchange between the putative sister species.
These approaches support the occurrence of genetically isolated, cryptic
species within the P. irregulare complex.
Viral population analysis by genomic sequencing
Z. XIONG (1), Z. Weng (1), D. W. Galbraith (1), W. O. Dawson (2)
(1) University of Arizona, Tucson, AZ, U.S.A.; (2) University of Florida,
Lake Alfred, FL, U.S.A.
Phytopathology 100:S154
Advances in sequencing technologies have enabled unprecedented analyses of
virus populations at the genomic level, to not only reveal the structure,
dynamics, and sequence polymorphisms of a viral population, but also map
recombination events across the entire genome. This approach is particularly
suitable for viruses that infect perennial hosts and that often coexist as a
complex of multiple, divergent strains, such as Citrus tristeza virus (CTV).
We developed and applied two high-throughput techniques to the genomic
analysis of CTV: an inexpensive high-density resequencing microarray, and
the high coverage approach of 454 pyrosequencing. The Affymetrix
resequencing microarray comprised nearly one million probes capable of
interrogating genomes of known CTV strains. Analyses of natural CTV
isolates indicated that the microarray readily determined major CTV strains
and their prevalence within a sample, but it was ultimately limited in
sequencing accuracy across the entire genome, due to cross hybridization in
regions highly conserved between strains. In contrast, 454 sequencing
generated high quality, full-length sequences and high resolution maps of
polymorphic sites for each strain within a CTV complex. Furthermore, it
identified recombinant sequences formed between constituent strains and the
positions of recombination hotspots across a genome. These data together
provide significant insights into the evolution and emergence of new viral
strains.
Next generation sequencing as a tool for studying virus ecology
U. MELCHER (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S154
Recent and ongoing developments in nucleotide sequencing enable asking
questions about the roles viruses play in ecosystems. Second generation
sequencing methods enable deep sequence coverage of single genomes and
metagenomes or wide sequence coverage of multiple specimens. Third
generation sequencing, already emerging, offers even more extensive
possibilities. A selection of newer sequencing strategies will be briefly
described and their potentials assessed. The general application of such
sequence approaches in ecology is to test for the presence of viruses in
ecological samples and to identify those present. Detection and identification
is performed bioinformatically and depends absolutely on some similarity
with sequences in the nucleotide or protein databases. Strategies for detecting
sequences from completely novel viruses still need to be tested. Identifying
what viruses are where and when they are there is important knowledge for
assessing may questions about the roles of viruses in ecology. These will be
considered at three levels: within the plant, in plant and vector communities,
and around the globe.
Bioinformatic analysis of microarray and next generation sequencing
data
K. F. FISCHER (1)
(1) University of Utah School of Medicine, Salt Lake City, UT, U.S.A.
Phytopathology 100:S154
Unbiased direct interrogation of the nucleic acid sequences in complex
biological specimens can reveal the presence of novel or unexpected agents
(e.g. viruses, archaea, bacteria, or other microbes). The complexity of
metagenomic and metatranscriptomic data requires efficient analysis
techniques that utilize what is already known about biological sequence
diversity. However, current and foreseeable sequence databases contain
enormous redundancy that can bog down analyses. Furthermore, non-random
distribution of organisms found in public databases presents significant
challenges. Designing and interpreting experiments that use these rich
resources requires an understanding of the biases present in the existing
databases and how they can affect our results. Metagenomic pathogen
detection microarrays can be designed and tuned to use microarray capacity
efficiently with specificity tailored to discovery, diagnostics or a compromise
between those roles. Experimental bias is necessarily introduced in selecting
the probes for an array. This bias can be avoided by using deep sequencing,
but very similar effects are seen in the post-sequencing analysis of
metagenomic DNA and RNA studies. Understanding these bioinformatics
challenges has allowed broad experimental platforms to address some of the
shortcomings of traditional high-specificity diagnostics.
Epidemiology/Ecology/Environmental Biology
Assuring the Safety of Fresh Produce: Issues
and Strategies
Genes, genomes, and microbes; food safety research as a plant pathologist
J. BARAK (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S155
Bacterial pathogens, both plant and human, colonize crop plants and can share
environmental niches. The colonization of plants by enteric animal pathogens
causes a unique human public health concern. In addition to the direct cost of
human illness, impacts of fresh produce-linked epidemics of food borne
illness have reached the tens to hundreds of millions of dollars for each
industry implicated. These outbreaks have eroded the public’s faith in the
healthiness of fresh produce. Plant pathologists bring a collective knowledge
and experience to unravel the synergy between the plant and human pathogens
on plants. Discoveries have been made in the identification of mechanisms
used by the bacteria to attach to and colonize plants, as well as factors that
impact the nature of such interactions. In contrast to other scientific
disciplines that study food safety, the perspective of the plant pathologist
centers on plants, pathogens, and the environment. The training and
experience routinely applied by plant pathologists to understand how
microbes colonize plants, are dispersed in the environment, and how plants
defend themselves will be critical elements of a balanced program to
minimize food borne illnesses.
Assessing vegetable producers’ beliefs regarding food safety issues
M. LEWIS IVEY (1), J. T. LeJeune (2), S. A. Miller (2)
(1) Ohio State University, Wooster, OH, U.S.A.; (2) Ohio State University,
OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S155
Foodborne disease outbreaks caused by contaminated fresh produce continue
to be a concern despite widespread efforts to reduce their incidence.
Knowledge gaps, misconceptions and emerging perceptions among growers
regarding their decision-making processes and practices to prevent and
respond to pre- and post-harvest contamination were identified using
responses to a survey mailed to Midwestern vegetable growers (n=621).
Returned surveys (n=261) were coded and responses analyzed using nonparametric statistical tests. Only growers who self-reported being very
familiar with good agricultural practices (GAPs) implemented them
consistently. There was no significant correlation between frequency of GAPs
implementation, such as water and equipment sanitation, and GAPs
familiarity amongst growers who claimed any lesser degree of familiarity.
Growers agreed that pre-harvest plant diseases and pre- and post-harvest
insects were sources of microbial contamination but were unsure if transplants
and post-harvest plant diseases were contamination sources. Most growers
disagreed that seeds were a source of contamination. Eminently, there is a gap
in perceived knowledge between familiarity and implementation of GAPs.
Growers’ beliefs that plant diseases can be sources of contamination warrant
further studies in plant-human pathogen interactions on produce. These
findings support the development of target-specific methods of
communication and response.
Seed industry challenges
R. L. DUNKLE (1)
(1) American Seed Trade Association, Alexandria, VA, U.S.A.
Phytopathology 100:S155
Food-borne illness outbreaks linked to fresh produce have led to extensive
investigations by federal and state agencies to identify exact source(s) of
human pathogens. To this date, no studies or investigations have linked seed
planted for fresh produce production to any outbreaks in the U.S. In the wake
of the outbreak of E. coli 0157:H7 in California in 2006, many dealers and
growers felt compelled to test spinach, lettuce, and other types of vegetable
seed for human pathogens prior to sale or planting in the U.S. Since testing
began in late 2006, ASTA has not been made aware of any seed that has tested
positive for human pathogens. Seed used for the production of sprouts
continues to pose a risk primarily because of the potential for post harvest
seed contamination during the sprout production process and the
environmental conditions which favor survival and incubation of
microorganisms that may have been introduced. In 2007 ASTA released and
in 2010 updated a statement on testing seed for human pathogens, concluding
that there is no significant value in testing seeds for the presence of human
pathogens. ASTA continues to closely monitor research literature as well as
all outbreaks to ensure that seed remains a negligible risk. Most seed
companies have intensified their quality management programs to provide
further assurrances. This presentation will discuss efforts being implemented
by the seed industry to prevent seed from becoming a pathway for human
pathogens.
Ground Zero: Food safety research and extension in California’s Salinas
Valley
S. T. KOIKE (1)
(1) University of California Cooperative Extension, Salinas, CA, U.S.A.
Phytopathology 100:S155
Coastal California and the Salinas Valley are home to the nation’s most
extensive leafy greens vegetable industry, where huge quantities of high
quality, fresh salad commodities are grown year round. While food safety
concerns are not new to this industry, the 2006 E. coli O157:H7 outbreak on
coastal spinach significantly altered this leafy greens world. This historic
event brought to light critical and unsettled issues. Foundational
epidemiological gaps, such as the sources of E. coli and its ability to survive
in the field, remain unfilled. Unproven assumptions, such as the belief that
wild animals are an important source of E. coli O157:H7, are used to establish
field practices and devise regulations. Proposed policies are based on research
generated in laboratories, growth chambers, and greenhouses. To fill gaps in
our knowledge of pre-harvest foodborne pathogen dynamics, county-based
extension researchers in the Salinas Valley teamed with campus-based
personnel and the leafy greens industry to conduct E. coli field studies placed
in Salinas Valley fields. Soil, lettuce, and spinach were inoculated with
generic and attenuated O157:H7 E. coli strains, plots were handled according
to commercial practices, and the survival of these organisms was studied. This
field-based approach demonstrated a role for off-campus extension in
addressing the needs of the farming clientele and highlighted the
appropriateness of field studies, conducted in real-world environments, in
contributing to food safety solutions.
Collaboration, cooperation, and engagement across agencies
L. L. SKELTON (1)
(1) FDA, CFSAN, Office of Food Safety, Produce Safety Staff, College Park,
MD, U.S.A.
Phytopathology 100:S155
Staff throughout USDA and FDA have been collaborating for years on
various projects. However, it has recently been brought to the attention of
policy-makers and administration officials that this collaboration is not well
known. The presentation will focus on highlighting some of the history of past
collaborative efforts, current activities and a new emphasis on engaging of
other Federal and State agencies throughout government specifically related to
the work FDA is undertaking on developing a produce safety rule.
The APS produce safety interagency initiative
J. FLETCHER (1)
(1) Oklahoma State University, Stillwater, OK, U.S.A.
Phytopathology 100:S155
Plant pathologists, who investigate the complex relationships between
microbes and plants, have much to contribute to the discovery and design of
effective solutions to microbial contamination of food plants. How microbes
colonize plants and are dispersed in the environment, and how plants defend
themselves, are crucial elements for developing intervention strategies and a
balanced program to minimize foodborne illnesses. The effectiveness of
specific risk reduction and prevention strategies is unclear since we have
insufficient knowledge about the interactions of food borne pathogens with
Vol. 100, No. 6 (Supplement), 2010
S155
one another, with plants, and with nonpathogenic microflora. Effective
solutions will require the application of emerging research tools and
strategies, as well as creative cross-disciplinary research efforts. The APS
Public Policy Board proposes a focus on research to gain fundamental and
practical knowledge of human pathogen-plant interactions. This should
include: (1) adding fundamental and applied research to the priorities of the
White House Food Safety Working Group; (2) an interdisciplinary workshop
to bring all relevant members of the food safety community (agency leaders;
academic, government and industry researchers; funders and regulators)
together to prioritize research needs and actions; and (3) establishment of a
new interagency funding initiative for fundamental and applied research on
the association of human pathogens with plants.
Plant Disease Epidemics and Food Security
in Globally Changing Agricultures and Environments
Seeking impact on food security of the poor through phytopathological
science
R. J. NELSON (1)
(1) Cornell University, Ithaca, NY, U.S.A.
Phytopathology 100:S156
Food security and plant disease epidemics: Modeling potential epidemics
on rice, potato, and wheat
S. SAVARY (1), E. Duveiller (2), G. Forbes (3), L. Willocquet (1), R.
Hijmans (4)
(1) IRRI, Manila, PHILIPPINES; (2) CIMMYT, Mexico, MEXICO; (3) International Potato Center (CIP), Lima, PERU; (4) UC Davis, Davis, CA, U.S.A.
Phytopathology 100:S156
Climate change and increased climate variability will affect the vulnerability
of major world food crops to disease epidemics. We present on-going research
to link epidemiological models with crop deployment models in a spatial
framework to quantify the risks that such changes are posing. We attempt to
produce a coherent set of models that are robust, transparent (simple), yet
which can incorporate the effects of host plant resistance characteristics,
chemical protection, and other aspects of crop management. A major
difficulty in this work is the paucity of actual disease observation data that
could be used to validate models across large areas. Predicting future crop
management is perhaps the intellectually most difficult challenge. In this
presentation, we report recent progress with Potato, Wheat, and Rice, and
indicate some key directions for future research.
Estimates of global crop losses
E. OERKE (1)
(1) University of Bonn, INRES - Phytomedicine, Bonn, GERMANY
Phytopathology 100:S156
Productivity of crops is at risk due to the incidence of pests, especially weeds,
pathogens and animal pests. Crop losses to pests can be substantial and may
be reduced by various control activities. Estimates on the loss potential and
actual losses - despite of current crop protection practices – are given for
major food and cash crops on a regional level as well as for the world-wide
total. Among crops the total loss potential of pests world-wide varies from
about 50% to >80%; actual losses vary from 25 to 40%. Overall weeds have a
higher loss potential than animal pests and pathogens. Efficient control of
pathogens and animal pests is more complex than weed control which can be
managed mechanically or chemically, and largely relies on the use of
synthetic chemicals. The efficacy of crop protection is higher in cash crops
than in food crops; differences among regions are more pronounced for food
crops. Despite an increase in pesticide use crop losses have not been
significantly decreased during the last decades. Pesticide use has enabled
farmers to modify production systems and to increase crop productivity
without sustaining higher losses likely to occur from an increased
susceptibility to pests. Minor losses often are economical acceptable,
however, an increase in crop productivity without adequate disease control is
not cost-effective, because a raise of the site-specific yield potential is often
associated with an increased vulnerability to damages inflicted by pathogens.
Plant Pathogen Population Genetics: An Essential
Tool for Crop Biosecurity
How can population genetics inform crop biosecurity efforts?
N. J. GRUNWALD (1)
(1) USDA ARS, Corvallis, OR, U.S.A.
Phytopathology 100:S156
In an ever more interconnected world, bioinvasions of new species, clones or
populations of plant pathogens are increasing in importance. Novel migrant
plant pathogens have repeatedly emerged as threats to agricultural, forest and
other ecosystems. Population genetic theory and tools have an important role
to play in characterizing the pattern and process of bioinvasions. Appropriate
molecular marker systems need to be combined with suitable methods for
analyzing population structure given the data. Tools for asking specific
evolutionary questions, particularly using coalescent theory, are expanding
rapidly. Population genetic approaches can for example assess if immigrant
populations are sexually recombining, subject to a bottleneck, or migrating
from one or several source populations.
S156
PHYTOPATHOLOGY
Plant diseases reduce crop productivity and quality on a chronic or
intermittent basis, and have sometimes contributed to famines. With changes
in climate, threats to food security may be exacerbated. This presentation will
take a comparative view of the challenges and opportunities for disease
management in relation to the food security of resource-poor farmers, based
on the author’s experience with pathosystems involving staple crops in Asia,
Latin America and Africa. The features of several pathosystems (rice blast,
potato late blight and northern corn leaf blight and mycotoxin-producing ear
rots) will be considered in relation to the use of host resistance and other
measures. The problematic links between genetic analysis, resistance breeding
and deployment of resistance will be considered for the three diseases.
Management of Aspergillus ear rot in maize, which is associated with a potent
mycotoxin (aflatoxin), is particularly difficult since it can have substantial
public health implications without presenting obvious symptoms. Lessons
learned across the pathosystems, both shared and disease-specific, will be
highlighted.
The role of pest risk analysis and quarantine measures in food security
P. H. BERGER (1), C. Devorshak (2)
(1) Center for Plant Health Science & Technology, Raleigh, NC, U.S.A.; (2)
Center for Plant Health Science & Technology, Plant Epidemiology & Risk
Analysis Laboratory, Raleigh, NC, U.S.A.
Phytopathology 100:S156
Food security for much of the world remains a precarious situation. Threats to
food security include economic, political and biological factors. Plant diseases
have the potential to impact food security, by reducing food supply, and also
through affecting livelihoods and economic stability. Diseases may spread
from one place to another naturally, or through human mediated spread.
Global trade in agricultural products and the movement of staples food aid
present some of the biggest challenges in the spread of plant diseases to new
places. One of our most important defenses in protecting our food supply is
our ability to identify and analyze threats—both existing and emerging, to
determine pathways of spread, model infection, predict impacts and formulate
recommendations for appropriate actions, in advance of a potential
introduction or epidemic. We are constantly refining analytical techniques
used to analyze risks associated with both existing and emerging plant
diseases, including economic and biological modeling, climate based
mapping, and surveillance. At the same time, improved access to scientific
information, in some cases through “real time” feeds, further aids our ability
to analyze potential threats, and in some cases prevent serious impacts from
occurring, or allowing us to take more effective action to lessen the magnitude
of impacts.
Application of comparative genomics for the identification and
monitoring of the highly virulent African race, Ug99, of Puccinia graminis
J. CROUCH (1), S. Sakthikumar (2), C. Cuomo (2), S. Stoxen (3), Z. A.
Pretorious (4), L. J. Szabo (1)
(1) Cereal Disease Laboratory, USDA-ARS, St. Paul, MN, U.S.A.; (2) Broad
Institute, Cambridge, MA, U.S.A.; (3) University of Minnesota, St. Paul, MN,
U.S.A.; (4) University of the Free State, Bloemfontein, SOUTHWEST
AFRICA
Phytopathology 100:S156
Throughout human history, wheat stem rust caused by Puccinia graminis f.
sp. tritici (Pgt) has been one of the most destructive diseases of cereal crops.
Stem rust has been well controlled in the U.S. for nearly a half a century, but
with the appearance of a new, highly virulent race of Pgt in Uganda (“Ug99”),
this disease has reemerged as a serious threat to global food supplies. Ug99
has already spread throughout northeastern Africa, the Arabian Peninsula and
recently into Central Asia, and is predicted to move into North America
within 10 years, likely through human mediated activities. To date five
members of the Ug99 lineage have been found in Africa, showing variation in
virulence to the stem rust resistance genes Sr24, Sr31 and Sr36. Over 1
million SNPs were identified by mapping Illumina sequence data from five
members of the Ug99 lineage as well as three additional isolates against the
assembled Pgt reference genome. This SNP database was used to selectively
design 38 real-time PCR hydrolysis probes, each containing at least two
homozygous SNPs. By screening a worldwide collection of 270 Pgt isolates,
we found that individually the probes were not capable of uniquely
discriminating between Ug99 and other races. Nevertheless, when a suite of
probes was used in combination, a distinct Ug99 fingerprint pattern was
generated. Ultimately, this technology will be a key component in monitoring
the movement of Ug99.
Inference of Phytophthora ramorum migration pathways
E. M. GOSS (1)
(1) USDA ARS, Corvallis, OR, U.S.A.
Phytopathology 100:S157
Population genetic analysis can reveal important insights into the movement
of plant pathogens, such as the connectivity of populations, migration events
among geographic regions, and the tracing of new epidemics to an inoculum
source. Phytophthora ramorum, the sudden oak death pathogen, was first
detected in California and Europe in the early 1990’s. The pathogen has
caused extensive mortality to coast live oak and tanoak in coastal Northern
California and has been costly to ornamental nurseries found to have P.
ramorum-infected plants. Human activities have repeatedly contributed to the
spread of this pathogen and long-distance migration of the pathogen is linked
to the plant trade. Population genetic analysis has been integral to tracking its
movement to date. Population genetic-based inferences on domestic and
global P. ramorum migration events will be discussed.
The population genomics of mycotoxin diversity in Aspergillus flavus and
Aspergillus parasiticus
I. CARBONE (1), B. W. Horn (2), G. G. Moore (1), R. A. Olarte (1), C. J.
Worthington (1), J. T. Monacell (3), E. A. Stone (4)
(1) Department of Plant Pathology, North Carolina State University, Raleigh,
NC, U.S.A.; (2) National Peanut Research Laboratory, USDA, ARS, Dawson,
GA, U.S.A.; (3) Department of Plant Pathology and Bioinformatics Research
Center, North Carolina State University, Raleigh, NC, U.S.A.; (4) Department
of Genetics and Bioinformatics Research Center, North Carolina State
University, Raleigh, NC, U.S.A.
Phytopathology 100:S157
Mycotoxins, and especially the aflatoxins, are an enormous problem in
agriculture, with aflatoxin B1 being the most carcinogenic known natural
compound. The worldwide costs associated with aflatoxin monitoring and
crop losses are in the hundreds of millions of dollars. Aspergillus flavus and A.
parasiticus are the most common agents of aflatoxin contamination of oil-rich
seed and grain crops. In addition to aflatoxins, A. flavus produces another
unrelated mycotoxin, cyclopiazonic acid (CPA). Populations of A. flavus show
a high level of variation in mycotoxin production, with individuals producing
both aflatoxins and CPA, aflatoxins alone, CPA alone or neither mycotoxin.
We performed array-Comparative Genome Hybridization (aCGH) for
multiple sexual crosses in A. flavus and A. parasiticus whereby each cross was
represented as a parent-offspring trio comprising two parents and one
offspring. Examination of aCGH data for each parent-offspring trio revealed
that meiotic recombination is highly structured along chromosomes, with
recombination hotspots in the aflatoxin, CPA and other predicted secondary
metabolic gene clusters. Crossovers in the aflatoxin cluster of F1 progeny
coincided with recombination hotspots observed in natural populations.
Population genetic data show that a combination of ecological factors,
asexual/sexual reproduction and balancing selection may influence mycotoxin
diversity in these agriculturally important fungi.
A population genetics framework for understanding aggressiveness and
toxigenicity of Fusarium head blight pathogens
T. WARD (1)
(1) USDA ARS, Peoria, IL, U.S.A.
Phytopathology 100:S157
A 14-fold increase in the frequency of 3ADON-producing F. graminearum
occurred between 1998 and 2004 in western Canada. Significant population
structure associated with trichothecene chemotype differences was observed,
and isolates from the 3ADON populations were found to accumulate
significantly more trichothecene than 15ADON populations. 3ADON
populations also exhibited higher fecundity and growth rates. Expanded
molecular surveillance based on 4,266 F. graminearum isolated from seven
Canadian provinces between 2005 and 2007 demonstrated the trichothecene
chemotype distribution in Canada was characterized by two longitudinal
clines. The frequency of 3ADON isolates continued to increase significantly
in western Canada between 2005 and 2007. However, similar changes in
chemotype frequency among isolates from eastern Canada were not observed.
These data suggest a difference in selective pressure acting on FHB pathogens
in eastern and western Canada or an uncoupling of the 3ADON chemotype
from the trait or traits under selection in some eastern Canadian FHB
populations. Analyses of the global population structure of F. graminearum
revealed a very close genetic relationship between a Japanese 3ADON
population and the novel 3ADON populations in North America. Additional
evidence of transcontinental movement of populations followed by limited
genetic exchange between resident and introduced populations is presented.
Molecular/Cellular/Plant-Microbe Interactions
Broad-Spectrum Resistance: Molecular Mechanisms
Involved in Pathogen Reception and Resistance
Signaling
Pattern recognition receptors in plant innate immunity
C. J. RIDOUT (1)
(1) John Innes Centre, Norwich, UNITED KINGDOM
Phytopathology 100:S157
Plants have pattern recognition receptors (PRRs) that detect pathogen
associated molecular patterns (PAMPs) such as fungal chitin and the bacterial
protein EF-Tu. Since PAMPs are highly conserved across microbial species,
taxa and kingdoms, PRRs could potentially offer durable broad-spectrum
resistance. We are investigating how PAMP-triggered Immunity (PTI)
contributes to partial resistance in agricultural crops. Working with wheat,
barley and oilseed rape (canola), we are evaluating natural variation in PTI
responses including the induction of early reactive oxygen species (ROS),
defence genes and callose formation. Wide variation in ROS production has
been established in Brassica parental mapping lines responding to chitin.
Pathology tests will now be performed to establish whether there is a
relationship between PTI and quantitative disease resistance loci. ROS burst
also varies in barley accessions, but has not been detected in wheat. EF-Tu
receptor (EFR) is a PRR first identified in Arabidopsis (Zipfel et al, 2006, Cell
125: 764). ELF18, an 18-amino acid peptide from EF-Tu, is sufficient to elicit
PTI responses. EFR was successfully transferred to tomato, which gained the
response to ELF18 (Lacombe et al, 2010, Nature Biotech in press). We have
now transformed EFR into wheat, and testing whether there is a gain in
ELF18 response. Our research will enable us to evaluate the potential of PRRs
for developing broad-spectrum disease resistance in agriculture.
Dissecting QTL: The genes that contribute to disease resistance revealed
J. E. LEACH (1), R. M. Davidson (1), J. Snelling (1), M. Bruce (1), H. Leung
(2), C. M. Vera Cruz (1)
(1) Colorado State University, Fort Collins, CO, U.S.A.; (2) International Rice
Research Institute, Manila, PHILIPPINES
Phytopathology 100:S157
Incorporation of quantitative trait loci (QTL) that control disease resistance
into elite germplasm could help reduce crop losses, and may even confer
broad-spectrum and durable resistance. Applied breeding programs, however,
have not enthusiastically pursued QTL for crop improvement. A key reason
for this lack of adoption is that reliable markers for accumulation of the QTL
are not readily available because the genes that contribute to the quantitative
trait are not known. Identifying these genes that function in QTL phenotypes
has proven difficult partly because of the imprecision of QTL mapping and
because the effects are small and can vary with environment. In rice, the
availability of a high quality genome sequence and the ability to associate
several types of phenotypic data, including transcript information, to the
physical map, is allowing new approaches to link complex phenotypes to
genomic regions and even genes. Progress in the identification of genes
contributing to rice disease resistance QTL, and description of future
approaches to expedite gene and marker identification for use in breeding
programs will be discussed.
Genetical genomics of Ug99 stem rust infection identifies master
regulators of defense in barley
M. Moscou (1), N. Lauter (2), B. Steffenson (3), R. WISE (2)
(1) Iowa State University, Ames, IA, U.S.A.; (2) USDA-ARS / Iowa State
University, Ames, IA, U.S.A.; (3) University of Minnesota, St. Paul, MN, U.S.A.
Phytopathology 100:S157
Vol. 100, No. 6 (Supplement), 2010
S157
Stem rust (Puccinia graminis f. sp. tritici) is a devastating fungal disease with
a host spectrum that includes wheat, barley, and its alternate host, common
barberry. Resistance in barley to P. graminis f. sp. tritici race TTKSK (Pgt
Ug99), a race with novel virulence, is mediated by the Rpg-TTKSK locus on
chromosome 5H that contains the known resistance genes rpg4 and Rpg5.
Variation in resistance observed on young plants of the Q21861 x SM89010
(QSM) doubled haploid (DH) population is predominantly a qualitative
phenotype, with little of the remaining variance explained by loci other than
Rpg-TTKSK. In contrast, infection phenotypes assayed on adult QSM DH
plants infected by field inoculum of Pgt Ug99 in Njoro, Kenya found several
additional quantitative trait loci that contribute to resistance. To molecularly
characterize these loci, Barley1 GeneChips were used to measure expression
of 22,792 genes in the QSM population after treatment with Pgt Ug99 or with
mock-inoculation. By analyzing the changes in genomic distributions of
expression Quantitative Trait Loci (eQTL) between treatments, we identify a
chromosome 2H trans-eQTL hotspot that regulates the expression of hundreds
of inoculation responsive genes scattered around the genome. This 2H.17
locus coincides with an enhancer of adult plant R-gene-mediated resistance
discovered through the Njoro field trials and provides early transcriptional
targets of Rpg-TTKSK-mediated resistance to Pgt Ug99.
Targeting conserved effectors and effector motifs for broad spectrum
resistance against oomycetes and fungi
B. M. TYLER (1), S. D. Kale (1), B. Gu (2), D. G. Capelluto (3), D. Dou (4),
F. Arredondo (1), D. Deb (5), R. Anderson (5), V. Antigliani (1), I. Fudal (6),
T. Rouxel (6), J. McDowell (5), C. Lawrence (1), W. Shan (2)
(1) Virginia Tech, Virginia Bioinformatics Institute, Blacksburg, VA, U.S.A.;
(2) Northwest A & F University, Yangling, PRC PEOPLES REP OF CHINA;
(3) Virginia Tech, Dept. Biological Sciences, Blacksburg, VA, U.S.A.; (4)
Department of Plant Pathology, Nanjing Agricultural University, Nanjing,
PRC PEOPLES REP OF CHINA; (5) Virginia Tech, Dept. Plant Pathology,
Physiology and Weed Science, Blacksburg, VA, U.S.A.; (6) INRA-Bioger,
Versailles, FRANCE
Phytopathology 100:S158
Fungal and oomycete pathogens of both plants and animals produce effectors
and/or toxins that act within the cytoplasm of host cells to suppress host
More than Just Antibiotics: The Multiple Mechanisms
Leading to Biological Control and Plant Growth
Promotion
The multiple roles of auxin production and turnover in bacteria: Impact
on plant health
J. LEVEAU (1)
(1) University of California - Davis, Davis, CA, U.S.A.
Phytopathology 100:S158
Many plant-associated bacteria have evolved ways to tap into plant hormone
signalling pathways and to manipulate plant physiology accordingly and to
their own advantage. One of the mechanisms for this exploitation of the plant
hormone system is the bacterial synthesis or destruction of one or more of the
five classical plant hormones, i.e. auxin (indole 3-acetic acid or IAA),
ethylene, abscisic acid, cytokinin and gibberellin. Our understanding of the
pathways, genes, and enzymes underlying bacterial tampering with plant
hormone homeostasis is biased towards what is known about a small number
of intensively studied cases, e.g. the synthesis of IAA, which may be
beneficial or detrimental to plants, or the activity of 1-aminocyclopropane-1carboxylate (ACC) deaminase, which lowers inhibitory ethylene
concentrations through degradation of the ethylene precursor ACC. Less is
known about other bacterial activities, such as the phenomenon of IAA
turnover which has long been recognized to exist but remains unexplained as
to its effects, if any, on plant physiology. Only recently, the first bacterial
genes for IAA turnover were discovered and characterized in a Pseudomonas
species. This presentation will cover the most recent findings in the field of
bacterial IAA turnover and explore the various ways in which this bacterial
phenotype might be exploitable towards the biological control of plant
pathogens and/or the promotion of plant growth and health.
Bacterial determinants of induced systemic resistance induced by
Pseudomonas chlororaphis O6
S. HAN (1), A. J. Anderson (2), B. McSpadden Gardener (3), K. Yang (1), Y.
Kim (1)
(1) Chonnam National University, Gwangju, KOREA; (2) Utah State
University, Logan, UT, U.S.A.; (3) Ohio State University, Wooster, OH,
U.S.A.
Phytopathology 100:S158
S158
PHYTOPATHOLOGY
defenses and cause disease. Effector proteins of oomycete plant pathogens
utilize an N-terminal motif, RXLR, to enter host cells. Genome sequences of
oomycete pathogens have revealed hundreds of RXLR effectors. Most of
these are rapidly evolving, but a small number are highly conserved,
suggesting they may be good targets for broad-spectrum R genes. By detailed
mutagenesis of oomycete RXLR motifs, we defined a new bioinformatic
model for these motifs and used it to identify candidate RXLR-like motifs in
fungal effectors. Several of these motifs were then confirmed experimentally
to be responsible for cell entry by these effectors. We have found that both
oomycete and fungal RXLR(-like) motifs are responsible for binding of the
effectors to phosphatidyl-inositol-3-phosphate (PI-3-P). Using this
information, we have succeeded in blocking entry of oomycete and fungal
effectors into host cells by using the head-group mimic inositol-1,3diphosphate or by using PI-3-P-binding proteins. These findings suggests that
agrochemicals that mimic inositol-1,3-diphosphate, or transgenes that encode
secreted PI-3-P binding proteins, may confer broad-spectrum protection
against oomycete and fungal infection.
The molecular basis of broad-spectrum powdery mildew resistance
R. PANSTRUGA (1), C. Consonni (1), M. Humphry (1), J. Lorek (1), K.
Becker (1), P. Bednarek (1)
(1) Max-Planck Institute for Plant Breeding Research, Köln, GERMANY
Phytopathology 100:S158
Loss-of-function mutant alleles of the barley Mlo locus are known to confer
durable, broad spectrum resistance against the powdery mildew disease
caused by the Ascomycete Blumeria graminis f. sp. hordei. This type of
antifungal immunity has been discovered 65 years ago and has been widely
used in European agriculture for more than 25 years. We recently showed that
powdery mildew resistance conferred by mlo alleles is not restricted to barley,
but also occurs in Arabidopsis, tomato and pea. The molecular basis of this
unusual type of disease resistance remains, however, mysterious. We exploit
the genetic and molecular tools available for the dicot reference species,
Arabidopsis thaliana, to get insights into the molecular mechanisms leading to
mlo resistance. We propose a model in which powdery mildew fungi exploit
MLO protein function for fungal pathogenesis by suppressing multiple
defence pathways in their plant hosts.
Root colonization by the beneficial rhizobacterium, Pseudomonas
chlororaphis O6, induces disease resistance in tobacco against two different
leaf pathogens. To identify the bacterial determinants involved in resistance,
we used mutational and biochemical analysis. By screening transposongenerated mutants in P. chlororaphis O6 for a reduced potentional to induce
resistance in tobacco to the soft-rot pathogen, a diverse set of genes involved
was implicated in resistance, including those involved in chemotaxis,
biosynthesis of purine, phospholipase C, transport of branched-chain amino
acids, an ABC transporter, and the two-component sensor kinase GacS.
Additional mutations were detected in the intergenic spacer regions between
genes encoding a GGDEF protein and fumarate dehydratase, and in genes of
unknown function. Biochemical studies indicated that a P. cholroraphis O6
produced 2R,3R-butanediol as an active compound to induced systemic
resistance against tobacco soft rot disease but not wildfire disease. Treatment
of tobacco with the pure compound also enhanced aerial growth and induced
tolerance to abiotic stresses, a phenomenon also seen in the plants that were
colonized by P. chlororaphis O6. The global sensor kinase, GacS, of P.
chlororaphis O6 was a key regulator for induced systemic resistance against
E. carotovora through regulation of 2R,3R-butanediol production. This is the
first report of microbial genes related the induced systemic resistance and a
volatile production by pseudomonad.
Differential roles of lipopeptides in plant host defenses and pathogen
suppression
M. ONGENA (1), G. Henry (1), P. Thonart (1)
(1) University of Liege, Gembloux Agro-Bio Tech, Gembloux, BELGIUM
Phytopathology 100:S158
Cyclic lipopeptides (cLPs) constitute a structurally diverse group of
metabolites produced by various fungal and bacterial genera. Recent advances
revealed that the panel of natural functions retained by these lipopeptides is
larger than previously suspected. Focusing on cLPs isolated from
Pseudomonas and Bacillus, we will provide an overview of this structurerelated versatility of their biological functions. cLPs from plant-associated
bacteria may be virulence factors synthesized by pathogenic isolates but may
also play a crucial role in the biocontrol activity of beneficial strains because
of their involvement in motility, biofilm formation and pathogen antagonism.
In addition, some rhizobacterial cLPs such as surfactin and massetolide tightly
interact with plant cells and stimulate the induced systemic resistance (ISR) at
the micromolar level. They constitute a novel class of bacterial elicitors with a
possibly specific mechanism of action that we wanted to further investigate.
Rather than a receptor-mediated recognition process, our results suggests that
surfactins prefererably interact with the lipid fraction of the plant plasma
membrane. These cLPs do not create irreversible pore formation but act in a
way sufficient to induce some disturbance or transient channelling in the
phospholipid bilayer that can in turn activate a biochemical cascade of
molecular events leading to defensive responses.
Bacterial signaling: Activation of secondary metabolites with multiple
roles in biological control
L. S. PIERSON (1), E. A. Pierson (2)
(1) Texas A&M University, College Station, TX, U.S.A.; (2) University of
Arizona, Tucson, AZ, U.S.A.
Phytopathology 100:S159
Most bacteria produce and detect small signal molecules. These signals allow
bacteria to coordinate specific behaviors such as swimming, swarming,
exopolysaccharide production, biofilm formation, and products that contribute
to either pathogenesis or symbiosis. In Gram negative bacteria, the most
common class of signal molecules are the N-acyl homoserine lactones (AHLs)
involved in a cell density-dependent form of gene regulation termed quorum
sensing (QS). Most QS systems are composed of a gene that encodes the AHL
synthase and a gene that encodes the protein that recognizes the AHL and
responds by activating or repressing target gene expression. These QS systems
are often integral members of complex regulatory networks themselves
subject to additional levels of regulation, including transcription rates, mRNA
stability, the rate of AHL synthesis and the signal threshold required for
perception. The root-associated bacterium Pseudomonas chlororaphis strain
30-84 produces two major phenazine (PZ) compounds, phenazine-1carboxylic acid (PCA) and 2-hydroxy-phenazine-1-carboxylic acid
(2OHPCA). The PhzR/PhzI QS system directly regulates PZ production in
strain 30-84 and is responsible for pathogen inhibition and persistence of
strain 30-84 in the rhizosphere. In nature, bacteria are usually located in
surface-attached communities termed biofilms. The PhzR/PhzI QS system is
absolutely required for biofilm formation by strain 30-84. However, it appears
PZ production under PhzR/PhzI control is a primary determinant of biofilm
structure, as the presence of extra copies of phzR/phzI failed to restore biofilm
Nature’s Molecular Biologist: Xanthomonas and TAL
Effector Function, Structure, and Diversity
Nature’s molecular biologist: Xanthomonas and the AvrBs3-related
family of transcription activation-like (TAL) type III effectors
F. WHITE (1)
(1) Department of Plant Pathology, Kansas State University, Manhattan, KS,
U.S.A.
Phytopathology 100:S159
Bacterial blight of rice represents a robust system for understanding the
interaction and co-adaptation process of a bacterial pathogen and the host.
Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight,
depends on a type III secretion system for effective invasion and colonization
of the host and is particularly noteworthy for the dependence on transcription
activation-type (TAL) type III effector genes. Considerable progress has been
made in our understanding of TAL effector function since the identification of
the type effector AvrBs3. The effectors are highly conserved, although each
member is distinguished by a unique configuration of the central repetitive
region. The repetitive region is associated with the phenotypic activity and
targeted DNA elements in the host genome. TAL effector genes are found
primarily in species of Xanthomonas, although related genes have been
identified in other species. This talk will address the basic properties of TAL
effectors, their involvement in a variety of plant diseases, and their position
within the pantheon of type III effectors.
Diversity of S and R genes in rice targeted by the TAL effector genes of
Xanthomonas oryzae pv. oryzae
B. YANG (1)
(1) Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S159
Bacterial blight of rice is controlled in large through the molecular interaction
between factors in host rice and pathogen Xanthomonas oryzae pv. oryzae.
The factors in the pathogen include a large repertoire of transcription activator
like (TAL) type III effectors which are, in a few cases, matched by the genes
in host. One aspect of the interaction involves the recognition of TAL
effectors (avirulence) by the cognate resistance (R) genes followed by the
onset of strong and rapid defense responses including hypersensitive reaction
(HR), so called gene-for-gene resistance, leading to an incompatible reaction
formation, while constitutive expression of the PZ biosynthetic genes resulted
in earlier biofilm formation. Many bacteria produce PZs, but often different
derivatives and in different ratios. To investigate why this might be, we
altered the overall ratios of PZs produced by strain 30-84. These changes
significantly affected cell adhesion, biofilm architecture and dispersal rates.
The PZ-altered derivatives also differed in their ability to inhibit plant
pathogens in vitro. These effects of PZs on biofilm development are probably
due to multiple mechanisms, including by changing cell surface properties and
by affecting changes in patterns of gene expression. We will discuss how PZs
may themselves serve as signaling molecules to alter gene expression and the
effect of non-PZ producers in mixed community biofilms.
Redefining the paradigm of biocontrol
B. MCSPADDEN GARDENER (1), S. Park (1), C. Chunxue (1), X. Rong (1)
(1) The Ohio State University - OARDC, Wooster, OH, U.S.A.
Phytopathology 100:S159
Contemporary research has indicated the multifunctional nature of various
microbial components in the phytosphere. At the molecular level, different
microbially-associated molecular patterns are known to influence host plant
physiology in rather dramatic ways. Lipopeptides and polyketides, once
thought to be nothing more than antifungal metabolites, have been shown to
be important factors in the induction of host resistance, plant stress resistance,
and biofilm formation. At the population level, some microbial populations
may affect disease development in contrasting ways, depending on the
structure of plant associated microbial communities and/or the prevailing
abiotic conditions they are experiencing. Given that plants are covered with
diverse and ever-changing populations of microbes, host plant health status is
clearly a dynamic property. Because such observations are inconsistent with
the classical definitions of biological control and plant disease, a new, more
holistic, perspective is required to understand the true nature of plant-microbe
interactions. Such a perspective needs to take into account the diverse and
dynamic nature of plant-associated microorganisms, the multifunctional
nature of microbial secretions, and portray plant health as an emergent
property. To that end, it is suggested that the relative importance of nonpathogenic plant-associated microbes to plant health be highlighted, a move
that will lead us towards a new paradigm for biocontrol.
of the disease. This incompatibility is exampled by the combination of Xa27/
AvrXa27. The interaction also involves targeting of host genes by some TAL
effectors (virulence) resulting in disease susceptibility provided plant lacks the
gene-for-gene resistance. In either case, the host gene is transcriptionally
activated by the corresponding TAL effector, and the induction is phenotypically associated with the disease susceptibility or resistance dependent on
the genetic context. In cases of some susceptibility (S) genes, rice has evolved
the alleles that are unresponsive to the corresponding TAL effector(s) and
occur as recessive resistance genes in certain cultivars. This is illustrated by
the recessive resistance gene xa13 and its allelic Os8N3 susceptibility gene
with the corresponding TAL effector PthXo1, as well as by other S genes
Os11N3 and Os12N3 with their respective TAL effectors.
TAL effector-driven host gene expression shapes Xanthomonas
interactions with crop plants
A. J. BOGDANOVE (1)
(1) Department of Plant Pathology, Iowa State University, Ames, IA, U.S.A.
Phytopathology 100:S159
Recent elucidation of the basis for specificity in host gene targeting by
transcription activator like effectors (TALEs) of Xanthomonas allows the
prediction of candidate targets in whole genome sequences. Combined with
genome-wide transcript profiling and functional analysis, this is a powerful
approach to discovering host genes and processes that can be subverted by the
pathogen to promote infection. Isolation of genes for resistance in some cases
can be expedited as well. Yet computational challenges to accurate predictions
persist. This presentation will describe ongoing efforts to identify important
rice genes in the “regulome” activated by the scores of TALEs present in two
strains of the rice pathogens X. oryzae pv. oryzae and X. oryzae pv. oryzicola.
The results of a bioinformatic study to improve binding site prediction will
also be presented. Finally, implications for discovery in other major crop
affected by Xanthomonas spp. that depend on TALEs will be discussed.
Exploiting TAL effector diversity for biotechnology
T. LAHAYE (1)
(1) Ludwig-Maximilians-University Munich, Martinsried, GERMANY
Phytopathology 100:S159
Plant pathogenic bacteria of the genus Xanthomonas inject TranscriptionActivator Like Effector proteins (TALEs) into plant cells. Upon injection,
Vol. 100, No. 6 (Supplement), 2010
S159
TALEs translocate to the plant nucleus, bind to defined DNA boxes and
activate expression of the downstream host genes. TALE-mediated activation
of plant promoters in most cases favours disease progression but triggers in
some plant genotypes activation of the plant immune system. Recent studies
uncovered the molecular basis of how the TALE DNA binding motif binds to
matching DNA boxes. Using this “TALE-CODE” we have generate a
designer TALE (dTALEs) that activate a desired target promoter. Recent
progress on the application of dTALEs will be presented.
Small Molecules in Phytopathology: From
Determinants of Disease to Modulators of Defense
leading to programmed cell death; of benefit of the pathogen. Our nonpathogenic OA-mutant strain is unable to alter host redox status, however,
chemical induction of reducing conditions in host cells with DTT, remarkably
restores its ability to cause disease. OA thus appears to have dual opposing
functions, by creating reducing conditions, OA inhibits the plant oxidative
burst defense response and cell death, and then subsequently promotes cell
death and disease via ROS. The reduction of the host cellular environment
may be a key strategy for establishment of necrotrophic fungal infection.
Unraveling the site- and mode-of-action of protein host-selective toxins
L. M. CIUFFETTI (1), V. A. Manning (1), I. Pandelova (1), M. Figueroa
Betts (1)
(1) Oregon State University, Corvallis, OR, U.S.A.
Phytopathology 100:S160
Some necrotrophic fungal pathogens produce host-selective toxins (HSTs) as
major virulence/pathogenicity determinants. Diseases caused by these
pathogens often follow an inverse gene-for-gene model where toxin
production by the pathogen and a single, corresponding, genetically dominant
locus in the host are both required for compatibility (the occurrence of
disease). Pyrenophora tritici-repentis, the causal agent of tan spot of wheat,
represents a model pathogen for studying the biological significance of the
inverse gene-for-gene interaction due to its production of multiple HSTs. Ptr
ToxA (ToxA) and Ptr ToxB (ToxB) are two structurally unrelated
proteinaceous HSTs that evoke different host responses, yet confer
pathogenicity. Comparative analyses of ToxA and ToxB reveal differences
and commonalities in the mode-of-action of these effectors and thus, provide
insights into the mechanism of P. tritici–repentis-induced disease. The
presentation will center on what is known about the site- and mode-of-action
of ToxA and ToxB.
Hijacking and manipulation of host responses by pathogen-derived
hormone mimics: An update on functional role of coronatine in bacterial
speck disease
R. UPPALAPATI (1), Y. Ishiga (1), T. Ishiga (1), T. Wangdi (1), C. Ryu (1),
K. S. Mysore (1)
(1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.
Phytopathology 100:S160
Plants utilize phytohormones to mediate local and systemically acquired
immune responses to defend against multiple pathogens. However, some
pathogens have evolved mechanism of virulence to highjack the hormonemediated signaling by producing hormone mimics to cause disease. Several
pathovars of Pseudomonas syringae produce a chlorosis-inducing virulence
factor coronatine (COR), which functions as a phytohormone mimic of methyl
jasmonate (MeJA) and JA-isoleucine (JA-Ile). A comparison of COR- and
MeJA-regulated transcriptomes revealed that COR and MeJA share similar,
but not identical activities and impact multiple phytohormone pathways in
tomato. COR, by structurally mimicking JA-Ile, hijacks an ubiquitin E3 ligase
of the SCFcoi1/jai1 complex to activate JA signaling and thereby suppress
salicylic acid (SA)-mediated defense responses. Furthermore, requires SGT1
(suppressor of G2 allele of Skp1) which associates with SCF complexes to
induce chlorosis. Interestingly, SGT1 also regulated disease associated
necrotic cell death. In an effort to elucidate the genes involved in CORinduced chlorosis and cell death, we utilized forward genetic screens and
identified a role for chloroplast localized Peroxiredoxin and NADPHdependent thioredoxin reductase in modulating COR-induced chlorosis
development and disease associated necrotic cell death. Thus, COR may
function at early stages of pathogenesis to suppress basal immunity and
targets chloroplast to regulate disease-associated necrotic cell death.
Sclerotinia sclerotiorum via oxalic acid creates a reducing environment in
the host which is required for pathogenic success
M. DICKMAN (1), M. Kabbage (1), B. Williams (1), H. Kim (1)
(1) Texas A&M University, College Station, TX, U.S.A.
Phytopathology 100:S160
Necrotrophs, which require dead host tissue in order to obtain nourishment,
were initially thought to cause disease symptoms and host cell death by sheer
brute force via the secretion of toxic metabolites. Recently however, emerging
data from several pathosystems have suggested that some necrotrophic fungi
are tactically more subtle in the manner by which pathogenic success is
achieved, though the mechanistic details are not known. Sclerotinia
sclerotiorum (Ss) is an extremely broad host range (>400 species)
necrotrophic fungal plant pathogen that produces the non-specific phytotoxin
and pathogenicity factor, oxalic acid (OA). Transgenic plants expressing a
redox-regulated GFP reporter, provided real-time evidence that Ss initially
induces reducing conditions that suppresses the host oxidative burst and
callose deposition, but subsequently Ss/OA promotes plant ROS generation
S160
PHYTOPATHOLOGY
Azelaic acid: A new player in priming plant defense
J. T. GREENBERG (1), H. Jung (2), T. Tschaplinski (3)
(1) University of Chicago, Chicago, IL, U.S.A.; (2) Dong-A University,
Busan, KOREA; (3) Oak Ridge National Lab, Oak Ridge, TN, U.S.A.
Phytopathology 100:S160
Plants produce numerous small molecules that function to regulate plant
defenses. In addition to directly activating defenses, some signals act by
priming defenses. In this scenario, when an infection occurs, defense
responses precede more rapidly and/or more strongly. Azelaic acid is made
upon infection and can translocate to distal leaves where it primes the
production of the key defense regulator salicylic acid (SA). New data suggests
that azelaic acid is effective both in Arabidopsis (where it was first described
as having a defense priming role) and solanaceous plants. Furthermore, at
least one additional small molecule is implicated as acting down stream of
azelaic acid in priming SA production. Thus, plants employ numerous small
molecules to initiate, activate and/or amplify defense signaling in response to
infection.
Networking by small-molecule hormones in plant immunity
A. LEON-REYES (1), D. Van der Does (1), A. Koornneef (1), S. C. Van
Wees (1), C. M. Pieterse (1)
(1) Utrecht University, Utrecht, NETHERLANDS
Phytopathology 100:S160
Plants live in complex environments in which they intimately interact with a
broad range of pathogens and insects. Genomics approaches expanded our
understanding of the molecular mechanisms by which plants tailor their
immune response. Hormones such as salicylic acid (SA), jasmonic acid (JA)
and ethylene (ET) play pivotal roles in the regulation of the defense signaling
network (Pieterse et al., 2009: Nature Chem. Biol. 5: 308-316). Their
signaling pathways cross-communicate, providing the plant with a powerful
capacity to finely tailor its immune response to the attacker encountered.
Research on the kinetics and mechanisms underlying SA-JA crosstalk
revealed that the SA-JA antagonism is conserved among Arabidopsis
accessions, highlighting the importance of this mechanism for plant survival.
The kinetics of SA and JA signaling appears to play an important role in the
outcome of the SA-JA interaction (antagonistic, synergistic, or neutral). The
antagonistic effect of SA on JA-responsive gene expression is linked to SAinduced changes in the cellular redox potential, suggesting that cross-talk is
redox regulated. Several key regulators involved in cross-talk have been
identified, including the redox-sensitive protein NPR1. ET appears to act as an
important modulator of NPR1 function in SA-JA crosstalk. Furthermore we
showed that SA-mediated suppression of JA signaling acts downstream of
SCF-COI1-JAZ components of the JA pathways and is directly targeted at
GCC-box containing promoters of JA-responsive genes.
Small molecule inhibitors of type III secretion systems in Gram-negative
plant and animal pathogens
H. B. FELISE (1), H. V. Nguyen (1), K. C. Barry (1), P. A. Bronstein (2), T.
Kline (1), S. I. Miller (1)
(1) University of Washington, Seattle, WA, U.S.A.; (2) USDA, Ithaca, NY,
U.S.A.
Phytopathology 100:S160
Bacterial virulence mechanisms are potential targets for drug discovery as
they are required for numerous global infectious agents to cause disease.
Gram-negative bacterial type III secretion systems, which are used by
numerous plant and animal pathogens to deliver protein virulence effectors to
host cells, may comprise one such therapeutic target. We have developed and
performed high-throughput screens of small molecule libraries to identify
broad-spectrum inhibitors of bacterial secretion systems by targeting
conserved components of these systems. Currently, we have identified and
characterized a class of compounds, 2-imino-5-arylidene thiazolidinones that
block secretion and virulence functions in a number of plant and animal
pathogens, including Pseudomonas spp., Yersinia spp., and Salmonella
enterica serovar Typhimurium. In addition, we have shown that these
compounds reduce virulence of the plant pathogen Pseudomonas syringae in a
whole organism infection model. Finally, we have demonstrated that these
compounds inhibit type III secretion as well as type II secretion-dependent
functions and type IV pilus assembly, providing a proof-of-concept that
inhibitors with broad-spectrum activity against Gram-negative secretion
systems could potentially be developed to prevent and treat plant and animal
bacterial diseases.
Plant Disease Management
The 2009 Tomato and Potato Late Blight Crisis:
The Interaction of the Urban Home Garden
and Commercial Agriculture—What Went Wrong
and What We Learned
Overview and impact for extension, grower, and gardener
M. MCGRATH (1)
(1) Cornell University, Riverhead, NY, U.S.A.
Phytopathology 100:S161
The 2009 growing season was unprecedented for plant pathologists and
anyone involved with producing tomato and potato in the northeastern U.S.A.
For the first time late blight was found on tomato plants being sold in garden
centers. Occurrence was widespread. Affected plants were not removed
immediately as store managers do not have this authority under the
consignment sale system and the pathogen is not regulated. Most gardeners
had not seen late blight before and thus did not recognize it initially. Above
average rainfall during June and July provided favorable conditions for
pathogen spread from gardens with affected plants to other gardens and farms.
Late blight occurs most years in the northeast, but mostly in the major potato
production area of Maine. The 2009 late blight outbreak occurred much earlier
and was more widespread than normal. The unusual source undermined the
regional approach to management that is based on identifying the farm(s)
where late blight starts to develop thus enabling other farmers locally and
regionally to be alerted. These facts meant farmers initially were unprepared
and many lost crops because late blight can be uncontrollable when fungicides
are not applied preventively. Late blight outbreaks continued to occur during
warm, low rainfall periods in Aug and in greenhouses in Oct. Many gardeners
experienced for the first time arguably the most destructive plant disease, and
learned they can have a devastating connection to agriculture.
Overview of the potted plant wholesale production business for the “Big
Box” retail stores
B. D. OLSON (1)
(1) Dow AgroSciences, Geneva, NY, U.S.A.
Phytopathology 100:S161
The container plant business has provided customers with potted vegetable
and flower plants for over 40 years. In the early 1900’s the industry began as
small local truck farms delivering field grown, bare-root, plants to the local
retail businesses. Beginning in the 1950’s, growers began growing potted
plants in greenhouses and selling to farm supply stores and independent
nurseries. Over time the industry has transformed dramatically to the present
operations that deliver potted plants to the mega “big box” stores throughout
the United States. The process of growing potted plants is a simple concept,
but the operations are far from simple when supplying the quantity and quality
of plants for today’s retail giants. Tomato, pepper, and herb plants are started
from seed. The seed is dropped by machine into plug trays. The trays vary in
size from 100 cells to 500. Once the plugs have established a root system and
reach transplant size, they are shipped to the growers for transplanting into larger
containers. It then takes two to four weeks for the plants to reach a final size,
at which point they are delivered to retail outlets for resale to home gardeners.
Perspective of the crisis from the state regulatory inspection service
S. KIM (1)
(1) Plant Disease Diagnostic Lab., Pennsylvania Department of Agriculture,
Harrisburg, PA, U.S.A.
Phytopathology 100:S161
In 2009, late blight on tomato and potato plants appeared in June - earlier than
in previous years - and was detected among samples from homeowners,
private fresh-market tomato- and potato-farms, and big box retail markets.
Under the Pennsylvania Plant Pest Act, the Pennsylvania Department of
Agriculture (PDA) routinely inspects tomato plants at retail locations and
production greenhouses. PDA inspectors issued Stop-Sale and treatment or
destruct orders for any infected plants being offered for sale. PDA additionally
inspects incoming out-of-state grown vegetable transplants each year.
Between May-June, 2009, 6.5 million tomato transplants were inspected and
found to be free of late blight symptoms. By September 2009, the
Pennsylvania State University and PDA identified late blight in 58% of PA
counties. The genotypes of Phytophthora infestans (US-8 >US-17>US-15 on
tomato and US-8>US-14>US-7>US-10>US-17 on potato) that occurred
between 1994 and 2004 have changed from predominantly resistant to
mefonoxam-sensitive. Between 2004 and 2006, two genotypes were
identified: US-14 (gpi 100/122; pep 100/100; A2; mefonoxam-sensitive) on
tomato and potato, and US-13 (gpi 100/100; pep 100/100; A2; mefonoxamsensitive) on tomato. In March 2010, PDA launched late blight targeted
inspection services in response to the 2009 late blight outbreaks.
Science of the epidemic
K. DEAHL (1)
(1) USDA-ARS, Silver Spring, MD, U.S.A.
Phytopathology 100:S161
Late blight caused by Phytophthora infestans is one of the most destructive
diseases of potato and tomato worldwide due to rapid asexual reproduction of
the pathogen under conducive weather conditions. A total of 155 isolates of P.
infestans were collected from the major crop production areas during 2007 to
2009 in the US. Isolates were characterized by their pathogenicity, mating
type, and in vitro metalaxyl sensitivity. They were also subjected to molecular
genotyping, by allozyme pattern, mitochondrial genomic haplotype, and DNA
fingerprinting using the multilocus RFLP probe RG57. Before 2007, isolates
collected from tomato and potato crops were mainly the US-8 or US-11 clonal
lineage. However, P. infestans populations in the U.S.A. underwent a
significant genetic shift in the 2007–2009 growth seasons; isolates with
unique genotypes and epidemiological parameters including increased
aggressive were detected in Florida and throughout the northeastern region of
the United States. The greatest concern relating to the introduced new A1 +
A2 populations was the potential impact of sexual recombination. Although
sexual recombination was not yet detected, a decrease in the percentage of the
US-8 clonal type implied that genomic diversity of the pathogen is changing
quickly. Despite introduction of both mating types, in many regions new, but
largely clonal populations have become established.
The 2009 potato and tomato late blight epidemics: Genealogical history,
multiple sources and migration events
J. B. RISTAINO (1)
(1) Dept. of Plant Pathology, North Carolina State University, Raleigh, NC,
U.S.A.
Phytopathology 100:S161
In 2009, potato and tomato late blight epidemics in the US were the worst in
modern history due to a “perfect storm” of widespread inoculum distribution
and conducive weather. Tomato late blight was found in the southeastern US
from March to June and was found in the northeast in June on tomato
transplants that were distributed through major garden center chains from MD
to ME and Canada. Some of those tomato late blight strains also migrated to
potato fields in several states. Prior to 2009, the US-20 and US-21 genotypes
were common on tomato in Fl and NC. The US-8 genotype is still common on
potato in NC and the NE but three new genotypes named US-22, US-23 and
US-24 were also found on potato. The US-22 genotype (A2; Gpi 100/122) is
the widespread strain that was found on tomato transplants. US-23 (A1, Gpi
100/100/111) also occurred on potato and tomato. We assessed the
genealogical history of P. infestans genotypes from 2002–2009. Migration
analysis suggested that gene flow occurred from tomato to potato in the
eastern US populations of the pathogen. Isolates from a home garden in TN,
commercial tomato fields in Long Island, NY in 2007 and FL in 2008 were
also US-22. We used coalescent analysis and documented that the 2009
isolates were derived from the 2007 population. The data suggest that the US22 genotype existed before the epidemics of 2009.
Policy meets practicality: Recommendations
Committee Task Force
M. A. DRAPER (1)
(1) USDA NIFA, Washington, DC, U.S.A.
Phytopathology 100:S161
from
an Extension
In 2009, the losses from late blight reached the highest levels in over a decade.
In response to this timely issue, several forums were held across the country to
Vol. 100, No. 6 (Supplement), 2010
S161
identify needs and consider the developing issues and grower readiness in
response to late blight. We have learned a great deal in recent years from
experiences with outbreaks of several high consequence pests. The late blight
epidemic provides one more cog in that gear. A common element among the
best responses to a plant disease outbreak is the importance of preparation, a
key to early detection and rapid response. Late blight is caused by a
genetically complex pathogen with a challenging epidemiology. Many wide-
area elements must be considered both tactically and strategically for a disease
with the characteristics of late blight. Recognizing the significance of new
disease records and developing a strategy for response, as well as determining
the research and infrastructural needs to accomplish the expected response are
critical pieces in a successful approach to recovery from a plant disease crisis.
Feedback loops and strategies for future development of late blight resources
from various producer and scientific forums will be discussed.
Biocontrol Beyond the Bench: Large-Scale,
Successful Biocontrol
features, advantages and benefits of the novel biocontrol product must be
compelling enough for the customer to switch from what they are currently
using to the novel biocontrol product. This presentation will address some of
the “normal” commercialization issues associated with introducing a new
biocontrol product in to the agricultural market.
Challenges and successes of registering microbial biopesticides
M. BRAVERMAN (1), D. Kunkel (1)
(1) IR-4 Project, Rutgers University, Princeton, NJ, U.S.A.
Phytopathology 100:S162
The IR-4 Project has had a regulatory assistance program since 1982 whereby
we help public sector researchers and small businesses in the EPA registration
process. The IR-4 Project also has been funding researchers through a small
efficacy grant program since 1995. Grants are divided into 3 stages which are
early stage projects on products in which no toxicology work has been
preformed, advanced stage projects which involve registered products looking
to expand the labeled uses and demonstration stage projects which are for onfarm demonstration trials of registered uses. The demonstration stage program
is co-funded and selected in cooperation with the Biopesticides and Pollution
Prevention Division of EPA. Some of the registrations IR-4 has assisted on
have included Aspergillus flavus AF36 for aflatoxin management in cotton,
pistachio and corn, Verticillium WCS 850 for Dutch elm disease and
bacteriophage for management of Xathamonas and Pseudomonas.
Registration under way include Trichoderma hamatum isolate 382, a viral coat
protein for the management of plum pox, and tobacco mild green mosaic
tobamovirus for management of the invasive weed, tropical soda apple. The
primary focus has been on U.S. registrations but cooperative work with
Canada and African countries has also developed.
Bringing a broad spectrum bioherbicide to market
A. WATSON (1)
(1) McGill University, Ste-Anne-de-Bellevue, QC, CANADA
Phytopathology 100:S162
There have been many bioherbicide research projects, but very few have
become commercial products. Dandelion is an important weed in North
America and represents one of the single largest target pests for application of
pesticides in North America. Chemical control using 2,4-D is the accepted
method of dandelion control but public awareness and concern about the
potentially harmful effects of lawn care chemicals lead provincial and
municipal governments in Canada to ban the use of pesticides on lawns
necessitating the discovery and development of alternative weed control
strategies. Now there is an effective biological option. SARRITOR is the first
bioherbicide developed and registered in Canada for control of dandelion and
other broadleaf weeds in turfgrass. The active ingredient of SARRITOR is a
naturally occurring broad host range fungal plant pathogen, Sclerotinia minor
(isolate IMI 344141). Commercialization of SARRITOR has been the
culmination of combined efforts and innovation stemming from McGill
University’s weed research laboratory in partnership with McGill’s
Technology Transfer Office, Natural Sciences and Engineering Research
Council of Canada, government ministries and laboratories, universities, and
industry collaborators. SARRITOR commercial was launched in 2008 while
SARRITOR domestic was launched in 2010. U.S. registration is pending.
Understanding your customer and delivering a quality product
B. FOSTER (1)
(1) BioWorks, Victor, NY, U.S.A.
Phytopathology 100:S162
While some believe a patented organism displaying biocontrol activity is the
final step to the successful commercialization of a new product, in reality it is
only the first step. The goal of a novel biocontrol product is no different than a
novel new synthetic active ingredient - to provide a unique, differentiable
solution that addresses specific, unmet customer problems or needs. The
Creating Possibilities for Sustainable Postharvest
Disease Control Through Integrated Approaches
to Both Pre- and Postharvest Fungicide Resistance
Management
S162
PHYTOPATHOLOGY
Lockdown: Collego bioherbicide gets a second act
K. CARTWRIGHT (1), D. Boyette (2), M. Roberts (3)
(1) ARI, Inc., Fayetteville, AR, U.S.A.; (2) Southern Weed Science Research
Unit, Stoneville, MS, U.S.A.; (3) Natural Industries, Houston, TX, U.S.A.
Phytopathology 100:S162
Collego™, originally registered with the EPA in 1982, was one of the first
two commercially available mycoherbicides. The product was used
successfully to control Northern jointvetch (Aeschynomene virginica) in midsouth rice for some 20 years. About 10 years ago, however, the mycoherbicide
was discontinued per the close of the marketing company and, as a result, the
product lost EPA registration status. In 2005–2006, Agricultural Research
Initiatives (ARI, Inc.-Fayetteville, Arkansas) pursued successful EPA
registration and has been awarded three section 3 (C) registrations for the
active ingredient (Colletotrichum gloeosporioides f. sp. aeschynomene) under
the trade name LockDown™ with three formulations applicable (retro, XL
and Liquid). Working with USDA and University scientists, extension
personnel, consultants and farmers, successful field and greenhouse trials
were completed in 2006 and 2007. Subsequently, manufacturing was scaled
up and optimized in 2008 with product launch during the summer, 2008. In
2009, Natural Industries (Houston, TX) began producing and marketing the
LockDown product. This year (2010) represents the third full year of market.
To date, production, quality control and overall efficacy for the product has
been excellent. In the future, we anticipate sales growth through more
aggressive marketing strategies and penetration into additional timing and
management slots for use of the product in rice.
Working together: Partnering with grower organizations from
development through distribution to make Aflatoxin Biocontrol a reality
in the US/Africa
P. J. COTTY (1), R. Bandyopadhyay (2)
(1) USDA/ARS, Department of Plant Pathology, University of Arizona,
Tucson, AZ, U.S.A.; (2) IITA, Ibadan, NIGERIA
Phytopathology 100:S162
Biocontrol products directed at competitive exclusion of aflatoxin producers
by atoxigenic strains of A. flavus are the only technologies registered for preharvest mitigation of aflatoxin contamination. These products provide useful
levels of efficacy both during crop development and postharvest. Lessons
learned with atoxigenic strains shed light on models through which public
sector organizations can partner with grower organizations to develop and
distribute biocontrol agents. In Arizona, cotton industry organizations
partnered with the USDA to field test, register, and manufacture the
biocontrol agent AF36. This experience was built upon by IITA in
development and registration of atoxigenic strains in partnership with
public/private and governmental organizations in Nigeria. Major obstacles to
success of such partnerships include requirements for initial capital outlay and
technical requirements imposed by the pesticide registration process. Farmers
readily accept aflatoxin biocontrol based on both experimental data and
personal experience with efficacy. However, products still need marketing and
distribution systems. In Africa, unlike the U.S. where market forces to meet
aflatoxin standards push adaptation, commercial incentives will combine with
government responsibility for public health to drive demand for biocontrol.
Several models for development and deployment are needed for biocontrol to
meet worldwide needs for aflatoxin management.
Resistance mechanisms in postharvest fungicides
U. GISI (1), H. Sierotzki (2)
(1) University of Basel and Syngenta Crop Protection, Stein,
SWITZERLAND; (2) Syngenta Crop Protection, Stein, SWITZERLAND
Phytopathology 100:S162
The most important pathogens attacking fruits like citrus, apples, peaches and
potato tubers after harvest include several species of Penicillium, Botrytis,
Glomerella, Monilinia, Alternaria, Fusarium and Helminthosporium. A range
of fungicide groups are used to control these pathogens such as phenylpyrroles (PPs, fludioxonil), anilinopyrimidines (APs, e.g. pyrimethanil),
quinone outside inhibitors (QoIs, e.g. pyraclostrobin, azoxystrobin), and sterol
biosynthesis inhibitors (SBIs, e.g. fenhexamid, several azoles). As with older
fungicides such as benzimidazoles (MBCs, e.g. thiophanate-methyl),
resistance can evolve after intensive use and limit pathogen control to a
certain degree. The extent and level of resistance evolution can be assessed
using a range of elements including intrinsic and extrinsic fungicide risks, as
well as pathogen and agronomic risks. Risk factors are assessed based on
molecular, genetic, biochemical, physiological and population aspects.
Resistance can be monogenic as in MBCs and QoIs or polygenic as in DMIs.
For PPs, resistance is claimed to be linked to os-2 and ABC transporter genes,
for APs by two mutations in the cystathionine-γ-synthase (cgs) gene.
Resistance management should include all available elements including good
agronomical practice, limitation of inoculum carry-over from field to packing
house, reduction in the number of fungicide treatments and use of mixtures
and alternations of fungicides with different modes of action.
Postharvest resistance management: An integration of strategies
encompassing the pathogen, fungicide properties, and epidemiological
principles
H. FORSTER (1), J. Adaskaveg (1)
(1) University of California, Riverside, CA, U.S.A.
Phytopathology 100:S163
Historically, resistance to postharvest fungicides has developed in pathogens
of fruit commodities that are stored for extended periods (e.g., citrus and
pome fruits). Strategies to minimize the selection for resistance begin with
simultaneous registrations of compounds with different modes of action
(MOA) that are highly effective in reducing decay (prevent selection) and
suppress sporulation of pathogens (prevent wild-type population displacement). Resistance management must prevent repeated exposure of high
pathogen populations to any fungicide. Ideally, rotations or mixtures of
fungicides with different MOAs reduce resistance potential because a lower
resistance frequency exists to multiple sites of action. Packinghouse practices
that increase the likelihood of resistance development include all methods that
lead to sub-optimal fungicide coverage, decreased fungicide residues, or
reduced fruit quality. Important chemical properties include systemic activity,
persistence, temperature and pH stability, compatibility with fruit coatings and
oxidizing sanitizers, or any property that facilitates integrated management
strategies. Sequential applications of fungicides as aqueous treatments for
decay control and in fruit coatings for sporulation control are ways to optimize
performance in a single process. Sanitation practices are essential to minimize
pathogen survivors and reduce the total population of pathogens that are
exposed to a fungicide.
Management of fungicide resistance in postharvest pathogens of pome
fruits: Integrated approaches from orchard to storage
C. XIAO (1), Y. Kim (1), Y. Yin (1)
(1) Washington State University, TFREC, Wenatchee, WA, U.S.A.
Phytopathology 100:S163
Recently registered pre- and postharvest fungicides pyraclostrobin+boscalid,
fenhexamid, fludioxonil, and pyrimethanil have provided new tools for
control of postharvest diseases in pome fruits. In the meantime, postharvest
pathogens Penicillium expansum and Botrytis cinerea are high-risk pathogens
for the development of fungicide resistance. Baseline sensitivities and
resistance-monitoring programs for key fungicides have been established.
Dual resistance to pyraclostrobin and boscalid had developed in B. cinerea in
apple orchards where the fungicides had been used. Resistance of P.
expansum to pyrimethanil was first detected in 2009 from decayed apple fruit.
While these fungicides are being increasingly used by the fruit industry,
strategies for management of fungicide resistance in these pathogens need to
Edible and Medicinal Mushrooms: Diversity,
Commercial Production, and Disease Management
in High-Volume Production Facilities
Global expansion in gourmet and medicinal mushroom cultivation and use
M. WACH (1)
(1) Sylvan Inc., Kittanning, PA, U.S.A.
Phytopathology 100:S163
Mushrooms have been held in fascination by humans dating back at least to
the era of the Caveman. Whether they were consumed as food, utilized as part
be developed and implemented. Management of fungicide resistance in
postharvest pathogens should involve both orchard and packinghouse
practices. Critical factors to be considered include disease cycles from orchard
to storage under different postharvest handling systems, inherent risk of both
fungicides and pathogens for resistance development, biological and
ecological characteristics of fungicide-resistance phenotypes, patterns of cross
resistance to other fungicides. Integrated approaches involving practices from
orchard to storage for management of fungicide resistance in postharvest
pathogens as well as for postharvest disease control will be discussed.
Resistance management strategies for new postharvest fungicides - Pace
International perspective
P. G. SANDERSON (1), D. Bylemans (2)
(1) Pace International, LLC, East Wenatchee, WA, U.S.A.; (2) Janssen
Pharmaceutica N.V., Beerse, BELGIUM
Phytopathology 100:S163
In 2004, two new fungicides, Penbotec (pyrimethanil) and Scholar
(fludioxonil) were registered in the U.S. for use on pome fruit. These were the
first new classes of chemical fungicides to gain postharvest registrations since
Benlate was registered in the early 1970’s. By 1978, it was reported that about
30% of isolates of Penicillium expansum (causal agent of blue mold)
recovered from packing houses were resistant to benzimidazole fungicides.
This level of resistance has persisted until the recent introduction of these new
chemicals. Hence, we have a good the model for fungicide resistance
development within pome fruit postharvest systems. In conventional packing
of pome fruit, fungicides may be applied either at harvest as a drench, or as
line sprays during packing. Studies on growth and dispersal of populations of
P. expansum clearly showed that field bins were the reservoirs of
benzimidazole resistant populations, and that drenching practices were
responsible for the accumulation and dispersal of those resistant populations.
Although these new fungicides are considered to have a somewhat reduced
potential for development of resistance compared to the benzimidazoles, we
can expect a similar loss of effectiveness unless changes are made in fruit and
equipment handling. The effects of fungicide alternation schemes, advances in
sanitation methods, and the effect of new application technologies on
resistance development will be explored.
Resistance management strategies for new postharvest fungicides Syngenta perspective
A. COCHRAN (1), E. Tedford (1), D. McKenzie (2)
(1) Syngenta Crop Protection, Greensboro, NC, U.S.A.; (2) Syngenta AG,
Basel, SWITZERLAND
Phytopathology 100:S163
Postharvest decay is one of the greatest challenges facing the fruit and
vegetable industries worldwide. Fungicides may be used to control post
harvest decays but their management is key, as continuous and exclusive use
may lead to development of resistance. Fungicide resistance management for
postharvest decay is challenging as some fruits and vegetables have to be
stored for a long time and/or transported long distances. There are many
factors that contribute to resistance development, including exclusive use of
fungicides having one mode of action, highly fecund pathogens such as
Penicillium spp., and storage conditions that are ideal for pathogen infection
and spread. Common strategies to offset resistance development in
postharvest pathogens include the use of sanitation, alternation of fungicides
having different modes of action, and/or the use of fungicide pre-mixtures. In
an effort to better manage fungicide resistance to storage diseases Syngenta is
combining good sanitation practices with the use of fungicide pre-mixtures in
fruit and vegetables. This pre-mixture approach is currently being employed
by Syngenta for citrus fruit and is being developed for other fruits and
vegetables. These mixtures represent a new and innovative approach to
postharvest disease control aimed at providing broad-spectrum decay control
built on a sound resistance management strategy when combined with proper
disease management in the field and use of postharvest sanitation when
possible.
of spiritual ritual or used as a poison, these higher basidomycetes play a
significant role in the evolution of mankind. In China, as early as 1245 A.D.,
Chen Yen-yu published details on the development, morphology, growing
methods and preparation of 15 different varieties of mushrooms. Commercial
cultivation can be traced back at least as far as 1313, when Wang Zeng
described the growing of Shiitake (Lentinula edodes). Cultivation of the
button mushroom Agaricus bisporus, was first described by Tournefort in
1707, wherein he describes the use of spent horse manure on which
mushrooms were growing for use as inoculum, facilitating continuous
production and spawning the modern mushroom industry. Today, some
14,000 mushroom species have been reported, of which about 60 have been
Vol. 100, No. 6 (Supplement), 2010
S163
commercially cultivated. While the majority of these cultivated species are
utilized as a food, a growing number of edible fungi are finding utility in other
ways. This presentation will explore the growth and development of the
commercial mushroom industry including a discussion of culinary, medicinal
and other uses.
Disease management in commercial mushroom facilities: Controlling the
fungus’ fungus
D. BEYER (1)
(1) The Pennsylvania State University, State College, PA, U.S.A.
Phytopathology 100:S164
Mushroom cultivation is an ideal model of the disease triangle, pathogen-hostenvironment interaction. Agaricus bisporus, the white and brown
commercially grown mushroom is exposed to many different types of
pathogens during its cropping cycle, insects, bacteria, nematodes viruses and
other fungi. Controlling these pathogens requires a solid integrated pest
management program and a good understanding of the biology for pests and
diseases. Better and safer methods must be found as alternatives to chemicals
and towards the environmental and cultural manipulation without lowering
yield or quality of mushrooms. This part of the symposium will describe the
disease management strategies used in commercial mushroom farms. Several
major fungal pathogens, their signs and symptoms will be described and how
cultural management and to a lesser extend chemicals are used to manage
these diseases.
Developing mushroom cultivation curricula at the university
M. DAVIS (1)
(1) University of California, Davis, Davis, CA, U.S.A.
Phytopathology 100:S164
A course on the cultivation of edible mushrooms is a marvelous way to
connect students from all academic backgrounds with both biology and
agriculture. The annual undergraduate course ‘Mushroom Cultivation’ at UC
Identifying Quantitative Resistance Using Modern
Technologies—Challenges for Plant Breeding
Bioinformatic strategies for predicting candidate genes under disease
resistance QTL
R. M. DAVIDSON (1), P. A. Reeves (2), P. M. Manosalva (3), J. E. Leach (4)
(1) Department of Plant Biology, Michigan State University, Fort Collins, CO,
MI, U.S.A.; (2) USDA, National Center for Genetic Resources Preservation,
Fort Collins, CO, U.S.A.; (3) Boyce Thompson Institute for Plant Research,
Ithaca, NY, U.S.A.; (4) Department of Bioagricultural Sciences and Pest
Management, Colorado State University, Fort Collins, CO, U.S.A.
Phytopathology 100:S164
Recent advances in genome sequencing have resulted in a plethora of genetic,
genomic and transcriptomic data in many online databases. We have
undertaken a comprehensive study aimed at combining numerous sequence
and expression datasets of the large multi-member germin protein family that
is known to include genes involved with broad-spectrum disease resistance.
First, we examine phylogenetic relationships and functional diversity of
germins across diverse genera including monocots and dicots, and then focus
on rice (Oryza sativa) as a model. The comparative analyses allow us to
predict candidate germin gene lineages with possible relevance to disease
resistance across taxa. Comprehensive data for rice, including a wellannotated genome sequence, robust genome-wide expression data and
published QTL data provide us the platform to identify candidates at the gene
level. The use of bioinformatics to layer data types, detect candidates and
connect them across plant species will become more powerful as an increasing
number of crop genomes are sequenced and gene functions are determined.
This approach will expedite the prediction of genes underlying QTL that are
useful for crop improvement.
QTL use for development of host resistance and putting it to use-Industry
perspective
G. TABOR (1)
(1) Pioneer Hi-Bred, Johnston, IA, U.S.A.
Phytopathology 100:S164
The availability and affordability of high throughput molecular marker
genotyping has greatly facilitated the identification and utilization of disease
Quantitative Trait Loci (QTL) in commercial breeding. QTL-based marker
assisted selection has reduced cost and time needed for development of
disease-resistant varieties in many crops. A disease QTL has to meet several
criteria to be commercially useful and these criteria vary according to the crop
S164
PHYTOPATHOLOGY
Davis is extremely popular and always has a waiting list to register. The 11week course introduces methods of growing edible mushrooms, including
culture maintenance, basic mushroom substrate preparation, composting,
spawn generation techniques, inoculation methods, harvesting, and pests and
pest management. The students grow Oyster, Shiitake, Button, Lion’s mane,
and Reishi mushrooms. A field trip to a local mushroom farm is often
included in the curriculum. The history of mushroom production and recent
trends in the diversification of edible mushrooms are discussed. At least one
biology prerequisite course is recommended but is not required. The goals of
the course are to provide the students with an understanding of the vocabulary
used in the mushroom industry, the experience of growing mushrooms, an
understanding of the biology of fungi, and hopefully, a lifelong interest in
mushrooms. An appreciation for agriculture is one of the underlying premises
of the course.
Certifying mushrooms organic to the USDA National Organic Program
Standard
T. ELLOR (1)
(1) Phillips Mushroom Farms, Kennett Square, PA, U.S.A.
Phytopathology 100:S164
Certifying and growing mushrooms to the USDA National Organic Program
(NOP) crop standard is challenging on a number of fronts. There is no part of
the NOP standard that applies specifically to mushrooms, so applying a
standard that was written predominantly for field crops, orchards, and produce
grown in soil outside presents unique difficulties. Growing organic
mushrooms in the ‘Mushroom Capitol of the World’ AKA the ‘Mushroom
Disease Capitol of the World’ presents another set of problems. Familiarity
with the Organic Standards and how and where it does and does not apply to
mushrooms is critical to writing an effective organic system plan. The 2nd set
of problems can be approached by being familiar with pests and diseases that
affect mushroom production, excellent sanitation and management, and sound
IPM practices which include alternatives to conventional controls.
and disease in question. In general, a commercially viable disease QTL should
not have adverse effect, due to either pleiotrophy or tight linkage, on critical
commercial traits. Some disease QTL are of high effect and/or probably
encompass major gene(s) for disease resistance; these QTL tend to have
higher chance of being utilized in commercial breeding. Conversely, many
QTL are of minor effects and some of them may not be incorporated in
commercial products due to many considerations including cost. With the
advancement in genomics, high-throughput genotyping technology and
supporting technologies, one of the limiting steps in QTL identification has
become lack of reliable phenotypic assays. Disease phenotypic assays have
not advanced in parallel with genotyping technology. Limited research
emphasis on understanding the basic biology, epidemiology and genetics of
host-pathogen interaction for most pathosystems have limited advancements
in development of new phenotypic assays.
Proteomics in identifying potential markers for developing broad
spectrum resistance
Z. CHEN (1), S. Park (1), C. Zhang (2)
(1) Louisiana State University, Baton Rouge, LA, U.S.A.; (2) Ames, IA,
U.S.A.
Phytopathology 100:S164
Soybean rust, caused by Phakopsora pachyrhizi, was first reported in the
continental U.S. in late 2004. Since then it has been spreading steadily north
in the past years. Severely infected soybean plants can be quickly defoliated
and resulted in significant yield losses of up to 80%. There is no resistant
soybean cultivar yet available to growers. Recently, soybean lines with singlegene resistance (such as Rpp1-5) were identified. However, these lines
showed resistance to limited numbers of rust races and became ineffective
soon after they were identified. In an effort to identify potential markers for
developing durable broad-spectrum resistance to soybean rust disease, a
proteomics approach was used to compare protein profile differences during
compatible and incompatible interactions between soybean and P. pachyrhizi.
Differentially expressed proteins were identified and sequenced through
tandem mass spectrometry. The expressions of some of the proteins were
further examined during the time course of rust infection to determine how
early host responds to pathogen attack and how their expressions are changed
at different infection stages. Two of the infection-induced proteins,
pathogenesis-related protein 10 and chalcone isomerase 1, which were
upregulated during early and middle stages of infection, respectively, were
further investigated through VIGS to determine their roles in soybean
resistance to rust disease.
Using network biology to identify quantitative genetic variation altering
signaling in both plant host and generalist pathogens
D. J. KLIEBENSTEIN (1)
(1) University of California, Davis, Davis, CA, U.S.A.
Phytopathology 100:S165
Botrytis cinerea is a highly diverse pathogen with natural variation
modulating an extreme range of phenotypes that is central to being a
generalist pathogen. We are combining modern systems biology with natural
variation in Botrytis and Arabidopsis and Tomato to identify how the
pathogen and plant genetic variation interact. This is highlighting fundamental
mechanisms influencing broad host resistance. By investigating quantitative
trait loci that control differential resistance to Botrytis isolates we can link
Induced Resistance: Where Does This Fit in IPM
Programs
Induced systemic resistance by biological control agents
P. BAKKER (1)
(1) University of Utrecht, Utrecht, NETHERLANDS
Phytopathology 100:S165
The mechanisms that underlie biological control of plant diseases using
antagonistic microorganisms include competition for space and nutrients,
antibiosis, lytic activity, and induced systemic resistance (ISR). In the last
decades it has become clear that elicitation of ISR by biological control agents
is a widespread phenomenon. Many microorganisms that were previously
reported to suppress disease by competition, antibiosis or lytic activity were
also demonstrated to elicit ISR. Induced resistance is a state of enhanced
defensive capacity developed by a plant reacting to specific biotic or chemical
stimuli. ISR is typically studied in systems in which the biocontrol agent and
the pathogen are inoculated and remain spatially separated. Much progress has
been made in elucidating the signal transduction pathways in the plants
involved in ISR and the microbial elicitors of ISR. The diversity of
microorganisms, plant species, signal transduction pathways and microbial
elicitors in relation to induced resistance, and ways to improve the efficacy of
ISR will be discussed.
Seed or soil applied bacteria that induce resistance-Use in IPM programs
J. W. KLOEPPER (1)
(1) Auburn University, Auburn, AL, U.S.A.
Phytopathology 100:S165
Studies with many different bacterial biocontrol agents, including PGPR, have
demonstrated that one mechanism by which effective strains reduce plant
disease is elicitation of induced systemic resistance (ISR) in various model
systems. Applications of these findings in practical disease management
strategies for commercial agriculture are now possible using seed treatment
with aerobic spore-forming PGPR (the bacilli). A case study is Bacillus
pumilus strain INR-7 which was isolated from inside surviving cucumber
plants in a field with a high incidence of cucurbit wilt disease caused by
Erwinia tracheiphila. Subsequent research revealed that the strain elicited ISR
in cucumber against multiple pathogens in the field and reduced the feeding
preference of cucumber beetles, which was related to decreased plant
concentrations of cucurbitacin, a feeding cue for the beetles. In soybean, strain
INR-7 elicited increased plant cell wall lignification which was associated
with biocontrol. Strain INR-7 is registered by the EPA as a biological
fungicide and which is currently marketed as Yield Shield™ by Bayer Crop
Sciences for managing Fusarium spp. and Rhizoctonia solani on soybean and
beans. While we have an understanding of some plant biochemical changes
that occur during the signal transduction elicited during ISR by INR-7, we do
not yet know what component of the strain triggers ISR. Future directions
with integrated biocontrol strategies will be discussed.
Foliar applied Bacillus that induce resistance-Use in IPM Programs
B. J. JACOBSEN (1)
(1) Montana State University, Bozeman, MT, U.S.A.
Phytopathology 100:S165
Several foliar applied Bacillus-based biological control agents (BCAs) are
registered by the U.S. EPA for disease control. Bacillus subtilis isolate QST
713, B. mojaviensis isolate 203-7, and B. mycoides isolate BmJ have been
reported to effect disease control involving induced resistance. The BmJ
isolate provides control of a wide range of diseases caused by fungal, bacterial
and viral pathogens by inducing systemic resistance in plants. Induction is
salicylic acid independent and involves signaling through the NPR1 and
ethylene pathways. Induced resistance has been demonstrated in sugarbeet,
global variation in plant defense gene and defense metabolite expression with
altered Botrytis virulence. This is showing that jasmonic acid is only required
for resistance to a subset of Botrytis genotypes. Understanding what is
conserved and variable within a generalist pathogen identifies targets that will
not overcome by pathogen genetic variation. One complexity to systems
biology is the concept that only genomics experiments done in the presence of
the pathogen will be informative. Given the diversity within these pathogens
this supposition creates massively expensive experiments. We are testing the
concept that systems biological approaches can identify predictive signatures
of generalist resistance in the absence of the pathogen itself. We will present
evidence that we can identify mechanisms controlling pathogen resistance
without conducting the experiments in the presence of the pathogen.
cucumber, potato and tomato. When using induced resistance applications
must be made before infection for optimal results. Control using BmJ alone is
in the 30–90% range and in most pathosystems tested, inferior to the best
commercial fungicides. However, when used with host resistance, reduced
rates or applications of fungicides control is often equal to the best labeled
fungicides. Research on Cercospora leaf spot of sugarbeet (Cercospora
beticola), showed incorporation of BmJ in fungicide control programs resulted
in reduced fungicide resistance. Bacillus BCAs such as BmJ offer the
opportunity to control bacterial and viral pathogens for which alternatives are
only equally effective or are not available. For fungal pathogens, this BCA
offers an alternative in fungicide resistance management and alternatives for
both conventional and organic growers. BmJ is compatible with a wide range
of pesticides.
The role of Trichoderma in crop management systems
G. HARMAN (1), F. Mastouri (1)
(1) Cornell University, Geneva, NY, U.S.A.
Phytopathology 100:S165
Trichoderma spp. have been know for decades as biocontrol fungi. However
some strains are endophytic plant symbionts. They invade and colonize roots,
thereby inducing plant resistance, which results in localization of the fungi.
Some strains also can invade and colonize twigs and stems. Successful use of
Trichoderma requires that highly efficient strains be discovered or produced.
Earlier, we believed that antibiosis and mycoparasitism were principal
mechanisms of biocontrol, but we now know that induced systemic resistance
is probably more important. However, biocontrol is only a subset of the
advantages that effective endophytic strains confer. They also induce
resistance to a variety of abiotic stresses, including water deficit, temperature,
salt and osmotic stress. They also increase nitrogen use efficiency (NUE) in
plants; it is anticipated that we can reduce nitrogen use for selected crops by
30% without reducing yields. These applications have major implications for
plant agriculture. NUE can, for example, reduce air and water pollution from
agriculture and can improve food security for small holders who cannot afford
sufficient nitrogen fertilizer to obtain maximum yields of plants. The fungi
create these numerous benefits because they alter plant gene expression.
Photosynthetic efficiency, especially in the presence of stresses such as
drought, is improved, as is plant ability to ameliorate effects of toxic reactive
oxygen species.
Chemical compounds that induce resistance-Use in IPM programs
A. H. TALLY (1)
(1) Syngenta Crop Protection, Greensboro, NC, U.S.A.
Phytopathology 100:S165
Disease protection through induced resistance has been known for many
years, but it was not until the development of acibenzolar-S-methyl
(ASM/Actigard/Bion) that researchers had a non-biological tool that induced
the plant response similarly to biological induction. Other substances are also
known to have similar effects – INA, salicylic acid, laminarin, harpin although the mechanisms may be different. What is evident, with a few
exceptions, is that the level of disease protection is more suppression of
disease rather than excellent control that is provided by many of the traditional
fungicides. Compounds such as ASM are useful tools as there are no
alternatives for some diseases, and in many cases, the compounds improve
performance of the fungicide and provide plant health benefits which make
them an excellent fit in IPM programs. Depending on the crop, these products
can suppress a broad spectrum of pathogens as well as provide another tool
for resistance management. There is also evidence that Actigard can help
maintain the utility of resistant cultivars longer. Rate ranges and use patterns
are being studied more to be able to more fully realize the additional benefits
in an IPM program.
Vol. 100, No. 6 (Supplement), 2010
S165
Kasugamycin: The Risks and Benefits of Introducing
a New Antibiotic
Re-discovery of kasugamycin for managing fire blight and other bacterial
diseases of plants in the United States
J. ADASKAVEG (1), H. Forster (2), L. Wade (3)
(1) University of California, Riverside, CA, U.S.A.; (2) University of
California, Davis, CA, U.S.A.; (3) Roseville, CA, U.S.A.
Phytopathology 100:S166
Kasugamycin was first discovered and produced as a commercial
fermentation product of Streptomyces kasugaensis in Japan in the 1960s. In
2006, after the US-EPA allowed import tolerances on food commodities, we
requested a U.S. registration through the federal IR-4 program for managing
fire blight, walnut blight, and other bacterial diseases (e.g., bacterial canker).
Although classified as a systemic aminoglycoside antibiotic, kasugamycin is
an antimicrobial with a unique mode of action from antibiotics, has
fungicidal/bactericidal activity, and is not used in animal or human medicine.
The proposed registration is based on a need for new compounds for
managing bacterial plant diseases. Streptomycin-resistant strains of the fire
blight pathogen Erwinia amylovora are widespread in the U.S. and recently,
strains less sensitive to oxytetracycline were detected and associated with crop
losses in CA. Copper-resistant strains of the walnut blight pathogen
Xanthomonas juglandis are also widespread in CA. These pathogens are
sensitive to kasugamycin and sensitivity baselines were developed. Because of
the potential of resistance developing in microbes to any single-site mode of
action material, disease management studies using rotations and mixtures of
kasugamycin with antibiotics, biologicals, and broad-spectrum compounds
(e.g., copper, mancozeb, captan) were conducted, shown effective, and are
suggested with the introduction of kasugamycin.
Kasugamycin – A novel antibiotic for North American agriculture
V. J. SPADAFORA (1), G. Orr (1), L. Wade (1), M. Wiglesworth (1)
(1) Arysta LifeScience North America, LLC, Cary, NC, U.S.A.
Phytopathology 100:S166
Kasugamycin (KM) is a novel aminoglycoside antibiotic being developed by
Arysta LifeScience in the U.S.A. and Canada, in conjunction with Hokko
Chemical Industry and leading research institutions under the trademark
Kasumin®. New antibiotics, while effective, have not been registered due to
concerns that resistance developed in populations of plant pathogens will be
transferred to human and animal pathogens. The lack of new agricultural
antibiotics has limited options for bacterial disease control in plants has
increased selection pressure and the incidence of resistance to existing
materials. Kasumin is an effective, useful new tool for North American
Agriculture that carries minimal intrinsic risks for use in plant protection and
to human and veterinary medicine. KM is relatively inactive against human
and veterinary pathogens and is not used for these purposes. It’s site of action
within the bacterial protein synthesis pathway is different from that of other
aminoglycoside antibiotics, allowing KM to be effective against strains of
plant pathogens that are resistant to other antibiotics, making it an effective
resistance management tool. The efficacy of KM has been proven globally,
and it is sold in 30 countries since it’s introduction in 1966. In North America,
efficacy has been proven in numerous research trials, under emergency use
exemptions, and under commercial conditions in Michigan and Mexico.
®Kasumin is a registered trademark of Hokko Chemical Industry.
EPA approaches for evaluating antibiotic uses in the context of FIFRA
L. ROSSI (1)
(1) EPA, Crystal City, VA, U.S.A.
Phytopathology 100:S166
EPA’s risk assessment approaches in the consideration of new requested
uses of antibiotic materials under FIFRA will be discussed. The components
Restoring Forest Ecosystems Impacted by Invasive
Pathogens
Can whitebark pine be saved?
E. M. GOHEEN (1), J. Schwandt (2)
(1) USDA Forest Service, Forest Health Protection, Central Point, OR,
U.S.A.; (2) USDA Forest Service, Forest Health Protection, Couer d’ Alene,
ID, U.S.A.
Phytopathology 100:S166
Whitebark pine (Pinus albicaulis), a five-needled pine of mountainous regions
in western North America, is considered a keystone species in the fragile high
elevation ecosystems it inhabits. The future of whitebark pine is of substantial
S166
PHYTOPATHOLOGY
of the EPA’s safety analysis, which includes food and drinking water
exposures, MRL establishment, as well as evaluations for worker exposure and risk, and ecological effects will be presented. EPA assessments
relative to evaluating the safety of new drugs from the standpoint of resistant bacteria will be covered. In this regard, EPA is using an
approach similar to that of FDA to characterize the safety of antimicrobial
drugs, which will be described. Milestones around the public participation process EPA applies to first domestic food uses will be explained as
well as plans for direct dialogue and coordination with partner agencies, such
as FDA and CDC. Regulatory mechanisms that may be available through
FIFRA labeling to enhance the safety and utility of antibiotics used in
agriculture will be discussed. In addition, post registration considerations such
as monitoring for the development of resistant bacteria will be summarized for
discussion.
Kasumin: Field results for fire blight management and evaluation of the
potential for resistance development in Erwinia amylovora
G. W. SUNDIN (1), G. C. McGhee (1)
(1) Michigan State University, East Lansing, MI, U.S.A.
Phytopathology 100:S166
The antibiotic Kasumin (Arysta Corp.; Cary, NC) was evaluated under field
conditions for control of the blossom blight phase of fire blight, caused by
Erwinia amylovora. Orchard studies were conducted on the highly-susceptible
varieties ‘Gala’ and ‘Jonathan’, and the trees were inoculated with a
concentrated suspension (1 × 106 cfu/ml) of the virulent strain E. amylovora
Ea110. Incidence of fire blight disease, as measured by the occurrence of
blossom blight symptoms in inoculated plots, was relatively high in
nontreated control trees in all six experiments conducted between 2006 and
2009, ranging from 35.6 to 72.5 percent infection. Application of Kasumin to
trees at least 24 h prior to and at least 24 h following inoculation with strain
Ea110 resulted in high levels of disease control that were not significantly
different from the standard streptomycin treatment in any experiment. We
assessed both the potential development of spontaneous mutants of E.
amylovora with kasugamycin (Ks) resistance and the possibility of acquisition of a transferrable Ks-resistance gene(s). Spontaneous Ks resistance
was only detected when E. amylovora strains were incubated in broth
medium containing progressively larger concentrations of Ks. These mutants
harbored mutations in the ksgA gene, and most were reduced in virulence
in an immature pear assay. We are currently attempting to isolate Ksresistance genes from the nontarget microflora present in apple trees and
orchard soil.
Resistance management strategies for bacterial pathogens: What works?
K. B. JOHNSON (1)
(1) Dept. Botany and Plant Pathology, Oregon Sate University, Corvallis, OR,
U.S.A.
Phytopathology 100:S166
With fungicides, proven strategies to slow development of resistance in the
target pathogen include utilization of appropriate material dose, of mixture
and/or rotation partners, and of limits to material use. With bacterial plant
pathogens, documented successes in resistance management are rarer, perhaps
because bacteria more readily acquire resistance and/or because there are so
few effective bactericides. Using Kasumin as a case study, potential resistance
management strategies for the fire blight pathogen, Erwinia amylovora, will
be deliberated and contrasted with other bactericides used for fire blight
control. Data demonstrating effective fire blight suppression with mixtures of
Kasumin and oxytetracycline, and with Kasumin integrated with biological
control will be presented. These resistance management strategies will be
discussed in relation to the mechanisms and heritability of resistance, to the
potential costs of resistance to reproductive fitness, and to the degree of
selection pressure imposed by the bactericide treatment.
concern due to the species acute vulnerability to the non-native fungus
Cronartium ribicola (cause of white pine blister rust), its susceptibility to
infestation by mountain pine beetle (Dendroctonus ponderosae) which may
kill trees that harbor natural resistance to blister rust, its risk of being
destroyed in large and intense wildfires, its likelihood of being replaced by
fire intolerant species due to fire exclusion, and the potential impacts of
warming temperatures at high elevations. Implementation of a conservation
and restoration program to protect and enhance existing populations, provide
regeneration opportunities, and increase the proportion of trees with natural
resistance to white pine blister rust can reverse this trend. Restoration projects
underway include: rust surveys and monitoring to determine host status,
collecting and storing whitebark pine seed, identifying and testing trees for
natural resistance to white pine blister rust, planting blister rust-resistant seed
or seedlings, using silvicultural methods to reduce competing vegetation and
create planting sites, encouraging natural regeneration, and treating blister
rust-resistant trees to prevent beetle attacks.
Management of Port-Orford-cedar (Chamaecyparis lawsoniana) in the
presence of the non-native pathogen Phytophthora lateralis
R. A. SNIEZKO (1), D. J. Goheen (2)
(1) USDA Forest Service, Cottage Grove, OR, U.S.A.; (2) USDA Forest
Service, Central Point, OR, U.S.A.
Phytopathology 100:S167
Port-Orford-cedar (POC), a unique and valuable tree native to SW Oregon and
NW California, is affected by a virulent, non-native pathogen, Phytophthora
lateralis. Infection results in death of hosts of all ages. The goal of the POC
management by the Forest Service and BLM is to maintain POC as an
ecologically and economically significant species on federal lands. The
integrated strategy developed seeks to maintain POC where risk of infection is
low, reduce disease spread and severity in high risk areas, protect uninfested
watersheds, and reestablish the tree species where appropriate. Techniques
such as road closures, sanitation treatments and washing vehicles are routinely
used. Successful breeding of POC with various degrees of resistance to P.
lateralis has been an encouraging recent development. The species’ range has
been divided into breeding zones, and seed orchards of resistant parents have
been established for some. Field trials have been established on a range of
cooperator’s lands. For some zones, seed for reforestation and restoration is
now available, and planting has begun. As plantings of genetically resistant
POC reach reproductive maturity, dispersal of pollen and seed will help
increase number and frequency of resistant trees in neighboring forests. A
current challenge involves finding opportunities to deploy resistant stock on
federal lands where planting has declined due to decreased harvests and
increased dependence on natural regeneration in silvicultural prescriptions.
Restoring a fallen giant – The American chestnut
W. L. MACDONALD (1), R. B. Mann (2)
(1) West Virginia University, Morgantown, WV, U.S.A.; (2) The American
Chestnut Foundation, Mount Sterling, KY, U.S.A.
Phytopathology 100:S167
The early 20th century introduction of Cryphonectria parasitica into eastern
North America forests resulted in a host-parasite interaction that eliminated
the American chestnut (Castanea dentata) as a forest tree and had
unparalleled ecologic, economic and sociologic consequences. Fortunately,
the American chestnut has been saved from extinction by its ability to sprout
from surviving roots. Restoration of the American chestnut, although a
daunting task, may be possible as a result of a concerted breeding program
and research designed to diminish the virulence of the fungus. For over 25
years, The American Chestnut Foundation (TACF) has conducted and
fostered research to employ a back-cross breeding approach to generate hybrid
chestnuts that possess phenotypically-like American chestnut but carry
resistance genes from their Oriental relatives. As these trees are developed,
they will be released to selected sites across the original chestnut range. State
TACF chapters also will contribute locally adapted germplasm for this
purpose. These plantings represent small interbreeding populations that will
generate resistant seed. The discovery of hypoviruses that debilitate C.
parasitica may be instrumental to the survival of the small developing
populations, especially if the trees do not possess adequate levels of
resistance. Any introduction program would be shortsighted if it failed to
acknowledge the constant threat posed by C. parasitica.
Sudden Oak Death and the future of California coastal forests
D. RIZZO (1), M. Garbelotto (2)
(1) University of California-Davis, Davis, CA, U.S.A.; (2) University of
California-Berkeley, Berkeley, CA, U.S.A.
Phytopathology 100:S167
Sudden oak death (SOD) is an emerging infectious forest disease caused by
the recently discovered generalist pathogen Phytophthora ramorum. Lethal
infections are concentrated in several ecologically important species,
including tanoak (Lithocarpus densiflorus) and various oak species (Quercus
spp.). The disease has killed potentially millions of trees in coastal forests of
California and may be changing the ecological dynamics and biodiversity of
these systems. Understanding the ecology of SOD and its long-term impacts
on forests requires integrating knowledge of feedback among hosts, the
pathogen and the environment. Which plant species will be successfully
recruited in the face of SOD and how subsequent successional patterns will
develop are important questions for forest managers and conservation
biologists. Development of short-term and long term management strategies
for SOD in California and Oregon coastal forests is still in the early stages.
Options being tested include tree removals, fire, chemical treatments, and host
resistance. These management strategies must be evaluated at different spatial
scales and in context with long-term management goals and policies.
Professionalism/Outreach
The APS Public Policy Board: New Challenges
for Phytopathologists
Opportunities for plant pathology funding and regulatory policy priorities
K. EVERSOLE (1)
(1) Eversole Associates, Bethesda, MD, U.S.A.
Phytopathology 100:S167
Public policy affects virtually all aspects of the diverse scientific enterprise
from providing education and training for careers in science to funding
research projects to applying results from fundamental research to the field.
Changes in program priorities by national and international funding bodies
and modifications in regulatory requirements affects the everyday world of the
plant pathology, sometimes making it easier for conducting science and
sometimes more difficult. While agricultural research funding faces many
challenges, many opportunities exist for plant pathologists to influence policy.
The APS has a diverse tool kit for understanding the interrelationship between
public policy and the science of plant pathology as well as for providing
advice, suggestions, and assistance to improve the conditions under which
scientists must operate. To gain the level of funding and breadth of
participation necessary to achieve success for APS priorities, efforts are
underway as well for building national and international collaborative efforts
that leverage scarce research funding across multiple agencies and countries.
An overview of these opportunities will be presented along with several
examples of how individual scientists can become involved.
The National Plant Microbial Germplasm Collection
A. BENNETT (1)
(1) University of Arkansas, Fayetteville, AR, U.S.A.
Phytopathology 100:S167
Culture collections of plant-associated microbes represent an essential
foundation for U.S. science. Microbial collections are a resource used to solve
a myriad of practical challenges to our agricultural and environmental systems
and play diverse and critical roles in understanding plant resistance to diseases.
Collections provide a critical link between past and present disease epidemics,
facilitate identification of emerging diseases, and are useful in developing
strategies to control plant diseases that impact the vitality of the U.S. agricultural sector. Our microbial culture collections are at risk however because the
United States lacks a coordinated national system to protect, preserve and
enhance these valuable resources. The APS Public Policy Board (PPB) supports
the formation of a National Plant Microbial Germplasm System (NPMGS).
This emphasis comes after two workshops and a strategic plan developed by
the APS ad hoc committee on culture collections. An overview of the proposed NPMGS structure, its administrative framework, and its searchable common cyber-database will be presented. Success stories resulting from proactive characterization of culture collections and “missed opportunities” will
be presented by APS members. This special session is a joint effort sponsored
by the APS-PPB and the APS Collections and Germplasm Committee.
Looking ahead in genomics of plant-associated microbes
S. H. HULBERT (1), J. E. Leach (2)
(1) Washington State University, Pullman, WA, U.S.A.; (2) Colorado State
University, Fort Collins, CO, U.S.A.
Phytopathology 100:S167
In recent years, the APS Public Policy Board has helped funding agencies
prioritize and gain support for microbial genome sequencing projects. Funding
agencies will likely be devoting fewer resources to these types of projects in
the future. Rapid advances in sequencing technology are making microbial
genome sequencing more feasible for much less money. Individual sequence
reads keep getting longer and cheaper, and other advances, like the ability to
read both ends of DNA fragments, are allowing for more affordable assembly
into relatively complete genome sequences. Some fungal and oomycete genome
sequences can now be derived for tens, instead of hundreds, of thousands of
dollars, and bacterial genome sequences can be completed even more cheaply.
Genomic sequencing is now included in biology oriented, individual
investigator grant proposals instead of just proposals focused on completing
Vol. 100, No. 6 (Supplement), 2010
S167
the sequence. With the growing amount of microbial sequence available and
other types of information associated with the sequence (e.g. polymorphism,
expression data, etc.), the PPB considers processing, storage, accessibility and
utilization of the data to be an important issue in the near future. The PPB is
sponsoring a symposium at this meeting ‘Integrated Microbial Bioinformatics’
to discuss the development of integrated databases and bioinformatic support
to help plant pathologists access and use this information.
Microbial-plant interactions: Human pathogens on plants
J. BARAK (1)
(1) University of Wisconsin, Madison, WI, U.S.A.
Phytopathology 100:S168
Fresh produce has become the most likely contaminated food leading to
human disease, negating the paradigm that food-borne human pathogens are
associated primarily with animal products. Recalls and litigation cost the
produce industry millions and impact every industry sector. Uncertainties
about our ability to prevent future contamination throughout the supply chain
haunt U.S. producers, processors, retailers, and regulators. As a result,
increasing pressure is being placed on the government to institute improved,
science-based food safety standards and audit compliance programs.
Fundamental and practical research is needed to identify best management
practices and to determine the contamination routes, environmental survival,
and interactions between human pathogens and plants. Plant pathologists are a
valuable scientific resource that can drive discovery and design of effective
solutions to microbial contamination of food plants. PPB has actively sought
to bring the expertise of plant pathologist to the attention of FDA officials
grappling with a mandate to create food safety regulations on farms. Through
this effort, FDA officials attended the 2009 Annual Meeting and subsequently,
hosted a listening session of APS members currently involved in food safety
research in Nov 2009. A joint effort between FDA-APS/PPB has planned
successive special symposium to be held at the 2010 Annual Meetings of APS
and the International Association of Food Protection.
Policy making up close: Reflections of the APS - Office of Science &
Technology Policy Fellow
M. PALM (1)
(1) USDA APHIS PPPQ, Molecular Diagnostic Laboratory, Beltsville, MD,
U.S.A.
Phytopathology 100:S168
Prepare for Your Future: Career Opportunities After
Graduate School: Part 2 - Extension
Is extension right for you?
J. L. BECKERMAN (1)
(1) Purdue University, West Lafayette, IN, U.S.A.
Phytopathology 100:S168
Extension is a model of technology transfer where university-developed
knowledge is delivered directly to people where they live and work; Extension
plant pathology focuses on providing growers with research-based
information to guide their disease management decisions. Successful
implementation of a research-based extension program requires a unique skill
set that allows the extension specialist to communicate with university and
industry scientists, crop protection companies, and growers of varying skill
levels and education backgrounds. Many of the valuable habits, traits, and
skills necessary for effective extension programs are not acquired through
traditional graduate education. However, all of them can be learned and can
improve the likelihood of developing a successful extension program.
Extension jobs from MS to PhD: Acquiring the skills and developing the
resume to get the job you want
B. J. JACOBSEN (1)
(1) Montana State University, Bozeman, MT, U.S.A.
Phytopathology 100:S168
Extension Plant Pathology has provided me a wonderful and diverse career for
37 years. During my Ms degree, I determined that an Extension/Research
faculty position was to be my chosen career path. During my graduate training
I took courses in communications (public speaking, print, radio and TV) and
worked with faculty extension plant pathologists on development of fact
sheets, recommendation guides, and extension education programs. Equally
important was the development of diagnostic skills both in the field and
laboratory. Experience provided by faculty mentors in appraising field
situations and understanding clientele needs has been critical to a successful
career. If you want a job in extension work with extension professionals, get
S168
PHYTOPATHOLOGY
The Office of Science and Technology Policy (OSTP), in the Executive
Office of the President, was established in 1976 to advise the President and
Executive Branch on science and technology effects on domestic and
international affairs, to ensure that the Executive Branch policies are informed by sound science, and to coordinate the implementation of the
President’s science and technology policies across agencies, states, and
stakeholders. The APS Public Policy Board worked with OSTP to establish an
APS sponsored OSTP fellow and Dr. John Sherwood served as the first
Fellow in 2008–2009. The second Fellow at OSTP, starting in April 2010, is
Dr. Mary E. Palm. Dr. Palm will discuss her activities at OSTP including
initiatives of interest to APS members such as food safety, scientific
collections, and education.
EPA from the inside: Report from the APS-EPA Fellow
F. P. WONG (1)
(1) University of California, Riverside, Riverside, CA, U.S.A.
Phytopathology 100:S168
There are a wide range of regulatory issues that affect plant disease
management in the U.S. and working with the EPA through APS and its
Public Policy Board is a mutually beneficial interaction that helps APS
members and our stakeholders. APS PPB is in the process of establishing a
Subject Matter Expert to serve as a resource for EPA on issues affecting plant
disease management. Historically, EPA has had the most impact on plant
pathology through the regulation of chemical pesticides; with new
development in technology, EPA is now faced with the challenges of
regulating the use of biopesticides and biocontrol agents and well as the
deployment of genetically modified organisms for crop protection. Issues that
APS Public Policy board has identified recently as those of interest include:
new pesticide spray drift regulation, the use of fungicides for plant health
promotion, the withdrawal of maneb and review of other EBDC fungicides for
re-registration, the status of demethylation inhibitors (DMIs) as endocrine
disruptors, fungicide resistance impacts and data requirements for plantincorporated protectants. APS meeting symposia involving EPA scientists
addressing issues such as regulatory processes that affect pesticide registration
and the use of crop protection tools and fungicide resistance development are
also being planned for the near future.
practical experience and show you have a love for extension teaching.
References from these extension professionals will be critical in getting an
extension position. It is important to understand that while in graduate school
you cannot anticipate what career opportunities will be available either upon
graduation or during your professional career. My consul is to be well
prepared by thoroughly understanding plant disease diagnosis and control and
have a firm foundation regarding the biology of the different types of plant
pathogens, epidemiology, pesticides and pesticide application. In addition,
understanding the historical and practical basis for extension education and
4H can give you a “leg up” in obtaining that extension job.
A year in the life of a diagnostician
G. E. RUHL (1)
(1) Purdue University, West Lafayette, IN, U.S.A.
Phytopathology 100:S168
Do you enjoy mysteries and problem solving? Are you interested in plant
health? Do you like to share what you have learned with others? Are you good
at creative writing and multi-tasking? If so, a career as a plant disease
diagnostician might just be your answer to a dream job. Today’s
diagnosticians utilize molecular, serological, biochemical and traditional
methods to sleuth out the causal agents of plant disease on all manner of plant
problems, on all commodities, for a diverse clientele. As members of the
National Plant Diagnostic Network (NPDN), Land Grant University (LGU)
diagnosticians are networked to assist in the detection and identification of
exotic and invasive plant diseases. NPDN diagnosticians are also provided
with opportunities to attend diagnostic training workshops provided by
USDA/APHIS/PPQ on detection methods for regulated pathogens.
Incorporating new diagnostic techniques into the clinic as well as providing
first reports on new diseases diagnosed in the lab encourages intellectual as
well as professional growth. Diagnosticians also often collaborate with
extension specialists and state regulatory personnel for transfer of knowledge
through teaching efforts and to develop first detector educational programs for
numerous groups of stakeholders including Extension Educators, growers,
consultants, inspectors, students, homeowners and others.
A year in the life of a county extension agent
T. C. STEBBINS (1)
(1) University of Tennessee, Chattanooga, TN, U.S.A.
Phytopathology 100:S169
Starting an extension specialist career from the ground up
L. J. DU TOIT (1)
(1) Washington State University, Mount Vernon, WA, U.S.A.
Phytopathology 100:S169
A County Extension Agent has come a long way since the character Hank
Kimball on the classic television show “Green Acres”. The extension agent is
the direct link between the county clientele and the University. The agent
organizes timely meetings and workshops to distribute new information. The
agent partners with many non-profit organizations to deliver programs
through existing channels. The agent trains volunteers to maximize the
outreach. Master Gardener training usually occurs in the winter so volunteers
are ready to help with spring and summer programs. Groups such as Habitat
for Humanity, area food banks, and local nature centers use the trained
extension volunteers. The volunteers also present seminars and talks that a
county agent might ordinarily give. They speak to garden clubs, church and
school groups. They help with community gardens and local beatification
projects that are visible to the community. The agent also advises other groups
like the landscapers, beekeepers and herb clubs. These groups hold regular
meetings that enrich public education. They spread university extension
information as well. The agent answers many phone calls and emails
regarding plant problems. A Master Gardener hotline helps with this outreach
as well. Hopefully an agent can also get out of the office and travel out into
the county at times. This personal contact is rewarding for both parties.
You recently completed your PhD degree in plant pathology and successfully
applied for a position as an extension specialist in plant pathology at a land
grant university – your dream job. However, you have not previously worked
with the crops for which you’ve been assigned responsibility. How do you
begin to establish a successful career as an extension specialist? How do you
determine areas of research on which to focus your program? Who are your
stakeholders? How do you effectively connect with (sometimes very diverse)
stakeholders? How do you set up productive collaboration with the research
community, county extension educators, growers, and private industry? What
sources of funding are available to support your research and extension
program? How do you resolve or mitigate conflicts of interest among
competing stakeholders and collaborators? What steps can you take to ensure
objectivity when dealing with politically sensitive issues? How can you
successfully adapt your program as new (and sometimes urgent) disease
problems develop? How do you seek constructive mentoring and professional
development opportunities to ensure continued growth professionally and
personally? This presentation will use specific examples to illustrate some of
the skills, resources, attitudes, and methods that contribute to building a
successful career as an extension specialist in plant pathology.
Vol. 100, No. 6 (Supplement), 2010
S169
2009 Southern Division Meeting Abstracts
The following abstracts were presented at the joint meeting of the APS Southern Division and the Southern Association of Agricultural Scientists (SAAS) in
Atlanta, Georgia, February 1–2, 2009. These abstracts are in addition to those published in the 2009 June Phytopathology Supplement. The abstracts are arranged
alphabetically, by first author’s name.
Association of fern distortion syndrome with endophytic bacteria and the
use of Benlate
J. W. KLOEPPER (1), J. A. McInroy (1)
(1) Dept. Entomology and Plant Pathology, Auburn University, Auburn, AL,
USA
Phytopathology 100:S170
Fern distortion syndrome is a wide-spread problem in commercial production
of Leatherleaf fern (Rumohra adiantiformis) in Costa Rica. Previous studies in
Florida suggested that the main symptom of frond distortion was associated
with history of Benlate use on this vegetatively propagated plant and
stimulation of deleterious bacteria. Field and greenhouse tests were designed
to confirm or refute the previous suggestion. Paired sampling of 10 ferns with
distorted and 10 with normally shaped fronds was done at 6 commercial
ferneries in Costa Rica. Populations of total bacteria and fluorescent
pseudomonads were assessed from the rhizosphere and from inside
(endophytic) rhizomes. Samples were also collected three times from two
ferneries in Florida, with and without Benlate history, and the populations of
bacteria in rhizosphere determined; in addition, at one time, populations of
bacteria on rhizomes and inside rhizomes were determined. Results from
Costa Rica revealed significantly greater populations of total bacteria inside
rhizomes of ferns with distorted fronds at 5 of 6 locations, and higher
populations of fluorescent pseudomonads at all locations. In Florida,
significantly lower populations of fluorescent pseudomonads were found at all
three sample times in rhizospheres of plants never treated with Benlate than in
distorted ferns propagated from sources treated with Benlate. Also, higher
populations of fluorescent pseudomonads and total bacteria were found on the
surface and inside rhizomes of ferns propagated from sources treated with
Benlate. Hence, our results support the previously published suggestion that
distortion of fronds is associated with use of Benlate and increased
populations of fluorescent pseudomonads.
Treatment of Leatherleaf fern with Benlate systemic fungicide increases
populations of total and alleopathic endophytic bacteria
J. W. KLOEPPER (1), J. A. McInroy (1)
(1) Dept. Entomology and Plant Pathology, Auburn University, Auburn, AL,
USA
Phytopathology 100:S170
Using molecular techniques, we previously reported that increased
populations of Pseudomonas spp. were associated with Benlate use on
Leatherleaf fern (2007 Phytopathology 97: S182). The current study was done
to confirm and extend the previous work, using isolation techniques. All
Rhizomes of Leatherleaf fern were collected from a commercial fernery in
Florida where Benlate was never used. Some rhizomes were planted and
grown until 3 fronds were present on each plant, and these plants were used in
a spray experiment containing 8 replicate plants of 3 treatments: Benlate WP,
Benlate DF, and water. Other rhizomes were directly used in a drench
experiment containing the same 3 treatments plus a 6-hr-old preparation of
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
S170
PHYTOPATHOLOGY
Benlate DF. In both experiments treatments, all applications of Benlate
resulted in significantly greater populations of total bacteria and fluorescent
pseudomonads in the rhizosphere, on the rhizome surface, and inside rhizomes
2–4 weeks after application. Benlate treatment also resulted in significantly
more deformed root hair tips and in enhanced populations of pseudomonads
inside petioles 7 weeks after treatment. The percentage of allelopathic
endophytic bacteria, based on testing whole bacterial cells and cell-free
metabolites on cucumber seedlings, was significantly increased by Benlate.
Hence, Benlate changes the microbial community and increases virulence of
the community in a perennial plant.
Applications of Benlate systemic fungicide on banana reduce plant
growth and increase endophytic bacteria
J. W. KLOEPPER (1), C. Ramírez (1)
(1) Dept. Entomology and Plant Pathology, Auburn University, Auburn, AL,
USA
Phytopathology 100:S170
Benlate systemic fungicide has been linked to increased populations of
endophytic bacteria in Leatherleaf fern. Banana, like Leatherleaf fern,
develops rhizomes which contain endophytic microorganisms that will persist
with the next crop. Two experiments were conducted to determine if Benlate
applications change endophytic bacteria and alter plant growth of banana.
Micro-propagated commercial banana plants were transplanted into field soil
three months prior to treatment. Each experiment consisted of 8 replications,
one plant each, with 6 treatments: spray with Benlate WP, Benlate DF, or
water; drench with Benlate WP, Benlate DF, or water. Six months after
Benlate application (experiment 1), all applications of Benlate resulted in
significant reductions in height, shoot weight, and root weight of banana
plants (P = 0.01). These reductions in plant growth were accompanied by
changes in the populations of endophytic bacteria. Benlate treatment consistently increased populations of total bacteria and 3 of the 4 Benlate treatments
increased populations of fluorescent pseudomonads inside pseudostems.
Experiment 2 was destructively sampled 15 months after Benlate application.
Compared to the appropriate controls, all Benlate treatments resulted in
significant reductions in stem caliper, stem diameter, height, and weights of
shoots, roots, and rhizomes. In addition, plants from all Benlate treatments
had higher populations of endophytic fluorescent pseudomonads and total
bacteria. Overall, the results indicate that Benlate increases populations of
endophytic bacteria in banana, as it did on Leatherleaf fern. However, while
the effect on Leatherleaf fern was distortion of frond shape, on banana, the
effect is an overall stunting of plant growth and development.
Evaluation of commercially available plant growth-promoting
rhizobacteria (PGPR) and plant extracts on sheath blight disease of rice
caused by Rhizoctonia solani
K. Vijay Krishna Kumar (1), S. Krishnam Raju (2), M. S. Reddy (1), J. W.
Kloepper (1), K. K. Lawrence (1)
(1) Dept of Entomology & Plant Pathology, Auburn University, Auburn, AL;
(2) Dept of Plant Pathology, Andhra Pradesh Rice Research Institute,
Maruteru, India
Phytopathology 100:S170
Sheath blight disease of rice caused by Rhizoctonia solani is a major production constraint in all rice producing areas of the world. The annual losses due
to sheath blight are estimated to be 25% under optimum conditions of disease
development. Disease management is currently focused on extensive use of
fungicides which has created concerns about environmental pollution,
pathogen resistance and escalating costs. Field trials were conducted during
rainy seasons of 2005 and 2006 in randomized block design with three
replications to assess the commercially available bio-pesticide products for
their effect on sheath blight. Products evaluated were Achook (Azadirachtin),
Biotos (Plant activator), Tricure (Azadirachtin), Ecomonas (Pseudomonas
fluorescens) and Bavistin (Carbendazim) in 2005 and Biofer (Plant extract),
Biotos, Defender (Plant extract), Ecomonas, Florezen P (P. fluorescens),
Trichozen (Trichoderma viride) and Bavistin in 2006. Products were applied
three times as foliar sprays after appearance of first symptoms initially and
repeated at 10 days interval. The disease severity was measured by adopting
Highest Relative Lesion Height (HRLH) at 90 days after transplanting. The
chemical (Bavistin) reduce disease severity 52% and 50% compared to the
control. Corresponding reductions in disease severity with the bio-pesticides
ranged from 22% to 48% in 2005 and from 15% to 31% in 2006. Specifically
with PGPR, the disease reductions ranged from 14% to 38% compared to the
control in both the years. Grain yields were assessed at 120 days after
transplanting and significantly increased grain yields (3,901 and 1,938 kg/ha)
over control (2,690 and 1,550 kg/ha) were obtained with PGPR in 2005 and
2006 respectively. Our results showed that there is a scope for effective
management of sheath blight disease with the use of the currently available
PGPR and other products that are available under the conditions evaluated.
Changes in chrysanthemum rhizosphere bacteria related to steam
treatment and reduced plant growth
A. Suárez (1), M. Ramírez (1), J. Pérez (1), N. Cardona (1), J. Calle (1), C.
RAMÍREZ (1)
(1) Institute of Biology, Universidad de Antioquia - Centro de Innovación de
la Floricultura Colombiana, Colombia
Phytopathology 100:S171
Steam treatment of soils is common in commercial production of
chrysanthemum in Colombia. Although highly effective, the beneficial effect
of steam shortly disappears, and reductions of plant growth and vigor occur
after the first harvest. Affected plants do not exhibit classical disease
symptoms, nor are pathogens isolated from them. In attempts to overcome the
growth reduction, growers re-apply steam frequently, thereby increasing
production costs and potentially damaging soil health. We hypothesized that
reduced plant growth, following steam treatment, is associated with increases
in deleterious bacteria. To test this hypothesis, populations of total culturable
and aerobic endospore-forming (AEFB) bacteria and fluorescent
pseudomonads were determined in rhizosphere soil during three different
planting cycles after steam treatment and compared to populations in a control
chrysanthemum-cultivated soil supporting satisfactory plant growth and
lacking any history of steam treatment. Significantly higher populations of all
three groups were recorded in the third round of planting. However, for the
second round, when the plant growth (high and fresh weight) was already
significantly reduced, only increases in fluorescent pseudomonads were
significant. For the first round after steam treatment, when plant growth was
optimum, the populations of total bacteria and AEFB were higher in treated
than in non-treated soil, whereas fluorescent pseudomonads were similar.
Significant negative correlations were found between plant growth and the
population of each of the bacterial groups evaluated, and the correlation
coefficient was greatest for fluorescent pseudomonads. Tests for potential
bacterial deleterious traits have revealed a higher proportion of indole-acetic
acid-producing morphotypes of fluorescent pseudomonads in treated soils,
whereas similar numbers were found for HCN production.
Vol. 100, No. 6 (Supplement), 2010
S171
2009 Caribbean Division Meeting Abstracts
Abstracts presented at the joint meeting of the APS Caribbean Division and The Florida Pathological Society in Orlando, Florida, May 16–19, 2009. The abstracts
are arranged alphabetically, by first author’s name.
Monitoring resistant populations of Xanthomonas citri subsp. citri and
epiphytic bacteria on young citrus trees treated with copper or
streptomycin
F. BEHLAU (2), J. B. Jones (2), J. H. Graham (1)
(1) Citrus Research and Education Center, University of Florida, Lake Alfred,
FL; (2) Plant Pathology Department, University of Florida, Gainesville, FL
Phytopathology 100:S172
Since Florida’s citrus canker (Xanthomonas citri subsp. citri, Xcc) eradication
program was halted in 2005 attention has focused on management strategies
that include the use of bactericides such as copper and streptomycin for
disease control. Widespread use of these chemicals in citrus industries
elsewhere in the world has led to development of resistant strains of Xcc. Cu
and Sm resistance were monitored in Xcc and epiphytic bacterial populations
on citrus trees repeatedly sprayed with these chemicals for control of citrus
canker. Copper hydroxide (Cu, Kocide 3000) or streptomycin sulphate (Sm,
Firewall) were sprayed on foliage of young ‘Ray Ruby’ grapefruit every 21
days from March to October 2008. Mature canker-symptomatic and nonsymptomatic leaves were sampled monthly to assay for resistant Xcc and
epiphytic bacteria, respectively. Leaves were washed with MGY broth + 1
mg/L of CuSO4 for 2 hrs using 10 mL of liquid medium/g of leaf and plated
on semi-selective medium MGY-KCC + Cu or Sm for isolation of resistant
Xcc or on MGY + Cu or Sm for monitoring resistant population of epiphytic
bacteria. No Cu or Sm resistant strains of Xcc were isolated. No major
differences in total epiphytic bacterial population were observed among
treatments over time in comparison to the check. However, Cu and Sm sprays
increased the ratio of epiphytic bacterial population with resistance to these
chemicals. Overall, the Sm resistant bacterial populations were proportionally
lower than Cu resistant bacterial population.
Survival of Xanthomonas citri subsp. citri on symptomatic fruit under
prolonged ambient and cold storage conditions
G. Bonn (1), E. TAYLOR (3), T. Riley (2), T. Gottwald (3), T. Schubert (1)
(1) Florida Department of Agriculture & Consumer Services, Division of
Plant Industry, Gainesville, FL; (2) USDA, APHIS, PPQ, Orlando, FL; (3)
USDA, ARS, US Horticultural Research Laboratory, Ft. Pierce, FL
Phytopathology 100:S172
Live cells of Xanthomonas citri subsp. citri (Xcc) were detected by excising
canker lesions from commercial fresh-packed grapefruit, macerating them in
phosphate buffer followed by dilution plating onto a semi-selective agar
medium (KCB). After 4–5 days incubation at 28°C, separate colonies were
counted at 100X using a dissecting microscope. Confirmation of Xcc was by
the use of Agdia’s ImmunoStrip® for suspect plate colonies and a bioassay by
needleless infiltration of leaf lamina of young ‘Duncan’ grapefruit seedlings.
Xcc was detected from lesions on fruit held at both ambient and 5°C for up to
100 days in storage. There was a general trend for the percentage of Xcc
positive lesions and actual bacterial populations to decrease with storage time.
Populations of Xcc decreased faster at ambient temperature than at 5°C,
possibly due to the higher metabolic activity of cells or microbial competition
at the elevated ambient temperature. The low numbers of viable canker
bacteria associated with peel lesions in grapefruit, especially over time,
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
S172
PHYTOPATHOLOGY
suggest that the risk of canker transmission from them is extremely low in
comparison to active lesions on the leaves, stems and fruit during the growing
season.
Distribution of Candidatus Liberibacter asiaticus in huanglongbing
infected citrus trees
R. H. BRLANSKY (1), K. S. Pelz-Stelinski (1)
(1) University of Florida, CREC
Phytopathology 100:S172
The distribution of the huanglongbing (HLB) associated Candidatus
Liberibacter asiaticus (Las) bacterium in mature field infected citrus trees was
evaluated. The number of tissue samples collected per tree ranged from 16–32
and included: fruit peduncles, bark phloem, and symptomatic leaf midribs and
petioles taken from throughout the tree canopy. Overall, the percentage of
Las-positive plant tissue samples obtained ranged from 23–44% based on realtime PCR results. Although samples taken from bark phloem varied, phloem
from one-year old bark consistently tested positive for Las. Similar variation
in the detection of Las occurred in samples obtained from leaf petioles and
fruit peduncles. The percentage of positive leaf petioles ranged from 0–100%,
while fruit peduncles ranged from 17–56%. Additional replicates continue to
be collected in order to firmly establish whether one-year old bark phloem is
consistently positive when another part of the tree has tested positive for Las
via PCR. These results indicate that distribution of the HLB-associated
pathogen varies widely within symptomatic, PCR-positive citrus trees and
thus illustrate the importance of obtaining multiple samples from trees where
an infection is suspected.
Infectious clones and characterization of a previously unreported beaninfecting begomovirus from Rynchosia minima (L.), an endemic legume
species from Puerto Rico
J. K. BROWN (2), M. Rehman (1), A. M. Idris (2)
(1) Department of Microbiology, Hazara University, Mansehra, Pakista; (2)
Department of Plant Sciences, The University of Arizona, Tucson, AZ, USA
Phytopathology 100:S172
R. minima plants exhibiting mild mosaic symptoms that were reminiscent of
begomovirus infection were observed in Puerto Rico during the summer,
1997. Total nucleic acids were extracted from symptomatic R. minima leaves
using the CTAB method. The viral single-stranded DNA was subjected to
rolling circle amplification. The SacI-linearized, multimeric DNA band was
ligated into SacI-digested pGEM7Zf+ and cloned. Eight clones bearing a ~2.6
kbp fragment were sequenced using primer walking. Analysis of the resultant
sequences indicated that five and three of the clones were DNA-A and DNAB components, respectively. The genome organization and number of open
reading frames (six) was typical of other bipartite begomoviral genomes from
the Western Hemisphere. Comparative analysis of the DNA-A (n = 5) and
DNA-B (n = 3) component sequences shared 98–99% and 99% nucleotide (nt)
identity, respectively. Inspection of the common region (CR) revealed that
they were cognate components, and shared an identical iteron. The DNA-A
component shared 80% nt identity with its closest relatives, Macroptilium
mosaic Puerto Rico virus and Rhynchosia golden mosaic virus. The DNA-B
component shared 64% and 62% nt identity with RhGMV and Cabbage leaf
curl virus, respectively. This previously undescribed begomovirus species is
herein named Rhynchosia mild mosaic virus (RhMMV). Clones of the
RhGMV DNA-A and DNA-B component were released with SacI and
biolistically inoculated to R. minima and bean (Phaselous vulgaris) seedlings.
R. minima seedlings developed mild mosaic symptoms like those observed in
field-infected bean plants, thereby fulfilling Koch’s postulates, and the bean
seedlings developed severe green-yellow mosaic symptoms, confirming that
the virus also infects bean. This previously undescribed virus could pose a
serious threat to bean crops in the Caribbean region.
Developing an effective international education program for management
of Ralstonia solanacearum Race 3 biovar 2
P. G. CHAMPOISEAU (1), J. B. Jones (1), C. Allen (3), T. M. Momol (2)
(1) University of Florida, Department of Plant Pathology, Gainesville, FL,
USA; (2) University of Florida, District Directors Office, Gainesville, FL,
USA; (3) University of Wisconsin-Madison, Department of Plant Pathology,
Madison, WI, USA
Phytopathology 100:S173
Because it threatens both potato and ornamental production, Ralstonia
solanacearum Race 3 biovar 2 (R3bv2) is considered a serious quarantine pest
in Canada and Europe and is listed as a Select Agent plant pathogen in the
United States, where it is subject to the strictest biosecurity regulations.
Although this pathogen is not known to be established in the US, import of
infected geranium cuttings from off-shore production sites has already proved
to be a possible pathway for introduction. Previous accidental introductions
resulted in multi-million dollar losses due to quarantine responses. Therefore
it is critical to prevent further re-introduction and possible spread of R3bv2 in
the US. This involves exclusion, early detection, and unambiguous
identification of the pathogen at both national and international levels. This
can be achieved by use of reliable diagnostic tools for the pathogen and
effective phytosanitary measures; however, this is not enough. It is also
essential to ensure preparedness and effective training of official regulators,
diagnosticians and other individuals responsible for first detection and
response to a possible R3bv2 discovery in the US. We have therefore
developed an integrated education and outreach program, as part of a USDAfounded project for a better management of R3bv2. This program involves
development of educational and training content by a team of experts, delivery
of educational materials to target audiences by diverse means including
current web-based technologies, as well as use of various evaluation tools to
assess program effectiveness. Monitored access of our Ralstonia/bacterial
wilt-dedicated website shows that stakeholders from diverse organizations
both within and outside the US regularly use this resource to obtain updated
and accurate information on R. solanacearum R3bv2 and bacterial wilt
disease management.
Nitrocellulose membranes as a solid matrix for Cucumber mosaic virus
immuno-detection and subgroup identification by RT-PCR
P. S. CHANG (1), S. A. Tolin (1)
(1) Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
Phytopathology 100:S173
Cucumber mosaic virus (CMV) is an important and widespread plant virus.
The strains and isolates of CMV are highly diverse and assigned to either
subgroup 1A, 1B or 2. Here we report the application of tissue blot
immunoassay (TBIA) followed by reverse transcription-polymerase chain
reaction (RT-PCR) for the immuno-detection and subgroup identification of
CMV from various hosts and locations. Freshly torn leaves were blotted onto
nitrocellulose membranes (NCM), which were used as sources of viral RNA
after processing by TBIA. CMV positive samples show a purple precipitate at
the blot site. A 3 mm disc was removed from the positive sites and cleaned by
rinsing with Triton X-100, followed by TE buffer, then dried and added
directly to RT reactions that included a reverse primer to the CMV coat
protein (CP) gene. The resulting cDNA was added to PCR reactions
containing forward and reverse primers for the CMV CP gene. Agarose gel
electrophoresis revealed amplicons of the expected size. The subgrouping of
CMV samples was predicted from sequences of PCR products and confirmed
by monoclonal antibodies specific to CMV subgroups 1 and 2. Successful
amplification was possible from NCM blotted and TBIA processed up to 15
months previously, but amplification levels from older blots were lower. The
PCR protocol was adjusted by increasing the number of cycles for consistent
results from blots older than 8 months. This method eliminates the need for
leaf tissue storage or costly RNA extraction when sampling for CMV
diversity or monitoring virus prevalence and incidence. NCM are thus a
suitable matrix for obtaining viral RNA for RT-PCR and archival storage of
viral nucleic acids, similar to Whatman’s FTA® Plant Cards.
Sugarcane orange rust, an emerging disease in the western hemisphere
J. C. COMSTOCK (1), N. C. Glynn (1), L. A. Castlebury (2)
(1) USDA-ARS Sugarcane Field Station, Canal Point, FL; (2) Yystematic
Mycology and Microbiology Laboratory, ARS, USDA, Beltsville, MD
Phytopathology 100:S173
Symptoms consistent with sugarcane orange rust were first observed in
Florida in June 2007, these were subsequently confirmed morphologically and
molecularly as being caused by Puccinia kuehnii, the causal agent of orange
rust. This was the first documented occurrence of sugarcane orange rust in the
Western Hemisphere. Since then it has been reported in Guatemala, Costa
Rica and Nicaragua and has been confirmed in several other Central American
and Caribbean Countries. A comparison of brown rust and its causal agent, P.
melanocephala and P. kuehnii, will be presented. Orange rust has impacted
both the commercial production and the cultivar development program in
Florida. One major difference in the epidemiology of the two pathogens is that
P. kuehnii tolerates warmer temperatures and orange rust severity continues
throughout the summer and early fall lasting much longer than brown rust.
This is significant as it means that commercial cultivars susceptible to both
pathogens are impacted by either one or both pathogens depending on the
month of the growing season. A cultivar that occupies 25% of the acreage in
Florida, CP 80-1743, is susceptible to the disease and has had reduced cane
yields. It is being withdrawn from production. Results from a comprehensive
approach towards developing sugarcane cultivars resistant to orange rust that
is being adopted in the Canal Point breeding program will be presented. This
involves identifying and discarding susceptible sugarcane clones as early in
the breeding program as possible, the development of novel screening
methods and the identification of sources of resistance for breeding.
‘Candidatus Liberibacter solanacearum’ on tomato and potential losses in
field production
R. D. FRENCH-MONAR (3), R. W. Wallace (2), J. A. Abad (1), T. A.
Wheeler (4)
(1) APHIS-PPQ-PGQP, Beltsville, MD, USA; (2) Dept. of Horticultural
Sciences, AgriLife Extension-Texas A&M System, Lubbock, TX, USA; (3)
Dept. of Plant Pathology and Microbiology, AgriLife Extension-Texas A&M
System, Amarillo, TX, USA; (4) Dept. of Plant Pathology and Microbiology,
AgriLife Research-Texas A&M System, Lubbock, TX, USA
Phytopathology 100:S173
In August 2008, tomato (Solanum lycopersicum) plots in Lubbock County,
TX that were utilized for a chemical test aimed at management of root knot
nematode became infected with ‘Candidatus Liberibacter solanacearum’.
Overall symptoms on tomato cv. Spitfire included leaf yellowing, lateral stem
dieback, upward leaf curling, enlargement of stems, adventitious roots, and
swollen nodes. PCR amplification was done using 16S rDNA OA2 and OI2c
primers for ‘Ca. L. solanacearum’ used for potato, tomato, and other
solanaceous crops in New Zealand, which amplifies a 1.1 kb fragment of the
16S rRNA gene of this new species. A 1.1 kb fragment was obtained,
sequenced, and found to be 99.9% identical in sequence to a ‘Ca. L.
solanacearum’ obtained last year from a potato production field in Texas. In
the tomato field, a total of 32 plots (one-row wide, 7.7 m long) comprised of
24 plants per plot were evaluated for disease symptoms and galling by rootknot nematode. Foliar disease incidence in plots ranged from one (4.2%) to 19
(79.2%) plants showing symptoms by the last harvest date. Regression
analysis was used to determine losses in yield associated with the bacterium
and with root-knot nematode. Percent galling by root-knot nematode only
explained 14% of the variability in yield, and 100% galling was predicted to
cause a 1.5% loss in yield, based on the regression model. In contrast, for each
1% incidence in plants with disease symptoms, there was a 0.9% loss in yield.
In essence, there was no yield contribution if a plant developed symptoms (R2
= 0.41). The potential exists for ‘Ca. L. solanacearum’ to be a detriment in
tomato production and a source for survival of this bacterium that has been
found to be associated with the Zebra Chip disease in potato, tomato, pepper
(Capsicum annuum), and other solanaceous crops.
Diverse tomato cropping systems affect arbuscular mycorrhizal fungal
community diversity and structure
E. G. JOHNSON (3), D. O. Chellemi (2), T. Wu (1), J. H. Graham (3)
(1) Division of Cell Biology, Microbiology, and Molecular Biology,
University of South Florida, Tampa, FL, USA; (2) USDA-ARS, US
Horticulture Research Laboratory, Fort Pierce, FL, USA; (3) University of
Florida, Citrus Research and Education Center, Lake Alfred, FL, USA
Phytopathology 100:S173
In conventional agricultural systems, AMF are greatly affected by factors
including soil disruption, intermittent lack of host root tissue, and plant
inhibition of AMF colonization due to high soil fertilization. To determine the
effect of diverse agricultural land management and crop production practices
on the AMF community structure and diversity, five tomato crop production
systems consisting of bahiagrass pasture cover, conventional, continuous
removal of vegetation (disk fallow), organic, and undisturbed (weed fallow)
were initiated. The plots were adjusted to the new management regime, except
for conventional, for three or four years followed by one or two years of
tomato cropping. Soil DNA samples were taken in the off season, at planting,
and after harvest. Phylogenetic analysis of AMF 18S rDNA sequence
combined with multivariate statistical analysis using PRIMER-E was used to
compare community structure and diversity. Initial analysis shows that
Vol. 100, No. 6 (Supplement), 2010
S173
bahiagrass, weed fallow, and organic land management practices support
different, diverse AMF communities, while disk fallow and conventional
practices greatly reduced detection of AMF sequences. Tomato cropping
caused the emergence of common sequences for the Glomus mosseae group,
in all cropping systems. Bahiagrass and weed fallow diversity were unaffected
by the emergence of the G. mosseae group, while organic, conventional and
disk fallow all converge on a low diversity community dominated by the G.
mosseae group. Current analyses will determine if the shift in AMF
community caused by tomato cropping in organic and bahiagrass plots is
seasonal or persistent, and if other factors such as soil fertility and disease
incidence correlate to these community changes.
Identification and characterization of powdery mildew caused by
Golovinomyces cichoracearum on sunn hemp
S. A. JORDAN (1), G. S. Maia (1), A. J. Gevens (1)
(1) University of Florida, Gainesville, FL, USA
Phytopathology 100:S174
Crotalaria juncea, or sunn hemp, is a warm season legume grown in FL as a
cover crop. In 2008, powdery mildew was observed on sunn hemp in a
research field in Hastings, FL. This disease is important because it has the
potential to impact the quality of sunn hemp and this powdery mildew can
infect cucurbits which are grown in north FL in late summer. Fungal growth
appeared first on lower, more mature leaves as white, powdery mildew
colonies initially seen on upper leaf surfaces and later moving to undersides;
petioles and floral parts were disease-free. As disease progressed, colonies
enlarged, coalesced, and covered entire leaf surfaces; heavily infected leaves
senesced and abscised. Mycelia produced white accumulations of
conidiophores and conidia. Hyphae were superficial with papillate appressoria
and produced conidiopohores with cylindrical foot cells that measured 48.5 ×
10.0 µm. Conidia were hyaline, short-cylindrical-ovoid, lacked fibrosin
bodies, borne in short chains, had sinuate edge lines with other immature
conidia, and measured 22.5-40.0 × 12.5-20.0 µm. The teleomorph was not
observed. The nuclear rDNA internal transcribed spacer (ITS) regions were
amplified by PCR and sequenced. On the basis of morphological
characteristics of the asexual state and ITS sequence data, the pathogen was
identified as G. cichoracearum. Pathogenicity was confirmed on healthy
plants. This is the first report of G. cichoracearum causing powdery mildew
on C. juncea.
Phytophthora cactorum a serious problem on prefinished Cattleya orchid
liners from Thailand
R. T. MCMILLAN (1), A. J. Palmateer (2), R. A. Cating (2)
(1) Kerrys, Homestead, FL; (2) University of Florida, Homestead, FL
Phytopathology 100:S174
The major pathogen on Cattleya orchids in Florida and in the New and Old
World countries is Phytophthora cactorum (Lebert & Cohn) J. Schroet.,
which causes Black Rot during the wet months of the year. All species of
Cattleya and their interspecific and intergeneric hybrids are susceptible.
Phytophthora cactorum infects leaves, pseudobulbs, rhizomes, and flower
buds. Shipments of prefinished Cattleya orchid liners from Thailand during
the monsoon season often infected with P. cactorum. Orchid plants with
visual symptoms of P. cactorum were removed from the shipment and
drenched with fungicides such as Banrot, Natriphene, Shield Brite, Truban,
and Phyton 27, Heritage, Shield Brite, Stature, Truban, Pentathlon, Aliette,
Subdue Maxx, and Insignia, in an effort to salvage some of the plants. In spite
of the effort to save the orchids, the level of Phytophthora over rode the
attempt to control the fungus resulting in destroying the shipments. Cattleya
liner shipments during the dry season are found to be P. cactorum free.
Controlling angular leaf spot in Florida annual strawberry
J. C. MERTELY (1), N. A. Peres (1)
(1) University of Florida-GCREC, Wimauma, FL, USA
Phytopathology 100:S174
Angular leaf spot (ALS) is a bacterial disease caused by Xanthomonas
fragariae that produces unsightly lesions on strawberry leaves and sepals.
Leaves with numerous spots and/or vein-following lesions become blighted
and die prematurely. During the 2008–2009 growing season, an epidemic of
ALS occurred in the principal Florida strawberry production area west of
Tampa. During the season, a replicated trial evaluating products for ALS
control was conducted at the Gulf Coast Research and Education Center in
Wimauma, FL. Treatments included the plant defense activator acibenzolar-smethyl (Actigard); copper fungicides Badge, Cuprofix, IRF070, Kentan,
Kocide, and Quint; hydrogen peroxide (Oxidate); and Streptomyces lydicus
(Actinovate) applied weekly throughout the season to the foliage with a CO2
back pack sprayer. A late-season evaluation of foliar symptoms showed that
Badge, IRF070, Kentan, Kocide, and Actigard significantly reduced the
proportion of leaves killed and partially blighted by X. fragariae. Alternating
applications of Actinovate and Cuprofix also showed this effect. However,
S174
PHYTOPATHOLOGY
only Badge and the low rate of Actigard significantly increased marketable
yield during a season with markedly high disease pressure. Future
experiments may target applications to periods favorable to infection and
disease spread, such as rain events associated with approaching cold fronts,
and periods of prolonged overhead irrigation for freeze protection.
Cultivar susceptibility, temperature and leaf wetness durations required
for lesion production by Alternaria alternata on tangerine and tangerine
hybrids
S. N. Mondal (1), L. Timmer (1), M. M. DEWDNEY (1)
(1) University of Florida
Phytopathology 100:S174
Alternaria brown spot, caused by A. alternata, is an important disease of
tangerines and their hybrids in many citrus-producing regions. A prediction
model for fungicide applications, the Alter-Rater, was developed previously,
but it was unknown whether the relationship between temperature and leaf
wetness duration (LWD) would be consistent across all tangerine hybrids. We
tested the LWD and temperature relationships on 5 tangerine and tangerine
hybrids: Dancy, Minneola, Murcott, Nova and Sunburst. The LWDs were 2,
4, 8, 16, 24 and 30h at temperatures of 20, 24, 28 and 32°C. The rating scale
for the number of lesions/leaf was: 0 = 0; 1 = 1-2; 2 = 3-5; 3 = 6-10; 4 = 1115; 5 => 15 lesions/leaf and the data were taken from 15 leaves. The
experiment was an incomplete block design. Cultivar differences were
observed; lesions formed on Minneola and Dancy with as little as 2h of leaf
wetness at all temperatures. Lesions were observed on Murcott, Nova and
Sunburst with 4h of leaf wetness. The optimal temperature range for lesion
production was 24 and 28°C for all LWDs. On the more susceptible Minneola
and Dancy, 24h LWD was required to reach the max. lesion rating, but that
level was never reached on Murcott, Nova and Sunburst even with 30h of leaf
wetness. The results should be incorporated into the Alter-Rater model so that
unnecessary sprays are not applied to less susceptible tangerine and tangerine
hybrids.
Identification of the Florida torreya canker pathogen
L. L. MOUNT (1), J. A. Smith (1)
(1) University of Florida
Phytopathology 100:S174
The Florida torreya (Torreya taxifolia Arn.) is a conifer of the Taxaceae. The
native population is restricted to within 20 miles of the Apalachicola River in
northern Florida and southern Georgia. Torreya wood is resistant to
decomposition. For this reason, it was lumbered for railroad ties in the 19th
century. Surveys conducted prior to 1970 detail a dramatic reduction of
population numbers, size and health. Historically, trees reached 18 m at
maturity. Of the 58 trees surveyed in November 2008, no individual surpassed
a height of one meter. Disease symptoms associated with this decline are leaf
spot, shoot tip dieback, and cankers. Fungal cultures were isolated from
symptomatic tissue and from the initial 58 trees tested, cultures of Fusarium
spp. and Botryosphaeria spp. were each isolated 20 times. The same tree
sample often produced both genera. Products were amplified by polymerase
chain reaction using internal transcribed spacer region rDNA (ITS-rDNA)
specific primers and the sequences obtained were compared to those deposited
in the GenBank database. Four unique sequences of Fusarium spp. and
Botryosphaeria obtusa were identified. Fusarium solani and F. lateritium
matched GenBank sequences to the species level. Fusarium lateritium was
previously identified as the causal agent of the leaf spot, but was cultured
during this study directly from cankered tissue. Two species of Phomopsis, as
well as Diaporthe, Lasiodiplodia, and Hypoxylon were also infrequently
isolated and identified in the same manner from dying shoots and cankers
sampled in November 2008 and January 2009. Inoculations of as many of
these genera as possible were conducted on torreyas grown using sterile tissue
culture at Atlanta Botanical Gardens in April 2009.
The impact of silicon soil amendments on cucumber anthracnose in the
greenhouse
J. PALENCHAR (1), S. Taber (1), L. E. Datnoff (2), and A. J. Gevens (1)
(1) Department of Plant Pathology, University of Florida, Institute of Food
and Agricultural Sciences, Gainesville, FL; (2) Current address: Dept. of Plant
Pathology and Crop Physiology, Louisiana State University
Phytopathology 100:S174
Cucumber is an economically important crop in Florida and in other parts of
the U.S. In Florida, one of the most common diseases on cucumber is
anthracnose, caused by the ascomycetous fungus Colletotrichum orbiculare.
Anthracnose can cause serious yield and quality losses, and produces
symptoms on all aboveground plant parts at any stage of growth. Organic
producers have limited options for anthracnose control. The use of silicon (Si)
as a tool for disease control has been established in other crop systems. In the
greenhouse, the control of anthracnose was demonstrated on 2-week-old
‘Straight Eight’ cucumber seedlings by amending soil (organic Fafard FOF
30) with a high rate of Si (Vansil W50). Treatments included cucumber seeds
planted into 1) Si-amended soil (600 kg Si/ha) + no C. orbiculare inoculation
2) Si-amended soil (600 kg Si/ha) with C. orbiculare inoculation, 3) nonamended soil with no C. orbiculare inoculation, and 4) non-amended soil with
C. orbiculare inoculation. Each treatment included 5 replications and the
experiment was repeated 4 times. Disease evaluations (Horsfall-Barrett scale)
were recorded for leaves at 3, 7, and 14 days post inoculation (dpi) and Si
levels in plant tissues were determined. Significant differences between
treatments were observed at 7 and 14 dpi. Si treatment reduced disease
severity on leaves by 20–60% when compared to the inoculated control. This
is the first study demonstrating the efficacy of soil-applied Si for the control
of cucumber anthracnose.
Diagnostics and emerging plant pathogens
M. E. PALM-HERNANDEZ (1)
(1) USDA/APHIS/PPQ, Molecular Diagnostic Laboratory, Beltsville, MD,
USA
Phytopathology 100:S175
Accurate identification of plant pathogens is essential for making management
decisions and determining appropriate regulatory actions. An accurate
identification is facilitated when a group is well understood taxonomically
based on robust systematics studies. Such studies provide information on
which morphological and molecular characters are taxonomically useful and
tools can then be developed to use in detection and identification.
Identification of emerging plant pathogens poses a particular challenge in that
they are often understudied and poorly characterized. Phytophthora ramorum,
the causal agent of sudden oak death and ramorum blight, exemplifies how
regulatory diagnostics evolve as scientific knowledge about the disease and its
causal pathogen is gathered and evaluated. The discovery of new and related
taxa, including P. foliorum that cross-reacted in the P. ramorum nested assay,
led to the development of additional assays. The use of new markers to
compare large numbers of P. ramorum isolates has provided a clearer picture
of the genetic diversity in the pathogen and a means of tracing origins of
newly found isolates. This case study is one of many examples of the
importance of a strong understanding of the systematics of a group as the
basis for accurate identification and the development of diagnostic tools.
Basil downy mildew in Florida: A disease of new importance
R. N. RAID (1), P. D. Roberts (4), P. F. Harmon (2), A. J. Palmateer (3), S. A.
Jordan (2)
(1) University of Florida, Belle Glade, FL, USA; (2) University of Florida,
Gainesville, FL, USA; (3) University of Florida, Homestead, FL, USA; (4)
University of Florida, Immokalee, FL, USA
Phytopathology 100:S175
Sweet basil (Ocimum basilicum L.) is one of the most important herbs
currently grown in Florida, with both commercial field and greenhouse
production. In addition, it is one of the most commonly propagated herbs in
home gardens. Fortunately, it has had very few foliar disease problems and
has, for that reason, required little or no disease management. During fall
2007, a new disease was first reported on field-grown basil in south Florida.
Symptoms initially appeared as a yellowing of the lower canopy, with
chlorotic areas frequently delineated by leaf veins. Gray, fuzzy fungal growth
was apparent on the abaxial leaf surface. The disease was subsequently
reported to be incited by a species of Peronospora. Yield losses during this
initial outbreak were near total, since preventative control measures were
formerly unnecessary, and therefore, non-existent. Since the initial outbreak,
basil downy mildew has become firmly established in Florida. It has been
observed from all regions within the state, as well as in numerous other states.
Although there is ample evidence that the disease may have been introduced
on infested seed, alternative sources (i.e. from closely related hosts) have not
been totally ruled out. With widely-scattered, year-round greenhouse and/or
field production providing a host continuum, it is very likely that basil downy
mildew will be a disease to contend with on a permanent basis. Nearly all
basil varieties or types appear susceptible at this time. Management programs
are currently under development. Aside from cultural practices to limit leaf
wetness and hence fungal infection, preventive foliar applications of
phosphonates and strobilurin fungicides have proven useful. Used together in
a program, these have provided economic but not total control.
Orange rust of sugarcane: Prospects for fungicidal control
R. N. RAID (2), J. C. Comstock (1), N. C. Glynn (1)
(1) USDA Sugar Cane Field Station, Canal Point, FL, USA; (2) University of
Florida, Belle Glade, FL, USA
Phytopathology 100:S175
Orange rust of sugarcane, incited by Puccinia kuehnii, was first observed in
Florida during June 2007 on one of the industry’s most important commercial
cultivars, CP80-1743. This was the first report of this disease in the Western
Hemisphere. It has since been reported in several other Central American and
Caribbean Countries. With host-plant resistance being a worthy long-term
goal, studies were initiated to investigate the feasibility of fungicides serving
as an interim or supplementary management strategy. Thirteen different
fungicide treatments were examined for their efficacy in controlling orange
rust during the 2008/2009 growing season. Experimental units consisted of
two rows of cane 15m in length replicated four times in a randomized
complete block design. Fungicide treatments consisted of select candidates
from two major classes of fungicides, the strobilurins (FRAC group 11) and
triazoles (FRAC group 3), alone, and in combination or alternation. Fungicide
applications were made using a CO2 backpack sprayer and were initiated
following canopy closure (approx. 1.5-m ht) at 21 day intervals. Rust severity
in the trial area was moderately severe, with severities in excess of 30% on the
distal third of the fourth leaf beneath the top-visible-dewlap leaf in the
untreated check. Results indicate that the strobilurin fungicides provided the
highest level of control, followed by strobilurin/triazole combinations, and
finally, the triazole fungicides alone. In separate trials using the strobilurin
fungicide pyraclostrobin, fungicide treatments were demonstrated capable of
reducing orange rust to levels sufficient to significantly reduce yield losses by
as much as 40%. While economic factors will ultimately be an important
consideration, levels of orange rust control obtained in these studies show
promise regarding prospects for fungicides as a potential management tool.
Fungicidal control of basil downy mildew
R. N. RAID (1)
(1) University of Florida, Belle Glade, FL, USA
Phytopathology 100:S175
Sweet basil (Ocimum basilicum L.) is one of the most common herbs grown
by home gardeners in Florida. Commercially, basil ranks as Florida’s most
important potted herb and the state ranks second nationally in field
production, shipping to the entire eastern seaboard. Since 2007, basil downy
mildew, incited by a species of Peronospora, has caused considerable losses
for commercial basil growers in the U.S. In the absence of control, total crop
failure is common. Two fungicide field trials were conducted to determine the
efficacy of various foliar applications for the control of this disease. The tests
included both registered and non-registered compounds. Experimental units
consisted of four rows 2m in length separated on the ends by alleyways and
replicated four times in a randomized complete block design. All experimental
compounds were topically applied on approximately a weekly basis using a
CO2 backpack sprayer equipped with 3 flat-fan nozzles mounted on a handheld boom. Treatment commenced at the 4-6 leaf stage, with mildew present
in the area at time of initial application. Disease severity was considered
extreme. Products tested in these trials included: acibenzolar, azoxystrobin,
Bacillus subtilis, chlorothalonil, copper hydroxide, cyazofamid,
cymoxanil/famoxadone, dimethomorph, fenamidone, mandipropamid,
mefenoxam, potassium phosphanates, propamocarb, pyraclostrobin/boscalid,
and Streptomyces lydicus. All products provided for significant suppression of
downy mildew early in the trials, but only a few provided for significant
control by the end of the tests. Dimethomorph, mandipropamid, cyazofamid,
fenamidone, and mefenoxam provided the highest levels of control, but are
not currently labeled for use on basil. Of currently registered products, only
azoxystrobin and the potassium phosphonates provided control levels that
could be considered acceptable from a marketing perspective. No compounds
licensed for use in organic herb production provided acceptable levels of
mildew control when sprayed on a weekly basis in these trials.
Current distribution of Texas Phoenix palm decline in Florida
D. A. RESTOM GASKILL (2), N. A. Harrison (3), M. L. Elliot (3), T. R.
Smith (1)
(1) Florida Dept. of Agriculture and Consumer Services, Gainesville, FL; (2)
USDA-APHIS-PPQ, Sarasota, FL; (3) University of Florida IFAS, Fort
Lauderdale, FL
Phytopathology 100:S175
Texas Phoenix palm decline (TPPD) is a fatal disease of date (Phoenix
dactylifera, P. sylvestris, P. canariensis, P. reclinata), queen (Syagrus
romanzoffiana) and cabbage (Sabal palmetto) palms caused by a ‘Candidatus
Phytoplasma palmae’-related strain belonging to subgroup 16SrIV-D. In
addition to the economic costs of disease management in nurseries and
landscapes, the potential ecological impact due to reduction in S. palmetto
populations is incalculable. TPPD was first reported and characterized in 2002
from samples obtained in Corpus Christi, Texas, and was detected in the
Tampa Bay area of Florida in 2006. Surveys were conducted by the
Cooperative Agricultural Pest Survey (CAPS) in 2008 and 2009 to determine
the current distribution in Florida. Palms displaying characteristic symptoms
of TPPD were sampled and analyzed by polymerase chain reaction assay.
Phytoplasma positive samples from new locations were sequenced. Samples
were also submitted by personnel from the University of Florida, Institute of
Food and Agricultural Sciences (UF-IFAS) and from private landscape
companies. TPPD was determined to occur at a high incidence in a three
Vol. 100, No. 6 (Supplement), 2010
S175
county area (Hillsborough, Manatee, Sarasota), while it was found to be of
limited distribution in seven additional counties (Pinellas, Polk, Hardee,
Desoto, Highlands, Lake, Duval). Spread of TPPD is likely occurring locally
through an unknown insect vector and over long distances through the
transportation of infected palms.
Biosafety regulation and biotechnology: How it affects public research in
Latin America and the Caribbean
M. M. ROCA (1)
(1) Zamorano University, Tegucigalpa, Honduras
Phytopathology 100:S176
While poverty in developing countries is usually linked with low agricultural
output, pest and plant diseases are major factors that contribute significantly to
this low productivity. Genetic engineering and transgenic’s have great
potential to improve crop production. However, the application and use of this
biotechnology has not materialized for the public sector because of the politics
associated with the regulatory process. As a result, the time, effort and
expense required for commercialization of transgenic crops are way beyond
what public-sector investigators can muster leaving only the private sector to
accomplish this task. Restrictive regulations were established when the
commercial use of transgenic crops was just beginning, and have not taken
into account the more than 12 years of extensive experience gained on crops
tested on more than 100 million hectares in 23 countries. This information has
scientifically demonstrated that crops obtained through biotechnologies do not
have risk profiles that are any different from those developed through more
traditional plant breeding methods. The potential health and environmental
risks originally foreseen have not materialized. Furthermore, it has been
demonstrated that this biotechnology provides environmental and economic
benefits. As it now stands, the current biosafety regulatory standards in place
continue to delay the development and use of transgenic technology. The time
has come where there is a great need to consider both the benefits and the
risks of this technology, and analyze them relative to those of the present
agricultural production systems especially in Latin America and the
Caribbean.
Low molecular variability of Potato yellow vein virus (PYVV) isolates of
Solanum phureja and Solanum tuberosum from Colombia
P. Rodriguez-Burgos (2), G. Chaves (2), L. Franco-Lara (1), M. GUZMANBARNEY (2)
(1) Militar University Nueva Granada, Bogotá-Colombia; (2) National
University of Colombia, Bogotá-Colombia
Phytopathology 100:S176
PYVV Closteroviridae-Crinivirus, is a quarentine, phloem-limited potato
virus, with a tripartite ssRNA(+). It causes yellowing of foliage with reduction
of yield. It is found in Colombia, Peru, Venezuela and Ecuador and is
transmitted by white fly Trialeurodes vaporariorum and tubers. To study
variability, CPg of 75 isolates of PYVV from Solanum phureja and 50 from S.
tuberosum, from 5 Colombian regions, was amplified by RT-PCR. Amplicons
were analyzed by SSCP and 32 were sequenced directly. Ten SSCP patterns
were observed (P1 to P10); P1 represented 78% of the isolates, P9 9.6% and
P6 4.8%. Phylogenetic analysis of 70% of CPg produced two groups: Group I
(29 isolates) and Group II (3 isolates). In group I, isolates 1084 of S. phureja
and 1114 of S. tuberosum, showed evidence of possible recombination within
CPg. No direct correspondence between the number of SSCP patterns and the
sequence clusters was found, but P1 was present in Group 1 and P6 was
found in all 3 isolates of Group II. The aa relationship dN/dS = 0.214
indicated negative selection, suggesting that PYVV has a tendency for low
mutation fixation. This might be related to a selection pressure coming
from the insect vector. According to variability of the CPg there are at least 3
virus variants circulating in the Country, although variability among them is
low.
Asparagus as host of Phythophtora species prevalent in Michigan and its
importance as rotational crop
L. RODRIGUEZ-SALAMANCA (1), M. Hausbeck (1)
(1) Michigan State University, East Lansing, MI, USA
Phytopathology 100:S176
Michigan ranks third in U.S. asparagus production, after Washington and
California, with 11.200 acres that produced 12 million kg of asparagus spears
in 2007. Phytophthora spear and crown rot has been recently identified in
Michigan fields as a major limiting disease of asparagus. Although different
species of Phytophthora have been reported in other production areas as
causing disease in asparagus, only one species has been found in Michigan
and has identified as P. asparagi. Phytophthora sp. isolated from vegetables
and ornamentals in Michigan were tested for their ability to infect asparagus
spears and cause lesions. A series of growth chambers studies were conducted
to determine; (i) an optimum inoculation point for detached asparagus spears
using three P. asparagi isolates when incubated at 15, 20 and 25°C, (ii) the
S176
PHYTOPATHOLOGY
ability of different Phythophthora sp. present in Michigan agriculture to infect
asparagus spears. All the studies were conducted three times. When detached
spears were wounded and inoculated at 2, 9 or 16 cm from asparagus tip and
incubated at 20°C, all the inoculation points developed similar-sized lesions.
However, when inoculated spears were incubated at either 15 or 25°C, the
size of the resulting lesion differed significantly among the inoculation points.
Among the select Phythophthora sp. used to inoculate asparagus spears, P.
capsici was able to cause small lesions. Further studies that investigate
pathogenicity of commonly encountered Phythophthora sp. is important in
determining appropriate crop rotation strategies.
Fungal diversity associated with rambutan (Nephelium lappaceum L.) in
Puerto Rico
L. M. SERRATO (2), L. I. Rivera (2), R. J. Goenaga (1)
(1) USDA TARS, Mayaguez, Puerto Rico; (2) University of Puerto RicoMayaguez Campus, Mayaguez, Puerto Rico
Phytopathology 100:S176
Rambutan (Nephelium lappaceum L.) is an exotic tropical fruit of increasing
importance in international markets, and that has awakened great interest from
farmers in Puerto Rico. During 2008 and 2009, fruit rot and lesions on leaves,
branches, and flowers were observed in rambutan orchards through the island.
To examine fungal diversity associated with rambutan, samples from different
organs were collected in symptomatic and asymptomatic trees. Plant tissue
was superficially disinfected and transferred to acidified potato dextrose agar
to promote the development of fungi. A total of 311 fungal isolates were
obtained, which include 19 genera. Based on morphology, the following
species have been identified: Beltrania rhombica, Botryodiplodia theobromae,
Botryosphaeria spp., Colletotrichum gloeosporioides, Colletotrichum spp.,
Curvularia spp., Cylindrocladium spp., Dolabra nepheliae, Fusarium spp.,
Gliocephalotrichum bulbilium, Lasmenia spp., Phomopsis spp., and Septoria
spp. Pathogenicity tests are in progress under laboratory and greenhouse
conditions, using seedlings and detached fruit. PCR amplification of the
rDNA ITS region and the beta-tubulin gene will complement morphological
identification of fungi.
Use of bio-enhanced organic mulches for integrated management of
nutsedge in tomato
Y. SHABANA (2), E. Rosskopf (5), R. Charudattan (2), A. Abou Tabl (1), W.
Klassen (3), J. Morales-Payan (4)
(1) Mansoura University, El-Mansoura, Egypt; (2) University of Florida,
Gainesville, FL, USA; (3) University of Florida, Homestead, FL; (4)
University of Puerto Rico-Mayaguez, Mayaguez, PR; (5) USDA, ARS,
USHRL, Fort Pierce, FL, USA
Phytopathology 100:S176
Purple and yellow nutsedges (Cyperus rotundus and C. esculentus,
respectively) are among the world’s most problematic weeds that impact
virtually every horticultural crop grown in Florida and the Caribbean. As an
alternative to conventional methods of control that includes the use of soil
fumigation with methyl bromide, we tested nine hay mulches (shoot straw of
bahiagrass, cogongrass, cowpea, millet, yellow nutsedge, sorghum
Sudangrass, sunnhemp, and rye) and three green mulches (shoot biomass of
cowpea, millet, and sorghum Sudangrass) as a means to suppress nutsedge
growth in a raised-bed tomato (cv. Tygress) field. In addition, two fungusinfested cogongrass hays (infested with the nutsedge pathogen Dactylaria
higginsii [Dh] or the saprophytic fungus Trichoderma sp. [Tri]), and two
plastic mulches (black and infra-red transmissible [IRT]) were tested. The
black plastic mulch and the Dh-infested cogongrass mulch consistently
reduced nutsedge emergence and growth more than the other organic mulches
and the IRT plastic mulch. Among the organic mulches, cogongrass infested
with Dh or Tri and cowpea, sunnhemp, Bahiagrass, and cogongrass provided
the highest levels of nutsedge suppression. No disease symptoms developed
on nutsedge plants when Dh- or Tri-infested cogongrass was used as the
mulch. Both plastic mulches (black and IRT) and Tri-infested cogongrass
enhanced tomato yield and the proportion of larger fruits. The highest yield of
extra large tomatoes per plant was obtained when these mulches were applied.
Laurel wilt of avocado: Management and mitigation research in Florida
J. A. SMITH (1), R. C. Ploetz (2), T. J. Dreaden (1)
(1) School of Forest Resources and Conservation, University of Florida,
Gainesville, FL, USA; (2) Tropical Research and Education Center,
University of Florida, Homestead, FL, USA
Phytopathology 100:S176
Laurel wilt, caused by the fungus Raffaelea lauricola and transmitted by the
exotic redbay ambrosia beetle, Xyleborus glabratus, threatens the U.S.
avocado industry with elimination if drastic measures are not taken in the near
future. Since its introduction in Georgia approximately 6 years ago, the
disease has spread on several hosts in the Lauraceae on the southeastern
coastal plain and now looms only 100 miles from commercial avocado groves
(7,500 acres worth $34 million/yr) in Miami-Dade County, FL. Within the last
2 years, door-yard avocadoes have been rapidly killed and serve as a source of
inoculum for the epidemic. Current research efforts include: examining extant
avocado germplasm for resistance; using a taxon-specific real-time PCR
technique to diagnose the pathogen and identify it in screening and
epidemiology studies; and fungicide efficacy trials. The results from these
studies will be presented and future work will be discussed. Laurel wilt
threatens avocado production worldwide. Thus, we will address its potential
impact and preventing its movement to new areas.
Combating the loss of red bay and other native species to laurel wilt
J. A. SMITH (3), M. A. Hughes (1), C. Anderson (3), R. C. Ploetz (4), A. E.
Mayfield (2)
(1) Department of Plant Pathology, University of Florida, Gainesville, FL,
USA; (2) Florida DACS Division of Forestry, Gainesville, FL, USA; (3)
School of Forest Resources and Conservation, University of Florida,
Gainesville, FL, USA; (4) Tropical Research and Education Center,
University of Florida, Gainesville, FL, USA
Phytopathology 100:S177
Laurel wilt, caused by Raffaelea lauricola, currently threatens all native and
some exotic species in the Lauraceae in the United States. Since 2003, the
disease has devastated native stands of redbay, Persea borbonia, and threatens
several other taxa in the family, including avocado. Two natives, pondspice
(Litsea aestivalis) and pondberry (Lindera melissifolia), which are on state
endangered and federal critically endangered lists, respectively, face
extinction. Despite sanitation and other efforts to slow the movement of laurel
wilt, it continues to move to new areas every year, largely due to the
efficiency of the disease vector, the exotic redbay ambrosia beetle (Xyleborus
glabratus). There are many gaps in our current state of knowledge about the
biology of the disease, and several studies are underway. Current research has
focused on: protecting existing trees via fungicides; identifying and utilizing
putative resistance in redbay and avocado; elucidating the disease’s
epidemiology and host range; and determining to what extent genetic and
pathogenic variation exist in the R. lauricola population. An update on the
results from these studies will be given and future research needs will be
discussed.
Effect of acibenzolar-S-methyl on bacterial leaf spot of shrub roses
caused by a Xanthomonas sp.
G. E. VALLAD (1)
(1) Gulf Coast REC, University of Florida, Wimauma, FL, USA
Phytopathology 100:S177
A bacterial leaf spot was recently identified on shrub rose varieties
‘Knockout’ and ‘Double Knockout’ caused by a Xanthomonas sp. and can be
problematic during vegetative propagation and nursery production.
Acibenzolar-S-methyl, the active ingredient of Actigard (Syngenta,
Greensboro, NC), is an elicitor of plant defenses that has demonstrated
efficacy in the control of several bacterial diseases of vegetable crops.
Greenhouse and nursery trials were established to test the effect of Actigard
on the severity of bacterial leaf spot on ‘Knockout’ and ‘Double knockout’
roses. While lower rates of 0.25 to 0.5 oz of Actigard per 100 gallons
were effective at reducing disease severity on potted roses and liners,
higher rates of 0.75 to 1.0 oz gave the best results. Multiple applications
of Actigard (1.0 oz/100 gal) prior to disease development improved
bacterial leaf spot control over single applications. Results demonstrate the
potential to use Actigard for disease management on ornamental and nursery
species.
Effect of acibenzolar-S-methyl on the management of early blight and
target spot of tomato
G. E. VALLAD (1)
(1) Gulf Coast REC, University of Florida, Wimauma, FL, USA
Phytopathology 100:S177
Acibenzolar-S-methyl, the active ingredient of Actigard (Syngenta,
Greensboro, NC), is an elicitor of plant defenses. While labeled for tomato,
usage is currently limited to the control of bacterial leaf spot (Xanthomonas
spp.) and bacterial speck (Pseudomonas syringae pv. tomato). In 2008, two
field trials assessed the performance of Actigard (8 weekly applications at
0.75 oz per acre) when integrated into a standard spray program that included
weekly applications of copper sulfate (2.1 lbs a.i. per acre) mixed with either
mancozeb (1.5 lbs a.i. per acre) or chlorothalonil (1.5 lbs a.i. per acre). The
addition of Actigard reduced the severity of early blight (Alternaria solani)
and target spot (Corynespora cassiicola) by 22 to 44% over the standard spray
program alone, and by 31 to 60% compared to the non-treated plots. In the
spring trial, plots treated with Actigard yielded 336 more cartons (25 lbs) of
marketable tomatoes per an acre than those receiving the standard alone, and
1,179 cartons more per an acre than the non-treated plots. No yield
improvement was observed in the fall trial, due to the development of disease
in the late season. Results demonstrate the benefit of including Actigard as
part of an overall spray program to manage common foliar diseases caused by
bacterial and fungal pathogens of tomato.
Salmonella outbreaks associated with vegetables: How high is the risk?
A. VAN BRUGGEN (1)
(1) Emerging Pathogens Institute and Plant Pathology Department, IFAS,
University of Florida, Gainesville, FL, USA
Phytopathology 100:S177
Gastro-enteritis outbreaks increased in the 1990s and have remained steady
since then. Most outbreaks have been associated with seafood, but most
individual cases with vegetables and fruits (38% of all cases). Salmonella
enterica is the most common pathogen involved in outbreaks associated with
vegetables. Several Salmonella outbreaks were traced back to contaminated
tomatoes. Salmonella enterica is very versatile: there are more than 2500
serovars, which occur in various environments, including many plant and
animal species. The main reservoirs are the intestines of birds, pigs, cattle,
wild mammals and reptiles, but they are also harbored by protozoa,
earthworms, nematodes and snails. They can multiply in the rhizosphere of
various plants and occur on plant surfaces as well as in the endosphere.
Because of the human as well as economic costs associated with Salmonella
outbreaks, it is important to study the risk of an outbreak to occur. However,
there are different kinds of risk: calculated probabilities as well as perceived
risks. These last risks are concerns voiced by consumers on a comparative
scale. Among various safety concerns, microbiological risks are ranked high,
due to some knowledge and experience and the feeling of not being able to
control exposure. Perceived risks do not necessarily coincide with calculated
probabilities, but may be more influential in terms of the response to an
outbreak. Quantitative microbial risk assessment consists of several steps:
hazard identification and characterization, exposure assessment and risk
characterization. In a project on risk assessment of enteric pathogens in
the vegetable production chain, we limited ourselves to exposure
assessment through lettuce contaminated from manure and soil. The
occurrence and survival of enteropathogens in cattle manure were primarily
determined by the feed given to the cattle: low-fiber feed resulted in more
shedding and longer survival in the low-fiber and low-pH manure. Other risk
factors were low numbers of nonpathogenic coliform bacteria and high
dissolved organic carbon contents. Constant temperatures and low oxygen
levels also contributed to long survival times in manure. Survival times in soil
were negatively correlated to microbial diversity and positively to dissolved
organic carbon contents. A probabilistic exposure model for E. coli O157:H7
resulted in a relatively low probability of about 1 contaminated head in 10,000
lettuce heads. The risk can best be reduced at the beginning of the production
chain, the cattle farm. There are several quantitative risk assessment models
for Salmonella, but most of them are for animal products, except for one
model for almond contamination and on for vegetable contamination from
irrigation water. No risk model was found for tomato production and
processing. In a tomato safety research workshop, research needs were
identified, but control at the source of the chain was not mentioned while this
was the crucial factor in our lettuce risk model. Risk models based on
calculated probabilities could be used to influence perceived risks by the
general public.
STAR-D: The NPDN accreditation program for diagnostic laboratories
A. B. VITORELI (1), C. L. Harmon (1)
(1) University of Florida, Gainesville, FL, USA
Phytopathology 100:S177
The Food and Agriculture Defense Initiative was established in 2002 to enable
the United States Department of Agriculture to develop a network linking
plant and animal disease diagnostic facilities across the USA. The National
Plant Diagnostic Network (NPDN) is the plant disease component of this
network. The mission of the NPDN requires quick and accurate diagnosis of
high consequence plant pathogens, weeds and insect pests that threaten
national security; communication of such information response authorities;
the ability to scale up and manage sample surge as needed; and diagnostic data security. To accomplish these objectives, the NPDN relies on
diagnostic data generated by laboratories in Land-Grant Universities, State
Departments of Agriculture, and USDA-APHIS. Traditionally, these
laboratories have provided diagnostic services at the State or regional levels at
a high level of competence. However, to accomplish the national objectives
listed above, a standardized approach to diagnosis is required, particularly if
the diagnosis has regulatory implications. The NPDN System for True,
Accurate, and Reliable Diagnostics (STAR-D) has been developed to enable
participating laboratories to meet standards of quality for laboratory
management, facilities, equipment, and trained personnel. The ISO 17025
Quality Standard can be adapted to testing done by NPDN diagnostic
laboratories by providing a basis for a quality system to meet the needs of the
NPDN STAR-D.
Vol. 100, No. 6 (Supplement), 2010
S177
Levels of P in Areca catechu leaves following phosphorous acid
application through adventitious roots
G. C. WALL (1)
(1) University of Guam, Mangilao, GU 96923, USA
Phytopathology 100:S178
Bud rot disease of betel nut (Areca catechu L) has been shown to be caused by
Phytophthora palmivora. Fosphite, or phosphorous acid, is recommended for
the control of Phytophthora, applied by injection to the trunk. After finding
evidence of damage associated with injection sites in the trunks of betel nut
trees, a decision was made to look for other ways of applying the fungicide in
order to avoid damaging the trees. A paired t test was devised to study the
effect of applying phosphorous acid solution via adventitious roots of betel
nut trees. A group of mature trees was sampled pre- and post-application.
Levels of P were determined from leaf samples collected from each frond per
tree. There were 12 pairs of trees in the study; one set of trees was treated with
the recommended rate (applied by absorption through an adventitious root)
and half were controls, treated only with sterile distilled water also via one
adventitious root. After the appropriate statistical analysis (NCSS, Kaysville,
UT), differences were found in the level of P in the leaf samples according to
treatment. Control trees had higher levels of P in their leaf tissue compared to
trees given phosphorous acid. The underlying hypothesis was that application
of the fungicide via adventitious roots of trees would result in a systemic
distribution of the fungicide throughout the tree. It was expected that all leaf
samples from treated trees would show higher levels of P compared to
untreated controls. Surprisingly, P levels were lower in treated trees, yet there
was no difference between fronds, suggesting an even effect throughout
treated trees. No explanation is known at this time for the reduced P levels
observed after treatment; however, results were consistent enough to yield
highly significant differences statistically.
Epidemiology of soybean rust (Phakopsora pachyrhizi) in soybean
(Glycine max) sentinel plots in Florida
H. M. YOUNG (2), J. J. Marois (2), D. L. Wright (2), D. F. Narvaez (1), G. K.
O’Brien (2)
(1) Monsanto, St. Louis, MO, USA; (2) University of Florida, NFREC,
Quincy, FL, USA
Phytopathology 100:S178
The overwintering of soybean rust (SBR) in the Southeastern United States
has been variable due to weather conditions which may influence disease
incidence and severity in the major soybean producing regions of the
Midwest, making it important to understand the epidemiology of the pathogen
in Florida. This study examined the incidence and severity of SBR in relation
to prevailing weather data, growth stage, and maturity group (MGIII, MGV,
MGVII) in 15 m square soybean plots across the Panhandle of Florida from
S178
PHYTOPATHOLOGY
2005 through 2008. Of the three maturity groups, the MGIII soybean became
infected first the least often. Plots became infected first at growth stage R4
(full pod) or later. On average, plots became infected 40 days earlier in 2008
than 2005. Precipitation was the principle factor affecting disease progress,
where disease increased rapidly after rain events and was suppressed during
dry periods. The area under the disease progress curves (AUDPC) for
incidence and severity was the lowest in 2007, most likely due to dry
conditions. In 2008, there was a significant increase in disease incidence and
severity as reflected in the AUDPC. This can be attributed in part to the
occurrence of Tropical Storm Fay, which deposited up to 290 mm of water in
the plot locations during the third week of August. Results from this study
may lead to a better understanding of the impact of weather on the
epidemiology of this pathogen.
Effect of rhizobacteria, acibenzolar and silicon on bacterial spot of tomato
S. ZHANG (1), T. L. White (1), W. Klassen (1), M. C. Martinez (1)
(1) Tropical REC, University of Florida, IFAS, Homestead, FL, USA
Phytopathology 100:S178
Bacterial spot, caused by Xanthomonas perforans, is one of the most
economically important diseases of tomato in Florida and other tomato grown
areas worldwide. Chemical controls have been only partially effective due to
the wet and warm climate in Florida and the development of resistance in
populations of this bacterial pathogen. It is imperative that practical
alternative strategies be developed to sustain the production of tomatoes.
Greenhouse and field trials have been conducted to investigate the effect of
plant growth-promoting rhizobacteria (PGPR), acibenzolar-S-methyl (ASM)
and silicon nutrient on bacterial spot of tomato. In the greenhouse, eight
bacilli PGPR strains were evaluated on two cultivars of tomato (FL47R and
Tygress). Tomato seeds were sown into pro-mix in 128-cell Styrofoam flats
and grown for 1–2 weeks when solutions of PGPR, Actigard 50WG (ASM)
and silicic acid were applied weekly as soil drenches. Tomato seedlings were
transplanted into 4-inch pots containing potting mix after 3–4 soil drenches,
and inoculated by foliar spray with suspensions of X. perforans at 1 × 108
CFU/ml. Results indicated that PGPR strain SE76 and INR7 significantly (P <
0.05) reduced disease severity of bacterial spot on both tomato cultivars
compared to the nontreated control. SE52 on cv. FL47R and SE34, IN937a
and IN937b on cv. Tygress each had a significant effect on disease reduction.
In the first field trial on tomato cv. FL47R, Actigard 50 WG at 30 mg/l
significantly suppressed bacterial spot rated at 8, 9, 10 and 11 weeks after
transplanting, whereas silicic acid at 0.15 and 1.5 mM did so only at 8 weeks.
In another field experiment, the same eight PGPR strains and Actigard 50WG
at 30 and 3 mg/l were tested on tomato cv. Tygress. Significant disease
reduction was observed on tomato plants treated with PGPR strains IN937a
and IN937b 3 weeks after transplanting.
2009 North Central Division Meeting Abstracts
Symposia Presentations
Abstracts submitted for presentation at the Symposia Presentations held at the 2009 North Central Division meeting in Ames, Iowa, June 21–23, 2009. The
abstracts are arranged in order of presentation.
Implications of Climate Change on Plant Pathogens
Climate impacts on agriculture: Implications for crop management
J. L. HATFIELD (1)
(1) National Soil Tilth Laboratory, Ames, IA, USA
Phytopathology 100:S179
Climate impacts on agriculture can be either direct or indirect in terms of
affecting production or quality of the product. The direct impacts result from
temperature, precipitation, or CO2 effects on crop growth and development
while the indirect impacts result from the climatic impacts on weed, insect, or
disease populations which in turn affect crop production. Climate has
changed, is changing, and will continue to change; however, the current state
of the climate relative to agriculture is one in which there is increasing
variability in temperature and precipitation and rising CO2 concentrations.
Increasing temperatures hasten plant development and can lead to plant stress
when there are limited soil water supplies. One aspect of rising CO2
concentrations is increased water use efficiency; however, this may not
overcome plant stress induced by lack of soil water recharge by variable
precipitation. Variations in climatic parameters will affect growth and
development of weeds which will make weed management an increasing
challenge. Changing temperature and precipitation patterns will affect the
overwintering of insects and diseases and increase the range of pests.
Development of management strategies to increase agricultural production
will have to consider all aspects of climate on the agricultural systems and
where the opportunities exist for improved efficiency in crop production and
potential for enhanced crop protection strategies.
Climate change and food security
L. ZISKA (1)
(1) USDA Crop Systems and Global Change Lab, Beltsville, MD, USA
Phytopathology 100:S179
Documented and projected changes in atmospheric carbon dioxide are likely
to alter agricultural productivity in two ways: directly, by supplying additional
carbon for photosynthesis and growth; and, indirectly, by altering climate,
specifically surface temperatures and precipitation. In this overview on the
impact of carbon dioxide and climate change on food security, I will present
data from a number of sources that document the likely changes in
temperature, temperature and carbon dioxide and water availability on crop
quality and production, and identify other biological interactions with pests,
Nematode Pests of the North Central Region
Insights into the mode of action of cyst nematode effector proteins
T. HEWEZI (1), T. J. Baum (1)
(1) Iowa State University, Department of Plant Pathology, Ames, IA, USA
Phytopathology 100:S179
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
weeds, and diseases. In addition, I will discuss possible opportunities, focusing
on exploitation of genetic and intra-specific variability within plant germplasm as a possible means to maintain agricultural production in the future.
Forecasting weather and climate for plant disease models: A western
perspective
L. COOP and the Western Weather Workgroup Integrated Plant Protection
Center (1)
(1) Oregon State University, Corvallis, OR, USA
Phytopathology 100:S179
Input requirements for many plant disease models currently challenge our
ability to incorporate short and long term climate change effects. In the Western
US, these challenges supplement ones incited by the mountain and coastal
influenced terrain. The Western Weather Workgroup has addressed some of
these needs via a series of meetings, grant projects, and on-farm field trials.
Some helpful technologies that address the problems of scale in disease
forecasting that we are using include PRISM climate mapping, mesoscale
weather forecasts, online error analysis, ingest of real time public and private
weather observations, and new methods for downscaling IPCC climate change
model projections. A proposed Swiss Needlecast model for PNW forests can
benefit from these climate change model projections, and so can degree-day
model forecasts by using modified climate “normals”. Many of the technologies presented have been incorporated into a nationally focused website, http://
uspest.org/wea, which addresses several IPM and plant biosecurity needs.
Modeling plant disease in a changing climate
J. RUSSO (1)
(1) ZedX, Inc., Bellefonte, PA, USA
Phytopathology 100:S179
Environment, along with host and pathogen, are the long-recognized key
components for disease development. A changing climate impacts the
environment component and, by their triangular relationship, host and
pathogen. The challenge to plant epidemiological modelers is to incorporate
the appropriate weather variables for quantifying the impact of a changing
climate on disease development. Furthermore, modelers must be sensitive to
the spatial and temporal scales represented by weather variable data and the
uncertainty associated with the data in predicted disease behavior. Lastly,
even after accounting for scale and uncertainty, the modeling of plant disease
in a changing climate must be evaluated in the context of an ecosystem.
Plant-parasitic cyst nematodes secrete proteinaceous effectors, which play the
central role in host infection and formation of their feeding sites. The majority
of these proteins are novel and their putative functions can not be assigned
due to the absence of significant sequence similarities to known proteins in
sequence databases. Elucidation of the mechanism of action of these effectors
is an absolute necessity for engineering resistance to these damaging plant
pests. Remarkable progress has been made recently in understanding the mode
of action of cyst nematode effectors. Two effectors targeting different
subcellular compartments have been functionally characterized. The first effector is a cellulose binding protein (CBP) that acts in cell wall modification.
Vol. 100, No. 6 (Supplement), 2010
S179
Transgenic plants expressing CBP revealed its vital role in mediating plant
susceptibility to cyst nematode infection. We identified pectin methylesterase
3 (PME3) as a strong and specific interactor of CBP. Our data indicate that
CBP interacts with PME3 thereby activating and potentially targeting this
enzyme to modulate the properties of the cell wall via modification of pectin,
and subsequently affecting plant growth and pathogen susceptibility. The
second effector is a cytoplasmic protein, which we term 10A06. 10A06 was
found to affect plant morphology and nematode susceptibility when expressed
in planta. 10A06 specifically interacts with a plant spermidine synthase
(SPDS). Our results collectively indicate that 10A06 functions in modulating
polyamine signaling to promote plant susceptibility.
Plant-parasitic nematodes in midwestern corn fields
T. A. JACKSON (1)
(1) University of Nebraska, Lincoln, NE, USA
Phytopathology 100:S180
Plant parasitic nematodes have historically been a costly challenge for corn
production in some areas, but some common cropping practices helped to
mitigate their impacts. During recent years, producers have utilized more
pyrethroid insecticides and transgenic insect resistant corn hybrids, neither of
which is as effective against nematodes as the organosphosphate and
carbamate soil insecticides used in the past. The recent increase in the
incidence of damage caused by nematodes, particularly in the Midwest has led
to questions about the prevalence of nematodes in corn fields and the potential
for further injury. In 2006, a preliminary survey, funded by Syngenta Seed
Care, of corn fields in Nebraska was initiated and expanded to include other
states in the corn belt during 2007. Three fields were arbitrarily selected to
represent each county with more than 20,000 acres of corn. Both soil and root
samples were collected from the fields and submitted to one of six
laboratories, five university and one private laboratory, for analysis. Results of
the analyses indicated that plant parasitic nematodes were present in
practically every field to varying degrees, with some population densities
Implications of Plant Diseases in Biofuel Production
Plant disease in Miscanthus and other cellulosic biomass crops
E. HEATON (1)
(1) Iowa State University, Dept. of Agronomy, Ames, IA, USA
Phytopathology 100:S180
Cellulosic biomass crops are slated for production on roughly 30 million acres
of U.S. farmland in the next 20 years but very little is known about the
implications plant disease might have for these crops. To date, the acreage of
dedicated energy crops is small, and disease issues have yet to cause concern.
What issues might we expect? What has been observed in small stands?
Reproductive growth is not important in cellulosic crops, therefore chief
concerns are expected to be foliar blights that reduce carbon assimilation, and
root and stalk rots that reduce harvestable yield. In the Midwest, sorghum
(Sorghum bicolor), switchgrass (Panicum virgatum) and Miscanthus
(Miscanthus × giganteus) are the leading herbaceous candidate biomass crops.
Sorghum is a familiar crop now being bred for dedicated biomass production.
Diseases currently problematic for forage sorghum including downy mildew,
(Peronosclerospora sorghi), Fusarium spp. and Anthracnose (Colletotricum
graminicola) will become increasingly important for biomass sorghum.
Switchgrass has been for studied as a biomass crop for decades, but still little
is known about its pests and pathogens. Principle diseases include rusts,
smuts, root rots and Panicum mosaic virus. Generally, diseases have not
caused major yield reductions and susceptibility varies among cultivars.
Miscanthus is still new to the US but has been studied and used in Europe for
nearly 20 years. A relative of sugarcane, M. × giganteus is sterile and
currently planted from rhizome pieces, leading to clonal fields with little or no
genetic variation over large areas. Even so, no economically important diseases have emerged in M. × giganteus. In Europe, Fusarium rots and Barley
yellow dwarf virus have been reported. A disease survey of M. × giganteus
recently conducted across the Midwest has observed 1 virus, 5 fungal diseases
and 10 different genera of plant parasitic nematodes present in fields.
Impact of diseases on biomass productivity in switchgrass
G. P. MUNKVOLD (1)
(1) Iowa State University, Dept. of Plant Pathology, Ames, IA, USA
Phytopathology 100:S180
Switchgrass (Panicum virgatum) is widely considered as a preferred feedstock
for lignocellulosic ethanol production, which could lead to a significant
increase in area planted to this crop. Current switchgrass production is
dominated by a few cultivars that have been developed for site adaptation but
not specifically for disease resistance. Increased density of switchgrass in the
S180
PHYTOPATHOLOGY
exceeding historic estimates of damage thresholds. Results also varied
markedly by state, indicating that the efficiency of the nematode extraction
procedures varied between laboratories. These results indicate that there is the
potential for yield loss in corn caused by nematodes warranting further research.
Pratylenchus penetrans is a common and persistent pathogen of potato in
the north central region
A. E. MACGUIDWIN (1)
(1) University of Wisconsin-Madison, Madison, WI, USA
Phytopathology 100:S180
Root lesion nematodes, Pratylenchus spp., are the most common nematode
problem in the North Central region. Pratylenchus penetrans is the species
most damaging to a wide range of crops including potato. Surveys showed 9
of 102 potato fields in Wisconsin surpassed the threshold for nematode
damage (200 per 100 cc soil). The same number of fields surpassed the
damage threshold for Verticillium dahliae (10 propagules per gram soil). The
majority of the remaining fields were infested with subthreshold densities of
both pathogens and at risk for the potato early dying disease (PED) caused by
the interaction of V. dahliae and P. penetrans. The potential for PED in the
surveyed fields was verified by a bioassay and corroborated our laboratory
research showing the interaction of these two pathogens. Historical data
shows that population densities of P. penetrans have increased in Wisconsin
over the last twenty years. One of the factors likely to have contributed to an
increase in P. penetrans is a change in crop rotations. Rotation crops vary in
their host status for reproduction by P. penetrans, but also important are root
system characteristics that impact the quantity and quality of dead root fragments
serving as reservoirs of nematode inoculum for the next crop. Our analysis of
many historical data sets showed that a significant proportion of a P.
penetrans population survives in detached root fragments when live hosts are
not available. Growers recognize the importance of P. penetrans to potato, but
are only beginning to appreciate the impact of P. penetrans on other crops and
the role that crop rotation plays in the root lesion nematode disease of potato.
landscape may exacerbate existing disease problems, which could present a
significant obstacle to biomass productivity. The diseases with the greatest
potential to suppress biomass yield are switchgrass smut, caused by Tilletia
maclaganii, and switchgrass rust, Puccinia emaculata. The smut disease
results in stunting, premature flowering, and replacement of seeds by fungal
sori; it has been reported from several states spanning from Kansas to New
York, and likely occurs elsewhere in the U.S. Studies in Iowa indicated that
the disease occurred in over 50% of the area planted to switchgrass, and that it
reaches high levels of incidence (up to 70%) in older stands. Smut incidence,
stand density, and yield were determined in 10 fields differing in disease
incidence (from 0.7 to 55.4%, mean 26%). Mean biomass/tiller was reduced
by 38 to 82% in diseased tillers compared to healthy tillers. Yield loss
estimates ranged from 1.7 to 40.1% among the fields. Disease incidence and
yield loss had a linear relationship (R2 = 0.95) and, based on regression
modeling, yield loss for all sampled fields was estimated at 17.0%.
Economically viable switchgrass production will require strategies to reduce
the impact of smut and rust. Cultivar development is the most promising
approach, and recent efforts include selection for P. emaculata resistance, but
resistance to T. maclaganii has not been identified consistently in any current
cultivars, and sources of resistance for use in breeding are not yet evident.
Concentration of Fusarium toxins in naturally contaminated corn and
corn processing co-products derived from ethanol production
A. W. Schaafsma (1), V. LIMAY-RIOS (1), and J. D. Miller (2)
(1) Ridgetown College, University of Guelph, Ridgetown, Ontario, Canada;
(2) Carleton University, Ottawa, Ontario, Canada
Phytopathology 100:S180
In north temperate areas such as Ontario, contamination by toxins from
Fusarium graminearum, particularly deoxynivalenol (DON) and zearalenone,
is common. Fumonisin contribution has been modest by comparison with
other corn-producing areas. In this study, three matrices [corn meal, distiller’s
dried grains with solubles (DDGS), and condensed distiller’s soluble (CDS)]
were sampled in sequence from a continuous dry milling processing plant for
the determination of mass balance of DON. LC-MS/MS was used as a
confirmatory method for determination of DON and other Fusarium toxins.
DON concentrations in the CDS and the final DDGS co-product were
significantly higher (P < 0.01) than in the starting material (corn grain). Toxin
concentration increased by a factor of 3 on a dry weight basis in DDGS
compared to the starting corn, and by 4 in CDS. Mean concentration of DON
in CDS was four times higher (7.1 mg kg–1) than in corn grains (1.8 mg kg–1)
and 1.4 times higher than in DDGS (5.24 mg kg–1). Mass balance calculations
show that CDS is the main source of contamination of DON comprising ca.
70% of the toxin found in the final product (DDGS). Most DON (87%) was
accounted for by this analysis. The presence of mycotoxins in DDGS and
CDS affects their utility as animal feed supplements. Our data indicate that
concentrations in the grain corn entering ethanol plants should be close to the
dietary values recommended for swine in Canada and the United States for
DON (1 mg kg–1). Aside from DON, small amounts of acetyldeoxynivalenol,
DON glucoside and zearalenone were found in corn, DDGS and CDS. Unlike
the situation for DON, the DON glucoside was not concentrated into DDGS
and CDS. This indicates that some DON glucoside may have been hydrolyzed
during the fermentation process.
Microbial characterization of distillers wet grains: Results and challenges
M. LEHMAN (1)
(1) USDA-ARS North Central Agronomy Research Laboratory, Brookings,
SD, USA
Phytopathology 100:S181
Distillers grains are co-produced with ethanol and carbon dioxide during the
production of fuel ethanol from the dry milling and fermentation of corn
grain, yet there is little basic microbiological information on these materials.
We have characterized the microbiology of distillers wet grains (DWG) over a
nine-day period following their production at an industrial fuel ethanol plant.
Potential Crop Biosecurity Risks that Threaten Agriculture in the North Central Region: Staying Ahead
of the Curve
Disease threats to natural and agricultural plant systems: Think locally,
act globally
J. STACK (1)
(1) Kansas State University, Manhattan, KS, USA
Phytopathology 100:S181
Plant health is the foundation for human health and wellbeing. Plant-based
agricultural systems are critical to the economies of many states in the
Midwest and Great Plains regions of the United States and the exports from
these regions contribute to global food security. Recurring and emerging
diseases pose direct and indirect threats to sustainability of plant, animal, and
human systems. Comprehensive plant biosecurity plans are necessary
prerequisites to sustainable plant health in the face of the long list of general
and specific threats that results from global trade, climate change, population
growth, and biocrime. A plant biosecurity strategy that minimizes the impacts
from plant diseases without compromising production efficiency and trade is
essential. Among the challenges to plant biosecurity are: 1) the ability to
accurately identify and prioritize pathogen threats and plant system vulnerabilities, 2) the ability to develop preparedness plans that are strong enough to
protect plant systems from identified threats while robust enough to protect
against unanticipated emerging disease threats, and 3) the development of
resilience in natural and agricultural plant systems. We need to think locally
(e.g., develop strong plant biosecurity plans and don’t import uninspected
plants or plant products) and act globally (e.g., support national and
international phytosanitary regulations and don’t export uninspected plants or
plant products). While the threats from bioterrorism are often overstated, the
threats from accidental introductions as a result of global trade in plants and
plant products are often understated. The large number of existing disease
threats to Midwest and Great Plains plant systems, the potential for newly
emerging yet unknown disease threats, and our poor ability to accurately
prioritize those threats requires a more general approach to plant biosecurity.
The uncertainty associated with the processes of threat identification,
vulnerability assessment, and impact prediction should be cause for concern.
Regional and national efforts to enhance detection and diagnostics
R. HAMMERSCHMIDT (1)
(1) Michigan State University, East Lansing, MI, USA
Phytopathology 100:S181
The threat of accidental and intentional introductions of new pathogens and
pests along with the potential for re-emergence of older disease/pest problems
illustrates the need for enhanced capacity diagnostics and detection. Two
programs, the USDA-CSREES sponsored National Plant Diagnostic Network
(NPDN) and ipmPIPE (Pest Information Platform for Extension Education),
which is sponsored by several USDA agencies and other private and public
groups, will be used to describe recent efforts to improve diagnostic and
detection capacity. Since its inception in 2002, the NPDN has, for example:
enhanced diagnostic capacity at land grant diagnostics labs; provided
diagnostic training for new disease problems; assisted in the development of
standard operating procedures for diagnostics; developed and deployed first
detector training programs; and conducted disease detection and diagnostic
This freshly-produced DWG had a pH of about 4.4, a moisture content of
about 53.5% (wet weight basis), and 4 × 105 total yeast cells/dry g, of which
about 0.1% were viable. Total bacteria cells were initially below detection
limits (ca. 106 cells/dry g) and then were estimated to be ~ 5 × 107 cells/dry g
during the first four days following production. Culturable aerobic heterotrophic organisms (fungi plus bacteria) ranged between 104 and 105 CFU/dry
g during the initial four day period and lactic-acid bacteria (LAB) increased
from 36 to 103 CFU/dry g over this same period. After nine days, total viable
bacteria and yeasts/molds topped 108 CFU/dry g and LAB approached 106
CFU/dry g. Community phospholipid fatty acid analysis (PLFA) yielded
limited data, but indicated a stable microbial community over the first four
days of storage. Thirteen morphologically-distinct isolates were recovered of
which ten were yeasts and molds from six different genera, two were strains
of the lactic acid-producing Pediococcus pentosaceus, and only one was an
aerobic heterotrophic bacteria, Micrococcus luteus. The microbiology of
DWG is fundamental to assessment of spoilage, deleterious effects (e.g.,
toxins), or beneficial effects (e.g., probiotics) in its use as feed or in alternative
applications. Significant challenges are encountered when applying cultureindependent analyses (DNA-based, PLFA, total protein, and direct observation techniques) to characterize the microbiology of wet distillers grains.
exercises. Through these efforts, there has been an improvement in our ability
to detect and diagnose as well as enhanced communication and cooperation
among the land grant university diagnosticians, state departments of
agriculture and USDA-APHIS. The ipmPIPE is a national warning system to
help growers protect their crops from the diseases and pests. The program was
initiated with funding from USDA and the soybean industry to assist in early
detection and diagnosis of Asian soybean rust. There are now four additional
ipmPIPE programs: soybean aphid, legumes, cucurbit downy mildew, and
pecan nut casebearer. In each of the ipmPIPE programs, field observations
and sampling are conducted by the land grant university in each state.
Samples are examined by university NPDN labs or state specialists, and the
results are entered into electronic databases. There is a public website that is
available for use by growers and others for obtaining current information on
disease/pest spread and management recommendations. For more information
visit: www.NPDN.org and www.ipmpipe.org.
The role of the seed industry in crop biosecurity
W. E. DOLEZAL (1)
(1) Pioneer Hi-Bred International, Inc., Johnston, IA, USA
Phytopathology 100:S181
The North Central Region of the United States is a rich agricultural production
region for several major commodities, including corn, soybean, wheat and
sunflower. It is also a major region for seed production, especially for corn
and soybean. The establishment and continued funding of the National Plant
Diagnostic Network, with its regional networks and the Soybean, Legume and
Soybean Aphid ipm-PIPE programs have greatly aided in the accurate
identification and monitoring of major economic pests which threaten crops in
the North Central Region. Efforts for building low cost, true partnerships with
seed industry personnel in existing federal and state pest monitoring
programs, plus a pilot public/private collaborative effort in monitoring
Puccinia polysora will be discussed.
Meeting the challenges of U.S. crop biosecurity: Pre- and post threat
introduction
F. W. NUTTER, JR. (1), N. Holah (1), N. Van Rij (2), D. Wright (3), J.
Marois (3)
(1) Iowa State University, Ames, IA, USA; (2) Cedara Department of
Agriculture, Pietermaritzburg, South Africa; (3) University of Florida,
Quincy, FL, USA
Phytopathology 100:S181
The global monitoring of exotic biotic plant pathogens, prior to their
introduction into the U.S. by natural, accidental, or deliberate means, remains
a key challenge in the effort to safeguard our nation’s agricultural biosecurity.
Remote sensing, GPS and GIS technologies are now being integrated and
utilized successfully to identify specific plant pathogens. This new paradigm
replaces less successful attempts to find and apply unique spectral signatures
for pathogen identification. Pathogen-specific temporal and spatial signatures
for Asian soybean rust and Cercospora leaf spot epidemics affecting soybean
crops grown in South Africa, Argentina, and the U.S. were extracted from
high resolution (<1.0 m2 per pixel) satellite images obtained by commercial
satellites. Such approaches offer the means to detect and correctly identify
biotic threats prior to (and after) introduction into the US, thereby serving as
both an early (pre-introduction) warning system and as a tool for postintroduction response.
Vol. 100, No. 6 (Supplement), 2010
S181
2009 North Central Division Meeting Abstracts
Abstracts presented at the APS North Central Division meeting in Ames, Iowa, June 21–23, 2009. The abstracts are arranged alphabetically, by first author’s
name.
Genetic diversity of Colletotrichum coccodes vegetative compatibility
groups using Fluorescent Amplified Fragment Length Polymorphism
markers
K. M. ALANANBEH (1), N. Gudmestad (1)
(1) North Dakota State University, Fargo, ND, USA
Phytopathology 100:S182
Colletotrichum coccodes (Wallr.) Hughes, is a cosmopolitan pathogen that has
wide distribution and host range. C. coccodes is an imperfect fungus and
vegetative compatibility serves as a means of genetic exchange and is useful
for measuring genotypic diversity. Seven vegetative compatibility groups
(VCG’s) have been identified for this fungus using nitrate nit mutants.
Vegetative incompatibility (vic) alleles present among continental populations
prevents anastomosis from occurring among these populations thereby
limiting VCG as a method to evaluate diversity of the global population. The
main objective of this study was to study the genetic diversity of the VCG’s
among the North American, European, and Middle Eastern isolates of C.
coccodes using Fluorescent Amplified Fragment Length Polymorphism
(AFLP) markers to obtain a better understanding of the genetic diversity of the
global population. A total of 526 isolates of C. coccodes were used in this
study, 311 were from North America (NA), 183 from Middle East (Israel),
and 32 isolates from Scotland. Three AFLP primer sets were used to generate
amplified fragments. The C. coccodes isolates were compared with 62 isolates
previously studied. All DNA fragments within the range of 100 to 620 bp
were scored manually for the three primer sets. The bands were scored for
presence or absence (1 = presence or 0 = absence). Binomial data was used to
create a similarity matrix using the WINDIST application of the WINBOOT
program and the DICE similarity coefficient. Analysis of the first primer set
showed that the NA–isolates were assigned to VCG’s 1, 2, 3, 4, 5, and 6. This
is consistent with previous findings. Israeli isolates were assigned to VCG’s 2
and 5, and Scottish isolates were assigned to VCG5. According to the banding
pattern on AFLP gels VCG2 and VCG5 had the highest frequency compared
to the other isolates in NA and Israeli isolates.
Temporal fluctuations in plant parasitic nematode population densities in
corn across various Nebraska cropping environments
J. L. BEHN (1), T. Jackson (1)
(1) University of Nebraska-Lincoln, Lincoln, NE, USA
Phytopathology 100:S182
Behavioral differences have been observed among genera of plant parasitic
nematodes with respect to movement within the soil profile. Nematode
migration through the soil complicates recommendations for sampling
strategies. It is not clear what environmental or biological conditions
determine why and which nematode genera migrate. Samples were collected
at monthly intervals, as weather permitted, from 8 locations with varying
irrigation practices, cropping history, and nematicide use. All four Fullerton,
NE sites showed ectoparasitic nematode genera population densities
(Xiphinema spp., Trichodorus spp., and Tylenchorynchus spp.) that increased
over the winter months from November 2008 to May 2009 in the absence of a
host crop. At one Ewing, NE location, the Xiphinema spp. population density
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
S182
PHYTOPATHOLOGY
trends were similar to Fullerton, increasing over the winter months. However,
at a second site in Ewing, the population densities of Xiphinema spp.
decreased, while at the remaining two sites in Ewing, Xiphinema spp.
populations held steady over the winter. Trichodorus spp. were found in only
locations 3 and 4 at the Ewing site, and the population densities decreased
over the winter for both locations, contradictory to Trichodorus spp. at the
Fullerton site. It is difficult to interpret the reasons for differences in these
population density trends since these preliminary data are inconclusive.
Continued sampling of the sites is planned over the next calendar year to
identify the trends of the nematode genera so that sampling recommendations
can be improved for nematodes of corn.
The effect of foliar fungicide timing on yield and grain fill in high and low
aphid pressure environments
N. R. BESTOR (2), D. S. Mueller (2), A. E. Robertson (2), R. Ritson (2), M.
O’Neal (1)
(1) Department of Entomology, Iowa State University; (2) Department of
Plant Pathology, Iowa State University, Ames, IA, USA
Phytopathology 100:S182
With the arrival of two invasive pests of soybean, the soybean aphid and
soybean rust, there is increasing interest in the use of pesticides for soybean
production. Recently, application of foliar fungicides, and to some extent
foliar insecticides, to soybean to increase overall “plant health” has been
promoted. But the economic benefits of such applications are inconsistent and
not well documented. In 2008, the effect of three foliar fungicides (a
strobilurin, a triazole and a premix of strobilurin and triazole) applied at
growth stages R1 or R3 on seed size and yield was evaluated at two locations
in Iowa, one in southeast and one in northwest. Foliar fungicides applied to
soybeans in northwest Iowa had a significant positive effect on yield and seed
size, while fungicides applied to soybeans in southeast Iowa did not affect
yield or seed size. These results were not expected since higher foliar disease
levels occurred in southwest Iowa. At both locations, an application of
fungicide at R3 resulted in significantly greater yields than an application at
R1. In northwest Iowa, no differences in yield were observed between
fungicides; however, seed size at this location was significantly greater when
a fungicide containing a strobilurin was used. Soybean aphid pressure in
northwest Iowa was very high (cumulative aphid days [CAD] = 92,281) while
in southeast Iowa, aphid pressure was over 100 fold lower (CAD = 695),
which may have been a confounding factor. We plan to investigate the effect
of foliar fungicide applications in combination with foliar insecticides under
different environmental conditions in subsequent years.
Genetic diversity of Cercospora sojina revealed by amplified fragment
length polymorphism markers
C. A. BRADLEY (2), A. Wood (3), G. Zhang (2), J. Murray (3), D. Phillips
(1), R. Ming (3)
(1) University of Georgia, Department of Plant Pathology, Griffin, GA, USA;
(2) University of Illinois, Department of Crop Sciences, Urbana, IL, USA; (3)
University of Illinois, Department of Plant Biology, Urbana, IL, USA
Phytopathology 100:S182
Cercospora sojina, a phytopathogenic fungus, causes frogeye leaf spot (FLS)
of soybean. Losses caused by this disease in the United States were estimated
to range from 6.9 million to 12.7 million bushels annually from 2004 to 2007.
The genetic diversity of C. sojina isolates collected from three countries was
estimated using amplified fragment length polymorphism (AFLP) markers. A
total of 64 isolates of C. sojina were analyzed by eight AFLP primer
combinations, generating 40 markers. The average genetic similarity of the 64
isolates was 0.56 on a scale between 0 and 1, indicating a high degree of
genetic diversity within the species. Cluster analysis resulted in two major
clusters and seven sub-clusters. Two isolates collected from Georgia were the
most closely related, sharing a genetic similarity of 0.97. Two isolates from
China were clustered together. Besides these four samples, no clear separation
of isolates based on origin was found. This suggests that genetic diversity
within a population is as great as between populations based on locations. Our
results provide evidence that substantial genetic diversity exists within the
species C. sojina and that selection for broad spectrum host-resistance should
be targeted in soybean breeding programs.
Progress towards generation of plant ant-FvTox1 antibodies against a
Fusarium virguliforme toxin that induces sudden death syndrome in
soybean
H. K. BRAR (1), M. K. Bhattacharyya (1)
(1) Dept. of Agronomy and Interdepartmental Genetics Program, Iowa State
University, Ames, IA, USA
Phytopathology 100:S183
Sudden death syndrome (SDS), caused by Fusarium virguliforme (Fv), is a
serious soybean disease. It is hypothesized that the foliar SDS symptoms are
caused by a phytotoxin(s), released to the roots by the fungal pathogen. A low
molecular weight protein (~13.5 kDa) has been shown to produce foliar SDS.
The proteinaceous toxin was named as FvTox1. Mice monoclonal antibodies
were generated against FvTox1. We have cloned two single chain variable
fragment (scFv) antibodies from the hybridoma cell lines that express the
mice monoclonal anti-FvTox1 antibodies. Through western blot analysis, we
have shown that the recombinant scFv’s anti-FvTox1 proteins expressed in
Escherichia coli can bind to the E. coli expressed recombinant FvTox1
protein. Two recombinant scFv’s anti-FvTox1 genes are currently being
expressed in transformed soybean calli. We will investigate if any of the
scFv’s anti-FvTox1 proteins (plant anti-FvTox1 antibodies) expressed in
transformed soybean calli can bind to the FvTox1 protein. If we can show
successful expression of the plant anti-FvTox1 antibodies in transformed calli,
stable transgenic soybean lines will be created to stably express the plant antiFvTox1 antibodies. The transgenic lines expressing detectible levels of the
plant antiFvTox1 antibodies will be then investigated to determine if the plant
anti-FVTox1 antibodies can suppress the development of foliar SDS.
Corn ear insect damage, fungal infection severity, and mycotoxin
concentrations across varying Bt resistance platforms in Nebraska corn
fields with natural insect infestation
K. BRAUER (2), R. Wright (1), T. Jackson (2)
(1) University of Nebraska Lincoln, Dept of Entomology; (2) University of
Nebraska Lincoln, Dept of Plant Pathology
Phytopathology 100:S183
Ear rotting fungi are common in field corn. While they may not drastically
reduce yield directly, secondarily they can contaminate grain with
mycotoxins, for which producers can be severely penalized. Ear feeding
insects play an important role in fungal colonization of the corn ear by
creating wounds that serve as infection points. Insects can be managed with
the use of Bacillus thuringiensis (Bt) proteins in corn. Field trials with two
planting dates were established at two locations in 2007 and 2008 to test this
hypothesis under natural insect infestation that included western bean
cutworm, corn ear worm, and European corn borer. Treatments consisted of
similar corn hybrids with genes for cry proteins Cry1F and Cry1Ab, stacked
with cry proteins for rootworm resistance, and their near isogenic line
counterparts. At crop maturity, ears were manually harvested and the severity
of insect injury and visible fungal infection was determined. Fungal infection
rates were recorded from kernels cultured on PDA and fumonsin levels were
analyzed with a competitive direct ELISA test. Insect damage severity in 2007
was minimal, but was greater in all later planting dates in both years. Variance
between treatments was not significant, but positive correlations between
insect damage, Fusarium ear rot and kernel infection, and fumonisin
concentration were identified. When analyzed in classes, Bt hybrids, stacked
Bt hybrids, and isogenic lines; Bt hybrids and stacked Bt hybrids provided
significant (P < 0.01) reductions in the severity of insect damage, ear rot
diseases, fungal kernel infection, and fumonisin concentration. Higher levels
of insect damage were observed in 2008 than the previous year, but insect
pressure was likely still not severe enough to detect a difference between cry
protein treatments.
Identifying pre-plant risk factors for Bean pod mottle virus in Iowa
E. BYAMUKAMA (1), A. Robertson (1), F. Nutter (1)
(1) Iowa State University, Ames, IA, USA
Phytopathology 100:S183
Integrated disease management requires a thorough understanding of
pathogen-plant-environment interactions in order to develop cost-effective
management programs. Knowing pre-plant risk factors associated with Bean
pod mottle virus (BPMV) would enable soybean producers to deploy
management practices that delay early season BPMV infection and spread to
minimize negative impacts on soybean yield and quality. Potential abiotic and
biotic BPMV risk factors identified by correlation analysis were evaluated
using regression analysis to quantify the predictive power of single and
combined factors at the county scale. We examined thirteen factors: county
centroid latitude, longtitude, and elevation; soybean planting date, number of
soybean farms, and soybean acres; number of alfalfa acres harvested; for the
period of October through April, number of days with daily mean temperature
< 0°C, number of days with snow cover, consecutive days with maximum
temperature < 0°C, consecutive days with snow cover, and accumulated snow
depth; and for March, number of days with mean temperature below 0°C.
Variables with highest predictive value for BPMV incidence were days with
mean temperatures < 0°C in March and number of soybean farms within Iowa
counties, with partial coefficients –4.03 (X1), and –0.012 (X2), respectively.
The multiple regression model explained 54.5% of the variation in countyscale BPMV incidence; higher BPMV incidence was associated with days in
March with mean temperatures < 0°C (X1) and fewer soybean farms per
county (X2). Thus, we suggest that using the March temperature data and the
number of soybean farms/county, potential BPMV incidence can be predicted
before planting. Pre-plant predictions can aid soybean growers and seed
companies in making management decisions, such as the need for seed and/or
foliar insecticide treatments, and selection of planting sites with reduced risk.
The inheritance of mefenoxam resistance in single-zoospore isolates of
Phytophthora erythroseptica
V. CHAPARA (1), R. J. Taylor (1), J. S. Pasche (1), N. C. Gudmestad (1)
(1) Department of Plant Pathology, North Dakota State University, Fargo,
ND, USA
Phytopathology 100:S183
Pink rot of potato, caused by a homothallic diploid Oomycete Phytophthora
erythroseptica, is reported to be an economically important disease in the
United States and known to vary markedly in its sensitivity to the
phenylamide fungicide mefenoxam. Previous studies using single-oospore
populations of P. erythroseptica suggested that mefenoxam resistance was
inherited quantitatively. A study was conducted with eight hundred singlezoospore isolates of P. erythroseptica, produced from the eight parental
isolates having varying sensitivity (2 resistant, 4 intermediately resistant and 2
sensitive isolates) to mefenoxam. In vitro assays were conducted with mefenoxam concentrations of 0, 0.01, 0.1, 1.0, 10.0 and 100.0 µg/ml for isolates with
sensitive and intermediate fungicide responses, for resistant isolates higher concentrations of 0, 1, 10, 100, 200 and 300 µg/ml were used. In all instances each
isolate was tested twice. The progeny of sensitive (EC50 < 1 µg/ml) isolates
had the same phenotype as the parents, with no major shift towards increased
insensitivity to the fungicide. Similarly, the progeny from resistant parents
(EC50 > 100 µg/ml) were also resistant to mefenoxam, however, the progeny
from one parent were less insensitive to the fungicide and the progeny from
the other parent were generally more insensitive. All of the single-zoospore
progeny derived from the four intermediately resistant isolates (EC50 values
range from 1 to 99 µg/ml) had the same phenotype as the parental isolates
with progeny of two parents trending towards increased insensitivity while the
progeny of another parent generally had decreased insensitivity to the
fungicide. These results on the inheritance of mefenoxam resistance using
single-zoospore progeny of P. erythroseptica do not support the conclusions
of previous studies that mefenoxam resistance is inherited quantitatively.
A survey of Venturia inaequalis fungicide resistance in Indiana and
Michigan apple orchards
K. S. CHAPMAN (1), K. L. Quello (1), J. L. Beckerman (1)
(1) Purdue University, West Lafayette, IN, USA
Phytopathology 100:S183
Apple growers rely heavily on fungicides to manage Venturia inaequalis, the
fungus that causes apple scab. Fungicide resistance has developed as a result.
To quantify and assess the levels of fungicide resistance, isolates of V.
inaequalis were collected from Indiana and Michigan orchards and fungicide
resistance was evaluated. Previously published works were used to determine
the baseline concentrations of fungicides and thresholds for growth.
Differences were found in the levels of resistance between the two states. In
Michigan, 2.0% of the isolates tested were resistant to Sovran (defined as 90%
relative growth in the presence of fungicide), but 52.9% were shifted and less
sensitive to the fungicide. 63.5% of MI isolates were resistant to Topsin M.
With respect to Dodine, 13.5% were resistant (90% relative growth), but
67.3% showed a shift in resistance. 42.3% of isolates tested with Nova were
resistant (80% relative growth), and resistance had shifted in 55.8% of
isolates. For Indiana, there was no indication of resistance to Sovran. 86.6% of
Vol. 100, No. 6 (Supplement), 2010
S183
isolates tested had resistance to Topsin M. Dodine testing showed that 7.3% of
isolates were resistant and 62.2% had shifted resistance. Of IN isolates tested
with Nova, 34.1% were resistant and 57.3% were shifted in their resistance.
On a state level this survey will provide the opportunity to educate growers on
the degree of fungicide resistance present in local orchards and prevent
ineffective fungicide applications.
Transient expression of MFSV genes in Drosophila S2 cells
F. M. CISNEROS (4), C. Tsai (3), A. E. Whitfield (2), S. A. Hogenhout (1),
M. G. Redinbaugh (5)
(1) John Innes Centre, UK; (2) Kansas State University; (3) National Taiwan
University, Taiwan; (4) The Ohio State University; (5) USDA-The Ohio State
University
Phytopathology 100:S184
Maize fine streak virus (MFSV) is a member of the genus Nucleorhabdovirus
that is transmitted by the leafhopper Graminella nigrifons. The virus
replicates in both its maize host and its insect vector. To determine whether
Drosophila S2 cells support the production of full-length MFSV proteins, we
inserted the open reading frames for the nucleoprotein (N), phosphoprotein
(P) and replicase protein (L) of MFSV into the pMT/V5-His-Topo vector to
produce V5 epitope/ 6X His tagged proteins. The S2 cells were transfected
with these plasmid constructs. When analyzed by western blot, antibodies to
the V5 epitope clearly reacted with proteins of ~55 and 43 kDa in cells
transfected with plasmids carrying the N and P genes, respectively, the sizes
expected for the full-length fusion proteins. No bands were detected in nontransfected Drosophila S2 cells. The expression of the N gene was also tested
with antibodies raised against MFSV virions, which detects the N protein as
well as several other viral proteins. MFSV virion antibodies detected a protein
of ~55 kDa in S2 cell protein extracts. Antibodies raised against a peptide
sequence from the deduced MFSV P protein reacted with a protein of ~ 43
kDa in transfected S2 cell protein extracts. The expression of the MFSV N
and P genes were detected over a period of 4 days after induction of gene
expression with CuSO4, but were not detected in cells not exposed to CuSO4.
Experiments are underway to asses MFSV L gene expression in S2 cells. Our
results indicate that Drosophila S2 cells can steadily express full-length N and
P proteins for at least 4 days. This finding is important in order to optimize
Drosophila S2 cell system conditions for construction of an infectious fulllength clone of MFSV.
Pantoea stewartii subsp. stewartii carries two type III secretion systems
required for adaptation to insect vector and plant hosts
V. R. CORREA (3), D. R. Majerczak (5), E. Ammar (2), M. Merighi (5), C.
Exner (3), D. L. Coplin (5), R. C. Pratt (3), M. G. Redinbaugh (1), S. A.
Hogenhout (4)
(1) ARS, USDA Corn and Soybean Research, Plant Pathology, OARDC/The
Ohio State University; (2) Entomology, OARDC/The Ohio State University;
(3) Horticulture and Crop Science, OARDC/The Ohio State University; (4)
John Innes Centre, Norwich, UK.; (5) Plant Pathology, The Ohio State
University
Phytopathology 100:S184
Animal and plant pathogenic bacteria interact with their hosts by injecting
virulence proteins into host cells via type III secretion systems (TTSS). The
Hrc-Hrp cluster of Pantoea stewartii subsp. stewartii (Pnss), the causative
agent of Stewart’s wilt in maize (Zea mays L.), was previously shown to be
important for pathogenicity in plants. Pnss has a second TTSS (PSI-2), that is
similar to the invasion-associated TTSS, typical of animal pathogens. We
hypothesized that PSI-2 is required for Pnss colonization of its vector, the
maize flea beetle, Chaetocnema pulicaria. The PSI-2’s psaN gene, which
encodes an ATPase essential for building the injectisome and secretion of
effectors, was inactivated with transposon insertions and frame-shift
mutations. Beetles were allowed to feed on plants infected with Pnss mutants
or wild-type bacteria. Insect colonization by Pnss mutants and wild type
bacteria was analyzed using immunofluorescence confocal microscopy of
dissected insect organs or using viable cell counts of insect homogenates. Pnss
carrying transposon insertions and frame-shift mutations negated bacterial
persistence in flea beetle guts and reduced subsequent transmission to maize.
Complementation of mutants with plasmids carrying the psaN+ gene partially
restored bacterial persistence and transmission. Pnss psaN mutants were fully
virulent on maize, indicating that PSI-2 was not required for plant
pathogenicity. Our results demonstrate that the multiple TTSS in Pnss are
functionally active and play different roles in adaptation of the bacterium to
insect and plant hosts.
Interactions between lesion nematodes and fungal pathogens on maize
seedlings
M. P. DA SILVA (1), G. P. Munkvold (1)
(1) Iowa State University, Ames, IA, USA
Phytopathology 100:S184
S184
PHYTOPATHOLOGY
Lesion nematodes (Pratylenchus penetrans), are well known to have
interactions with root rot pathogens on a wide variety of host plants. The
objective of this research was measure the effects of P. penetrans infestation
on seedling disease symptoms caused by fungal pathogens (Rhizoctonia and
Fusarium spp.), assess the impact of nematode control with abamectin on
above pathogens and evaluate potential added seedling disease management
benefit of abamectin combined with commercial fungicide seed treatment on
maize. In a greenhouse experiment, 150 ml pots filled with autoclaved sandsoil mixture with a layer of fungal inoculum (colonized corn meal/sand
mixture) on top of the seed. A suspension of 1000 P. penetrans (adults,
juveniles and eggs) was added to the pots at the time of planting. A factorial
experimental design was used including 8 seed treatments × 4 pathogen
treatments × 4 reps. Experiments were harvested 30 days after planting.
Emergence was evaluated at 8, 13 and 20 days after planting. Shoot lengths,
fresh and dry shoot weights, fresh and dry root weights and root health were
determined. Roots were scanned and image analysis conducted with
WinRhizo software; root length, root volume and root branching were
determined. The results suggest significant effects on root health with
interactions between fungal pathogens/root-lesion nematodes and between
seed treatment/fungal inoculation. Results also suggest significant effects on
root length and root branching for fungal inoculation. R. solani had a greater
effect on emergence than F. verticillioides. Further root health analysis will be
conducted with WinRhizo.
Development of a forecasting model to estimate risk of Sclerotinia stem
rot development on canola in North Dakota
L. E. DEL RIO (1)
(1) North Dakota State University, Fargo, ND, USA
Phytopathology 100:S184
Sclerotinia stem rot (SSR) is an important yield reducing disease that is
endemic to canola producing areas of North Dakota. SSR, which is caused by
Sclerotinia sclerotiorum (Lib.) de Bary, is managed mainly through the use of
fungicides. Weather conditions play an important role in development of SSR
epidemics and thus on the profitability of fungicide applications made to
control it. A warning system aimed at estimating the risk of development of
SSR epidemics was produced using logistic regression analysis, disease data
collected from more than 800 fields through field surveys and weather data
collected through a net of 27 weather stations. The selected model had a c
value of 0.79 and a Somers’ D value of 0.58, and identified rain and solar
radiation as independent variables of importance. When validated using a data
set of similar size that had not been used in model development, the model
produced a true positive fraction of 64% and a true negative fraction of 74%
and an overall accuracy of 72%. The model was available to canola growers
through a website in 2008.
Assessment of soybean genotypes for resistance to Pythium spp.: Key to
managing this seedling disease complex
M. L. ELLIS (1), P. A. Paul (1), A. E. Dorrance (1)
(1) Department of Plant Pathology, The Ohio State University, OARDC,
Wooster, OH, USA
Phytopathology 100:S184
Resistance to Pythium spp. is not well known in soybean cultivars, especially
for those species most prevalent in Ohio soybean fields. The objective of
this research was to begin screening for resistance to P. irregulare and P.
ultimum var. sporangiiferum. A greenhouse assay was used to evaluate 96
soybean lines for potential resistance to two isolates of P. irregulare, followed
by an evaluation of the top performing lines with two isolates of P. ultimum
var. sporangiiferum. For both assays, data for seed germination, total weight,
root weight, and a root rot score using an ordinal scale were collected. Based
on the results from the two assays, there were no significant interactions
between isolates within species and lines. There was a significant difference
between the two isolates of P. irregulare and among lines for the initial
screening. Thirty two lines were screened with P. ultimum var.
sporangiiferum and there was a significant difference between isolates for root
weight. PI 424354 had the highest weight following inoculation with P.
irregulare; however, it performed poorly, compared to the other lines, when
inoculated with P. ultimum var. sporangiiferum. Of the 32 lines screened,
none were resistant to one of the P. ultimum var. sporangiiferum isolates.
These results suggest that there is potential resistance to both Pythium spp.;
however, this resistance may not confer resistance to all isolates within and
across species.
De-acclimation and re-acclimation responses to sudden temperature
shifts in Lolium perenne
J. D. FARRELL (1), U. Frei (1), S. Fei (1), T. Lubberstedt (1)
(1) Iowa State University, Ames, IA, USA
Phytopathology 100:S184
Climate change has resulted in a higher variability in climate patterns;
exposing plants to frequent freeze thaw cycles especially during the late
winter and early spring. Perennial ryegrass (Lolium perenne) was chosen as
model for investigating cold acclimation and freezing tolerance in relation to
shifting temperatures. Perennial ryegrass is an important crop in Europe, Asia
and Africa as both forage and turf grass. In the United States perennial
ryegrass has the potential to become a cover crop in maize fields where
stover is removed. Recently, genomic resources have become available
including ESTs, microarrays and BAC libraries. Preliminary frost tolerance
assays, also known as ion leakage assay have revealed an interesting
pattern between two Mediterranean cultivars. One cultivar acclimated quickly
however as the cold temperatures continued the frost tolerance decreased
compared to the other cultivar, which acclimated slowly and was able to
sustain frost tolerance. The objective of this study is to determine the frost
tolerance of these two Mediterranean cultivars during cold acclimation, deacclimation and re-acclimation, simulating the typical pattern of a late winter
thaw cycle. In parallel mRNAs will be collected from each cultivar during
normal, cold acclimation, de-acclimated and re-acclimation conditions for
cDNA microarrays assays. Comparing gene expression between the two
Mediterranean cultivars during different temperature conditions will help
identify molecular mechanisms involved in acclimation, de-acclimation and
re-acclimation. The long term goal of this project is to identify the candidate
genes involved in these acclimation processes, to find the genomic location of
these genes and to extract the full length gene and promoter sequence; in the
hopes of expanding our knowledge to other crop species.
Low lignin (brown midrib) sorghum genotypes restrict growth of
Fusarium spp. as compared with near-isogenic wild-type sorghum
D. L. FUNNELL-HARRIS (1), J. F. Pedersen (1), S. E. Sattler (1)
(1) USDA-ARS, Grain, Forage and Bioenergy Research, University of
Nebraska, Lincoln, NE, USA
Phytopathology 100:S185
To increase usability of sorghum for bioenergy and forages, two different
brown midrib (bmr) genes, bmr-6 and bmr-12, were backcrossed into five
elite backgrounds, resulting in reduced lignin near-isogenic genotypes.
Field-grown grain from bmr-6 and bmr-12 plants had significantly
reduced colonization by Fusarium moniliforme sensu lato as compared with
wild-type grain. Fusarium isolates were identified to species using sequence
analysis of the translation elongation factor gene. Three of the most
commonly identified species, Fusarium thapsinum, Fusarium proliferatum
and Fusarium verticillioides, were members of F. moniliforme and included
sorghum pathogens. Three other commonly isolated species, Fusarium
bullatum, Fusarium pallidoroseum and Fusarium graminearum, likely
colonize sorghum asymptomatically. Chi-square analyses showed that the
ratios of Fusarium species colonizing bmr-12 grain were significantly
different from those of wild-type, indicating that bmr-12 affects colonization
by Fusarium spp. across genetic backgrounds. A thrice-replicated bioassay
was conducted in which peduncles of wild-type and near-isogenic bmr
genotypes in a single background were inoculated with fungi associated with
sorghum. F. thapsinum, F. verticillioides, Fusarium armeniacum and
Alternaria alternata were pathogenic on wild-type plants in most cases.
Lesion lengths were significantly reduced on one or both bmr genotypes
infected by F. verticillioides, F. thapsinum or A. alternata compared to lesions
produced on near-isogenic wild-type plants. These data indicate that bmr-6
and bmr-12 affect colonization by Fusarium spp. and A. alternata.
Evaluation of aggressiveness and host range of Fusarium acuminatum
and Fusarium redolens associated with root rot of dry beans
A. GAMBHIR (1), R. S. Lamppa (1), J. B. Rasmussen (1), R. S. Goswami (1)
(1) North Dakota State University, Fargo, ND, USA
Phytopathology 100:S185
Dry bean (Phaseolus vulgaris), a favored rotational crop with high nitrogen
fixing ability and food value is affected by a large number of fungal diseases.
Production of this crop in the US is primarily concentrated in the North
Central region of the country, where Fusarium root rots are a major concern.
Fusarium solani f. sp. phaseoli has been considered as the primary causal
agent of this disease. However, our findings suggest the involvement of other
Fusarium species. Among these, Fusarium acuminatum and Fusarium
redolens, were detected for the first time in 2007 in North Dakota and
Minnesota, on roots of dry bean plants collected from root rot afflicted fields.
Roots of the infected plants exhibited reddish brown lesions or discoloration
on hypocotyl and tap roots, characteristic of Fusarium root rot in dry beans.
Koch’s postulates were completed for these species. Variation in aggressiveness on dry beans among isolates and their ability to infect crops commonly
grown in rotation with dry beans was evaluated in greenhouse trials. Isolates
of F. acuminatum and F. redolens from dry beans exhibited pronounced
differences in the ability to cause disease on a highly susceptible kidney bean
cultivar. Some of the isolates evaluated were as aggressive as F. solani f. sp.
phaseoli. Aggressive isolates from both species were able to infect barley,
canola, chickpeas, corn, field pea, flax, lentils, potato, soybeans, sugarbeet,
sunflower and wheat. But the disease severity and symptoms developed varied
between hosts. These findings suggest a possible change in Fusarium species
causing root rots of dry beans in this region and highlight the potential threat
posed by them to production of dry beans and other crops grown in rotation.
Improving management of soybean cyst nematode through extension
demonstration and outreach
L. J. GIESLER (13), C. Bradley (10), A. Dorrance (8), T. Niblack (9), G.
Tylka (1), D. Jardine (2), D. Malvick (11), L. Sweets (12), S. Markell (4), L.
Osborne (7), P. Esker (14), G. Bird (3), J. Faghihi (6), A. Tenuta (5)
(1) Iowa State University; (2) Kansas State University; (3) Michigan State
University; (4) North Dakota State University; (5) Ontario Ministry of Agriculture, Food & Rural Affairs; (6) Purdue University; (7) South Dakota State University; (8) The Ohio State University; (9) USDA/ARS/University of Illinois;
(10) University of Illinois; (11) University of Minnesota; (12) University of
Missouri; (13) University of Nebraska-Lincoln; (14) University of Wisconsin
Phytopathology 100:S185
While soybean cyst nematode (SCN) is the most yield limiting pest of
soybean in the United States, soybean growers are not always properly
managing it. Recent surveys have demonstrated this in Iowa and direct
correspondence with growers and commercial agriculture professionals
quickly reveals that a major problem exists in that this pest is often ignored.
Extension plant pathologists and nematologists from the North Central states
are collaborating in this project to deliver a consistent message on
management of SCN. As a part of the project, a total of 28 replicated on-farm
strip trials were established to evaluate the influence of SCN resistance source
on yield and SCN reproduction across the North Central states. Soybean
yields were measured, and SCN populations were determined in the spring
and fall for all locations. In addition, each location was tested for SCN
population HG type, which identifies the ability of the population to reproduce
on each of the resistance sources used in the trials. Yield was consistently
higher in resistant cultivars compared to susceptible varieties, but response of
cultivar varied with location. The yields were highest for varieties utilizing the
Peking source of resistance, which had a 5.3 bu/A yield advantage over
susceptible varieties averaged over all locations. In fields with high SCN
populations (>3,000 eggs/100 cc soil), the average yield advantages of
varieties utilizing the Peking, PI 88788, and Hartwig sources of resistance
were 15.5, 11.8, and 6.3 bu/A better than the susceptible varieties,
respectively. In addition to research plots, team members developed extension
programs on SCN and delivered SCN information in all states. A total of 30
field days and 33 indoor education programs were delivered to over 5,000
participants in 50 hours of programming.
Recovery of Phakopsora pachyrhizi urediniospores from passive spore
trap slides and extraction of their DNA for quantitative PCR
J. S. HAUDENSHIELD (2), P. Chaudhary (2), G. L. Hartman (1)
(1) USDA-Agricultural Research Service, Urbana, IL, USA; (2) University of
Illinois, Urbana, IL, USA
Phytopathology 100:S185
Enumeration of rust spores from passive spore traps utilizing white petrolatumcoated slides by traditional microscopic evaluation can represent a serious
challenge. Many fungal spores look alike, and clear visualization on the
adhesive can be obscured by particulate debris or nonuniformities within the
adhesive layer; reports will commonly describe only the number of “rust-like”
spores. Molecular methods of P. pachyrhizi detection, utilizing both standard
PCR and quantitative PCR (qPCR), have been available for several years, but
extraction of fungal DNA from petrolatum-embedded spores remained
difficult. We now demonstrate the utility of a novel method for recovering the
petrolatum layer carrying trapped spores from slides using biodegradable
foam strips, with subsequent DNA extraction to yield material suitable for
quantification by qPCR. This method permits even single spores of P.
pachyrhizi to be recovered and detected. False-negative calls were minimized
by using a multiplexed exogenous control; no false-positives were observed.
This method was successfully employed to assess spore loads in passive traps
located at sentinel plots in the USA during the 2008 soybean growing season.
Evaluation of extraction methods for detecting Xanthomonas axonopodis
pv. phaseoli in common bean seed
Y. HE (1), G. Munkvold (1)
(1) Seed Science Center, Iowa State University, Ames, IA, USA
Phytopathology 100:S185
Xanthomonas axonopodis pv. phaseoli (Xap) and Xap var. fuscans are
important seedborne pathogens of Phaseolus vulgaris. In order to maintain
seed quality and meet phytosanitary requirements, accurate seed health testing
methods are critical. Currently accepted methods for these pathogens include
several variations on extraction methods; therefore our objective is to assess
Vol. 100, No. 6 (Supplement), 2010
S185
the influence of different extraction steps on the sensitivity of Xap detection in
P. vulgaris seeds. Seeds were inoculated with Xap to reach inoculum levels
from 101 CFU/seed to 105 CFU/seed and mixed with clean and healthy P.
vulgaris seeds. One contaminated seed was mixed into each 1000-seed
subsample. Thirty 1000-seed subsamples were tested for each different
extraction condition. Extraction methods tested included soaking whole seeds
in sterilized saline phosphate buffer overnight at 4°C and at room temperate
for 3h, soaking with and without vacuum, and concentrating the seed extract
by centrifuging. The seed extract dilutions were cultured on semi selective
agar media MT and XCP1. The proportions of positive subsamples were
recorded and compared to measure the effects of each extraction step on
detection sensitivity. The results showed that vacuum extraction and
centrifugation of seed extracts increased sensitivity, and soaking overnight at
4°C was more effective than soaking at room temperature for 3h. Our results
suggest that a centrifugation step would be a valuable addition to the current
method approved by the International Seed Testing Association (ISTA), but
these results should be confirmed using naturally infected seedlots.
Correlation between Fusarium head blight severity and deoxynivalenol in
three winter wheat cultivars
J. HERNANDEZ NOPSA (1), S. Wegulo (1)
(1) University of Nebraska-Lincoln, Lincoln, NE, USA
Phytopathology 100:S186
Fusarium head blight (FHB), caused by Fusarium graminearum, is a
damaging disease of wheat. In 2008, a field experiment was conducted to
identify relationships between visual assessments of FHB and deoxynivalenol
(DON) in three winter wheat cultivars. The cultivars Jagalene, Harry, and
2137 were planted following corn on 27 October 2007. In May 2008, plots
were inoculated with 1 × 105 spores/ml of F. graminearum at early anthesis
and were not irrigated. There also was heavy natural inoculum. Cultivars were
arranged in a randomized complete block design with three replications. FHB
severity was determined 21 days after inoculation on 20 heads tagged in each
of 13 disease severity categories in each plot: 0, 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 70, and 90%. There was a significant positive correlation between
FHB severity and DON in all three cultivars: Jagalene (r = 0.92, P < .0001);
Harry (r = 0.64, P = 0.0176); and 2137 (r = 0.88, P < 0.0001). DON
concentration was lower (P = 0.05) in 2137 than in Harry or Jagalene; it was
highest in Harry (32 µg/g) followed by Jagalene (29 µg/g) and 2137 (19 µg/g).
This study demonstrated (i) a positive correlation between FHB severity and
DON and (ii) differences among cultivars in the levels of DON they
accumulated. Similar results were obtained in 2007.
Evaluation of winter wheat cultivars for resistance to Fusarium head
blight and deoxynivalenol
J. HERNANDEZ NOPSA (1), S. Wegulo (1)
(1) University of Nebraska-Lincoln, Lincoln, NE, USA
Phytopathology 100:S186
Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, can
cause significant losses. The reaction to FHB and deoxynivalenol (DON) of
the winter wheat cultivars Jagalene, Harry, 2137, Hondo, Alliance, Infinity,
Goodstreak, Karl 92, Wahoo, Millennium, Wesley, and Overley was evaluated
in the field in 2008. In addition to natural inoculum, plots were inoculated with 1
× 105 spores/ml of F. graminearum at early anthesis and were not irrigated.
FHB index, the percentage of Fusarium-damaged kernels (FDK), DON, yield,
1000 kernel weight (1000kwt), and test weight (twt) were measured. Differences among cultivars were significant (P < 0.0068) for FHB index, FDK,
DON, 1000kwt, and yield. Ranges of measured variables were: FHB index:
13% (Harry) to 64% (Overley); FDK: 21% (2137) to 42% (Harry and Wahoo);
DON: 3.7 µg/g (Karl 92) to 9.9 µg/g (Harry); yield: 763 kg/ha (Wahoo) to
1,365 kg/ha (Karl 92); 1000 kwt: 24.8 g (Wahoo) to 30.8 g (2137). FDK and
DON were positively correlated (r = 0.59, P = 0.0442). There was a significant
((P < 0.05) negative correlation between FDK and yield ((r = –0.74), FDK and
twt ((r = –0.64), FDK and 1000 kwt ((r = –0.84), FHB index and twt ((r = –0.69),
and DON and twt ((r = –0.74). This study demonstrated differences among
winter wheat cultivars in their reaction to FHB and DON. Interestingly, Harry
had the lowest FHB index but the highest DON level, implying that cultivars
with resistance to FHB may be susceptible to DON accumulation.
accidentally introduced, or may be introduced by a natural event (e.g.,
hurricanes). In order to minimize injury to susceptible crops, a precise and
accurate early warning system is needed to detect, correctly identify, and
quickly respond to new plant pathogen threats. The integration of Global
Positioning Systems (GPS), Geographic Information Systems (GIS), and
remote sensing technologies offer tremendous opportunities for meeting U.S.
agricultural biosecurity needs. The objective of this study was to detect the
focal epicenters of Asian soybean rust where this plant pathogen was
deliberately introduced to soybean field plots. Pathogen-specific temporal and
spatial signatures were extracted from high-resolution satellite images of
soybean plots inoculated with Asian soybean rust in Quincy, FL. Disease foci
epicenters were determined using high resolution satellite imagery obtained
on 27 August, 21 September, and 29 September 2006. Image intensities were
extracted from plot images for each date. Contour maps and kriging were used
to map and identify the GPS locations of soybean rust disease foci and foci
epicenters. The use of integrated GPS, GIS, and remote sensing technologies
accurately determined the GPS coordinates where the pathogen was deliberately introduced into plots. The GPS coordinates of the predicted locations of
epicenters differed only 1.5 ± 0.92 m from stated inoculation points.
A cyst nematode effector protein appears to modulate numerous plant
molecular processes
P. J. Howe (1), T. Hewezi (1), T. R. Maier (1), R. S. Hussey (2), E. L. Davis
(3), M. G. Mitchum (4), T. J. Baum (1)
(1) Iowa State University, Ames, IA; (2) University of Georgia, Athens, GA;
(3) North Carolina State University, Raleigh, NC; (4) University of Missouri,
Columbia, MO
Phytopathology 100:S186
Cyst nematodes are some of the most destructive plant pathogens. These
biotrophic parasites form elaborate feeding sites, syncytia, in the roots of their
host plants and remove valuable nutrients from the plant. During the
formation of the syncytium the nematode secretes effector proteins into root
cells, which causes extensive molecular changes in the cell and allows the
parasite to manipulate cellular processes. In this study we have worked to
characterize the sugar beet cyst nematode effector protein 4D09 with the goal
to investigate its role in parasitism. For this purpose we assessed the timing of
4D09 gene expression and transferred this gene in the host plant Arabidopsis
to assess phenotypic plant changes that could reveal this effector’s functions.
Furthermore, when using a Yeast Two-Hybrid approach we found that 4D09
interacts with a plant protein that has been shown to be involved in a multitude of molecular processes. By targeting this key molecular regulator protein
the nematode might be gaining control of some of the plant cellular processes.
Aggressiveness of isolates of Phialophora gregata genotype B from
resistant and susceptible soybean monocultures
T. J. HUGHES (1), C. R. Grau (1)
(1) University of Wisconsin, Department of Plant Pathology, Madison, WI, USA
Phytopathology 100:S186
Many soybean accessions described as resistant to brown stem rot (BSR) are
preferentially colonized by isolates of Phialophora gregata genotype B (Pg
B). These isolates are generally considered less aggressive than isolates of Pg
genotype A because they cause mild or no foliar symptoms characteristic of
BSR. However, variation in aggressiveness has been observed among isolates
of Pg B. Monocultures of BSR-resistant or susceptible soybean accessions
were planted from 2000 through 2005 to determine if soybean accessions
influence the aggressiveness of isolates of Pg B. BSR-susceptible Corsoy 79
and BSR-resistant PI 567.157A were inoculated under greenhouse conditions
with a total of 39 isolates of Pg B obtained from the different monocultures.
BSR severity was determined as the percentage of symptomatic foliar and
internal stem tissue. Overall, BSR severity was low and did not exceed 20%.
Isolates of Pg B caused more severe foliar (P < 0.0001) and stem (P = 0.0008)
symptoms on PI 567.157A than Corsoy 79. Analysis of stem symptom
severity indicated an interaction (P = 0.0124) between soybean accession and
the origin of isolates of Pg. Isolates of Pg B obtained from the monoculture of
a BSR-susceptible or resistant accession were more aggressive than isolates
from a mixed culture of susceptible and resistant cultivars. The relationship
between the origin of isolate of Pg B and isolate aggressiveness was more
apparent for PI 567.157A than for Corsoy 79.
Spatial and temporal analysis to find the epicenters of soybean rust
disease foci using remote sensing, GPS and GIS technologies
N. S. HOLAH (1), D. F. Narvaez (2), J. J. Marois (3), D. L. Wright (3), F. W.
Nutter (1)
(1) Iowa State University, Plant Pathology Department, Ames, IA, USA; (2)
Monsanto Co, St. Louis, MO; (3) North Florida Research and Education
Center, University of Florida, Quincy, FL
Phytopathology 100:S186
Phytophthora root rot-like symptoms on soybeans containing Rps 1k in
Wisconsin in 2008
T. J. HUGHES (2), P. D. Esker (2), S. P. Conley (1)
(1) University of Wisconsin, Department of Agronomy, Madison, WI, USA;
(2) University of Wisconsin, Department of Plant Pathology, Madison, WI,
USA
Phytopathology 100:S186
Exotic plant pathogens have the potential to dramatically impact the U.S.
agricultural economy. New plant pathogen threats may be deliberately or
Cool and wet conditions during the early 2008 growing season in Wisconsin
were conducive for diseases like Phytophthora root rot (PRR), caused by
S186
PHYTOPATHOLOGY
Phytophthora sojae (Ps). While conditions had reversed by August and many
areas were drought-like, symptoms characteristic of PRR began to appear in
several fields. Since many of these fields were planted to cultivars containing
Rps 1k, serious concern arose over the breakdown of resistance conferred by
this gene. To determine if these symptoms were associated with colonization
by Ps, soybean plants were collected from 22 fields in 7 counties and assayed
for the presence of Ps. In all plant samples, Ps was neither isolated nor
observed. Instead, numerous isolates of Diaporthe phaseolorum var. caulivora
(Dpc), D. phaseolorum var. sojae (Dps), and Macrophomina phaseolina (Mp)
were obtained. Northern stem canker and pod and stem blight are caused by
Dpc and Dps, respectively, while Mp causes charcoal rot. Based on both field
observations and plant samples, the PRR-like symptoms observed in
Wisconsin in 2008 were thought to be the result of infection by Dpc, Dps, or
Mp. However, greenhouse inoculations with these fungi did not produce
symptoms similar to those observed in 2008 on two cultivars containing Rps
1k. Whether the PRR-like symptoms were the result of infection by a
combination of these fungi or if the plants defense response to Ps may have
increased susceptibility to Dpc, Dps, or Mp, still remain unknown.
Characterization of two Arabidopsis bHLH transcription factors that are
induced in cyst nematode syncytia
J. JIN (1), T. Hewezi (1), T. Baum (1)
(1) Department of Plant Pathology, Iowa State University, Ames, IA, USA
Phytopathology 100:S187
The soybean cyst nematode (SCN, Heterodera glycines) is a biotrophic
endoparasite that annually causes an estimated one billion dollar loss to the
United States soybean industry. The model system of Arabidopsis thaliana
and the sugar beet cyst nematode (BCN, Heterodera schachtii), a close
relative of SCN, has been used broadly to study the compatible interaction
between a cyst nematode and a plant. Successful cyst nematode parasitism
relies on the formation and maintenance of feeding sites (syncytia) in host
roots through processes that are highly regulated by the interaction between
the cyst nematode and the host. By using promoter::GUS fusion constructs,
we have discovered that two basic Helix-Loop-Helix (bHLH) transcription
factor promoters are induced in syncytia at 3 and 7 days after nematode
inoculation and that the syncytium appears to be the only location of
coexpression for both genes. We also detected that mRNA abundance of both
transcription factor genes was up-regulated in Arabidopsis roots following
BCN infection, corroborating our promoter data. Overexpressing bHLH genes
in Arabidopsis altered root morphology and changed susceptibility to BCN.
By using yeast-two-hybrid analyses and bimolecular fluorescent
complementation assays, we determined that the two bHLH transcription
factors studied here can form a heterodimer. We hypothesize that this
heterodimer specifically forms in the developing cyst nematode feeding site
and is involved in the reprogramming of root cells into syncytia. Expression
analyses are under way to identify target genes regulated by both transcription
factors.
Using BPMV and SMV vector systems to explore soybean cyst nematodeplant interactions
P. S. JUVALE (1), A. Eggenberger (1), C. Zhang (1), J. Hill (1), S. Whitham
(1), M. Mitchum (2), T. J. Baum (1)
(1) Department of Plant Pathology, Iowa State University, Ames, IA; (2)
Division of Plant Sciences and Bond Life Sciences Center, University of
Missouri, Columbia, MO
Phytopathology 100:S187
Soybean is one of the main sources of oil and an important source of complete
protein worldwide. Among the various pathogens that attack soybean, the
soybean cyst nematode (SCN) is especially devastating. In spite of sustained
research efforts, an elaborate understanding of plant-nematode interaction is
still lacking. Since the available methods to generate transgenic soybean
plants are time-, labor- and cost-intensive, there is a critical need for adapting
innovative approaches for rapid gene expression or gene silencing in soybean
roots in a high through-put manner to elucidate nematode-plant interaction.
Due to the rapid pace at which virus infection becomes established throughout
the plant and the high yield of viral encoded proteins, plant-virus based
vectors present promising tools for expressing foreign proteins in soybean. On
the other hand, virus-induced gene silencing (VIGS) is an exceptional reverse
genetics tool that can be used to generate mutant phenotypes for unknown
genes in soybean. We are using a soybean mosaic virus (SMV) vector for
expressing previously identified SCN parasitism genes in soybean and a bean
pod mottle virus (BPMV) vector for VIGS to elucidate gene functions in
nematode resistant soybeans varieties. Since the infection profile of soybean
roots by SMV and BPMV is not clearly understood, our primary goal is to
study and optimize viral infection of the soybean root system. Currently, we
are using reporter genes, GUS and GFP, to study virus movement to the root,
optimize conditions to maximize infected root volume and ensure viral
particle replication in the nematode feeding site.
Optimizing extraction of Fusarium virguliforme DNA from crop residue
and conidia
T. M. KOLANDER (1), D. K. Malvick (1), J. E. Kurle (1)
(1) University of Minnesota, St. Paul, MN, USA
Phytopathology 100:S187
Sudden death syndrome (SDS) of soybean (Glycine max), caused by
Fusarium virguliforme (Fv), can cause severe yield losses. Crop rotation is not
effective for managing SDS. One explanation is that Fv may survive and
possibly grow on residue from crops rotated with soybean. We tested three
methods for extracting Fv DNA from crop residue and Fv conidia for use in
PCR. Residue of soybean, corn (Zea mays), alfalfa (Medicago sativa), and
wheat (Triticum aestivum) was soaked in a suspension of Fv conidia. Residue
was buried in pasteurized field soil maintained at ~23°C for 3 and 6 weeks.
Modifications of the MoBio UltraClean™ Plant (UCP) kit, FastDNA® (FD)
kit, and the MoBio PowerSoil™ (PS) kit were used for residue extractions and
the latter two kits were used for extractions from 104 to 107 conidia. Standard
PCR (sPCR) and quantitative PCR (qPCR) were completed using Fv-specific
primers. The sPCR bands from residue were consistently more intense for the
FD kit, especially at 3 weeks post-burial. At 3 weeks, mean qPCR Ct values
for the FD kit were on average 0.9, 0.6, 2.8, and 6.5 cycles lower than the PS
kit for corn, wheat, alfalfa, and soybean, respectively. At 6 weeks, Ct values
resulting from the FD kit were lower only for alfalfa and soybean. The Ct
values for soybean, resulting from the FD kit, were 6.3 and 7.3 cycles lower
than the UCP kit after 3 and 6 weeks, respectively. Using sPCR and qPCR,
quantities of Fv DNA obtained with the PS kit correlated with the number of
conidia. Fv DNA from conidia was not detected with qPCR using the FD kit.
The FD kit was generally more effective at extracting Fv DNA from crop
residue, especially soybean and alfalfa, and the PS kit was superior for
extracting DNA from conidia.
Determining specificity of commercially available ELISAs for Clavibacter
michiganensis subspecies
K. A. KORUS (1), A. D. Ziems (1), A. K. Vidaver (1), T. A. Jackson (1)
(1) University of Nebraska, Lincoln, NE, USA
Phytopathology 100:S187
Clavibacter michiganensis (Cm) subsp. nebraskensis (Cmn), the bacterium
causing Goss’s wilt of corn, is currently diagnosed by symptom identification
and successful isolation onto CNS selective medium. An ELISA test kit
(Agdia®) specific to Cm michiganensis (Cmm) reportedly gives a crossreaction with Cm subspecies. This ELISA would provide a quick and
inexpensive method for diagnosis of Cmn. ELISA test kits were provided by
Agdia specific to Cmm, Cm tessellarius (Cmt), and Cm sepedonicus (Cms),
respectively. Also, an ELISA test kit (Neogen®) specific to Cmn was included
in the study. For each test kit 13 strains of Cmn, 3 Cmm, 5 Cmt, 3 Cms and 1
Cm insidiosus (Cmi) were tested for cross-reaction. Cultures were grown on
NBY medium for 24 hr, transferred to liquid nutrient broth and agitated for 72
hr, all at 27°C. The CFU/ml was calculated for each isolate and the optimal
concentration needed to produce a positive reaction for each strain.
Preliminary results conducted at a concentration of 104 CFU/ml from 2 of 4
replications indicate that all 5 subspecies (but not all strains) tested positive on
plates coated with antibodies specific to Cmm, Cmn, and Cmt but not on
plates coated with antibodies specific to Cms. ELISAs using antibodies
specific to Cmm, Cmn and Cmt could be used to give a cross reaction with
Cmn. Additional data will be presented on subspecies specificity of Cmm
ELISA test strips.
Optimization of inoculation methods with Fusarium virguliforme for
virus-induced gene silencing studies on soybean sudden death syndrome
L. LEANDRO (1), V. Silva (1)
(1) Department of Plant Pathology, Iowa State University, Ames, IA, USA
Phytopathology 100:S187
Plant age is important in soybean sudden death syndrome (SDS) since root
infection of mature plants may not be conducive to foliar symptoms due to
restricted xylem colonization. In order to conduct virus-induced gene
silencing (VIGS) studies, a method is needed that allows SDS symptoms to
develop in plants inoculated with Fusarium virguliforme two weeks after
inoculation with the virus. The objective of this study was to develop an
inoculation method for VIGS studies on SDS. Roots of 13-day-old soybean
plants were wounded by longitudinally splitting the tap root or by cutting the
tap and lateral roots 1.25 inches below the soil line, then replanting into soil
infested with conidia. To test the effectiveness of the inoculation at different
plant ages, roots of 13, 17, 21, 25 day-old plants were wounded with a
longitudinal split and replanted into infested soil. Plants were maintained in
greenhouse conditions and evaluated for foliar severity over time. In another
experiment, 10, 15 and 20 day-old plants with wounded and non-wounded
roots were introduced into a F. virguliforme cell-free toxin filtrate. Plants
were maintained in growth chamber conditions, and foliar severity was
evaluated over time. In soil assays, severity of foliar symptoms was similar in
Vol. 100, No. 6 (Supplement), 2010
S187
split root and cut root methods, and was negatively correlated with plant age.
In toxin assays, foliar severity did not differ among wounded and intact roots,
but was greater (P < 0.01) in the 10-day old plants than in 15 or 20 day-old
plants. Toxin assay with intact roots was identified as a simple and effective
inoculation method for VIGS studies. We also revealed that soybeans become
less susceptible to the F. virguliforme toxin as they mature, generating
intriguing questions about the role of plant age on SDS.
Effect of planting density, SCN population, and soil pH on soybean root
rot
K. Lim (1), C. GONGORA (1), P. Caragea (2), L. Leandro (1)
(1) Department of Plant Pathology, Iowa State University, Ames, IA; (2)
Department of Statistics, Iowa State University, Ames, IA, USA
Phytopathology 100:S188
Root health is essential for crop growth and productivity, but the factors
affecting root rot on soybeans are poorly understood. The objective of this
study was to investigate the effect of planting density, SCN population, and
soil pH on soybean root rot and yield. Field studies were established in 2006
and 2007 following a split-plot design with row spacing (15” and 30”) as the
main plot and plant population density (100K, 125K, 150K, 175K and 200K
seeds/acre) as the split plot. Soil pH and SCN density were assessed in each of
the 80 field plots. Roots collected at flowering were assessed for root rot
severity, root dry weight and colonization by fungal pathogens. Yield was
determined. Regression analysis was conducted accounting for spatial
dependence between the variables. A clustered (P < 0.01) spatial pattern was
found for pH, SCN, root rot and dry weight in 2006 and 2007, and for yield in
2007. Soil pH and SCN showed a similar spatial pattern in the field as root rot
severity. Plant population and row spacing did not affect root rot severity.
Root dry weight was affected (P < 0.05) by row spacing and plant population
in 2006, and by plant population in 2007. Soil pH was positively correlated
with root rot severity (r = 0.92, P < 0.001) and (r = 0.28, P = 0.02), in 2006
and 2007 respectively, and negatively correlated with root dry weight (r =
–0.6, P < 0.001) and yield (r = –0.3, P = 0.07) in 2007. SCN population was
positively correlated with root rot (r = 0.5, P < 0.05) both years. Fusarium was
the predominant fungus isolated from roots, and was more frequently isolated
from roots with >30% root rot than roots with less severe root rot. This study
suggests that soil pH plays an important role in soybean root rot and
productivity. The interaction between soil pH and root pathogens warrants
further research.
Quantifying and comparing the aggressiveness of Pantoea stewartii
isolates from Iowa
L. LIU (1), C. C. Block (1), F. W. Nutter (1)
(1) Iowa State University, Plant Pathology Department, Ames, IA, USA
Phytopathology 100:S188
Stewart’s disease, caused by Pantoea stewartii, can cause severe economic
damage to seed and sweet corn crops due to phytosanitary regulations that
prevent the export of seed, as well as cause direct reductions in yield. The
aggressiveness of thirteen Pantoea stewartii isolates was quantified and
compared by measuring incubation period (day), rate of lesion expansion/day,
and time to leaf death. Growth chamber experiments were conducted at the
optimal temperature of 30°C. Sweet corn plants (variety “Jubilee”) were
inoculated at the V8 growth stage with 12 wild-type Pantoea stewartii isolates
and a rifampicin-naldixic acid resistant isolate, Rif 9A. Both sides of the midrib of 4 leaves per plant were inoculated with 1 of the 13 isolates (1 × 108
CFU/ml). There were 5 corn plants for each isolate and 65 plants per
replication. Experiments were performed twice for each isolate. Acropetal and
basipetal lesion expansions were measured beginning when lesions were first
visible. Measurements continued at 24-h intervals until no further lesion
expansion was possible (leaves were dead). Our results to date show no
statistical difference among lesion expansion rates of the 13 Pantoea stewartii
isolates, which averaged 0.3984 cm/day acropetally and 0.4999 cm/day
basipetally. Of the 4 leaves tested, average expansion rates were fastest
(0.6018 cm/day acropetally and 1.0804 cm/day basipetally) on the eighth true
leaf. Incubation period was shortest on the seventh true leaf (7.7831 days).
There was no statistical difference between acropetal and basipetal expansion
rates. This study, the first to quantify the aggressiveness of Pantoea stewartii
isolates, serves as a baseline for detecting shifts in pathogen aggressiveness.
Effect of co-inoculation of Fusarium virguliforme and Phialophora gregata
on soybean
C. MATTUPALLI (1), P. D. Esker (1)
(1) University of Wisconsin, Madison, WI, USA
Phytopathology 100:S188
Fusarium virguliforme (Fv, causal agent of sudden death syndrome, SDS) and
Phialophora gregata genotypes A and B (PgA and PgB, causal agent of
brown stem rot, BSR) are two yield-limiting, soil-borne pathogens for
Midwest soybean producers. To evaluate the possible interactions between Fv,
S188
PHYTOPATHOLOGY
PgA, and PgB on disease development, a greenhouse study was conducted.
Two soybean cultivars, Jack (resistant to Fv and PgA) and Williams82
(susceptible to Fv and PgA) were planted in metromix+peat growth medium
that was amended with pathogen-infested vermiculite. There were eight
inoculum treatments: noninfested controls, Fv, PgA, PgB, Fv+PgA, Fv+PgB,
PgA+PgB, and Fv+PgA+PgB. Individual pathogens were added in equal
parts to yield 10,000 spores cm–3 of plant growth medium. Foliar symptoms
characteristic of either SDS or BSR were assessed during reproductive stages
(R1-R7) as the percentage of plant area infected. Mean area under disease
progress curve (AUDPC) ranged from 0%·days for noninfested controls to
518.84%·days for Jack inoculated with Fv. Results indicated that there was an
effect of variety (P < 0.0001), inoculum (P = 0.0092) and their interaction (P
= 0.0756). Multiple comparisons using a Tukey adjustment suggested that
Jack inoculated with Fv+PgA had greater disease development compared with
all Williams82 inoculum treatments. These preliminary results suggest that Fv
and PgA interact with each other and that their effect varies between cultivars.
First report of Fusarium root rot in soybean caused by Fusarium
tricinctum in Minnesota
P. W. Meyer (1), M. S. Clancey (1), I. E. Brose (1), J. E. KURLE (1)
(1) Dept. of Plant Pathology, University of Minnesota, St. Paul, MN, USA
Phytopathology 100:S188
Seed, seedling, and root rots of soybean caused by a complex of soilborne
fungi are possibly the most important diseases of soybean in Minnesota,
causing losses estimated at 380,000 tons in 2005. F. solani and F. oxysporum
are the predominant Fusarium species isolated from soybean taproots in
Minnesota. For soybeans grown in unamended field soil in a growth chamber
at 10 and 16°C, the predominant Fusarium species isolated from taproots
were F. solani and F. tricinctum. Three isolates of F. tricinctum were obtained
from these plants. One of the isolates produced lesions on soybean seedlings
after two weeks, using an inoculum layer method in inoculated sterile sand. F.
tricinctum has been previously reported as pathogenic on soybean in Ontario,
Canada. Its preference for lower temperatures might account for the low
frequency of isolation from Minnesota grown soybean. Its role in soybean root
rot in the field is not known. F. tricinctum could contribute to seedling rot
early in the season when the soil temperature is below 20°C.
Differential regulation of host mRNA translation initiation in the
Arabidopsis: TuMV interaction
J. MOELLER (1)
(1) Iowa State University, Ames, IA, USA
Phytopathology 100:S188
Viruses are known for their ingenuity in reprogramming the host processes of
transcription and translation, including use of non-canonical methods of
translation initiation. To assess virus-induced changes in host transcription
and translational processes, we used the Arabidopsis ATH1 GeneChip
oligonucleotide microarray to determine the mRNA species bound to 80S
ribosomes versus the mRNA species present in total RNA populations in the
Arabidopsis:Turnip mosaic virus (TuMV) interaction. The majority of genes
that are either well or poorly loaded onto ribosomes are consistent in their
loading behavior between non- and TuMV-infected tissues. However,
considerable differential regulation of translation initiation was also found
when non- and TuMV-infected tissues were compared. For example, there are
numerous genes that are up-regulated upon infection according to their mRNA
abundance in total RNA populations but show down-regulation according to
the genes’ translation initiation status and vice-versa. In support of this
finding, 1071 probe sets showed over 4-fold difference when contrasting
mRNA from total RNA to mRNA from 80S ribosomes in response to TuMV.
This study provides near genome-scale analysis of the regulation of translation
initiation in both non- and TuMV-infected states, and it suggests that analyses
of mRNA abundance in total RNA may lead to incorrect conclusions about
which genes are induced or down-regulated in response to viral infection.
Because mRNA associated with 80S ribosomes is expected to be more
predictive of the proteome, this approach may provide candidate genes with
greater relevance to the Arabidopsis:TuMV interaction.
Engineering payload designs for remote sensing applications for plant
pathology using latex weather balloons
M. E. NELSON (3), J. P. Basart (2), F. W. Nutter, Jr. (1)
(1) Department of Plant Pathology, (2) Electrical and Computer Engineering
Department, (3) Space Systems and Controls Lab, Aerospace Engineering
Department, Iowa State University, Ames, IA, USA
Phytopathology 100:S188
The High Altitude Balloon Experiments in Technology (HABET) program at
the Space Systems and Controls Lab (SSCL) at Iowa State University (ISU)
has been flying high altitude balloons in collaboration with the ISU
Department of Plant Pathology for over 10 years. Project goals are to obtain
real-time imagery of crops under stress from biotic and abiotic agents, as well
as to quantify the density of pathogen spore clouds above diseased crops.
These flights vary from ground tethered flights to flights reaching altitudes of
30 km or higher. Since 2007, the two teams have been working together to
design, build and fly hardware that is capable of acquiring both visible and
near-infrared digital images. The engineering design of such hardware
presents unique opportunities in building robust, yet accurate and reliable
equipment for the detection and accurate identification of various plant
diseases. The hardware we are using consists of 2 Digital SLR cameras
(Canon 5D cameras with 24-105 mm zoom lenses). However, one of the
Canon 5D cameras has been modified for near-infrared operation in the 830
nm near-infrared range. A Single Board Computer is used to remotely control
the cameras through a USB connection and allows us to take photographs as
well as adjust camera settings while the payload is in the air. The helium
balloon platform has also been used to quantify the horizontal and vertical
gradients of spore densities being released from disease plant canopies. We
have designed and built payloads that are capable of flying 6 Model 20
Rotorod spore collectors which are also remotely controlled from a ground
control station. This system allows spore collection at 6 different altitudes to
obtain a vertical profile of spore densities. Flights in the near future are being
planned for balloons to be released at pre-set altitudes to quantify spore
densities horizontally with respect to distance from the source (diseased field).
Effect of sclerotial moisture content on carpogenic germination of
Sclerotinia sclerotiorum
A. NEPAL (1), L. E. del Rio (1)
(1) North Dakota State University, Fargo, ND, USA
Phytopathology 100:S189
The effect of sclerotial hydration levels on Sclerotinia sclerotiorum
carpogenic germination (CG) was studied under controlled environment.
Sclerotia of S. sclerotiorum isolate WM031 was classified as large, medium or
small by sieving. Sclerotial water uptake in plain water and in three soil
textures set at four water content levels was characterized using four
replications and ten sclerotia per replication. Sclerotia were placed on petri
dish bottoms in moist chambers that kept them at 100%, 70–80%, 40–50%,
and 20–30% of their maximum hydration level using cool mist humidifiers.
Moist chambers were set at 18/14°C day/night for three months prior to CG
quantification. The experiment was replicated three times with 15 sclerotia per
replication. Water uptake rate by small sclerotia was significantly higher
(alpha = 0.05) than medium and large sclerotia in all moisture treatments.
Small sclerotia were fully hydrated in <5 hours, medium sclerotia in <15
hours, and large sclerotia in <25 hours, irrespectively of the texture and
saturation levels of the soil in which they were incubated. A significant
interaction (alpha = 0.05) between sclerotial hydration level and size was
observed for both, CG and the average number of apothecia produced per
sclerotium. At 100% hydration, large sclerotia had 1.7 and 2.9 times more CG
and apothecia per sclerotium, respectively, than medium and small sclerotia.
At 70 to 80% hydration, only 10% of medium and small sclerotia produced
apothecia while large sclerotia did not produce any. Sclerotia at 50%
hydration or drier did not produce apothecia irrespectively of size.
Is Rps8 alone? Evidence for different genes for resistance to Phytophthora
sojae on the chromosome 13 of soybean Pl 399073
M. A. ORTEGA (3), D. Tucker (1), S. A. Berry (3), S. St. Martin (2), S.
Maroof (1), P. Cregan (6), D. Hyten (4), R. Shoemaker (5), A. E. Dorrance (3)
(1) Department of Crop and Soil Environ. Sciences, Virginia Polytechnic
Institute and State University, Blacksburg, VA, USA; (2) Department of
Horticulture and Crop Science, The Ohio State University, Columbus, OH,
USA; (3) Department of Plant Pathology, The Ohio State University,
Wooster, OH, USA; (4) SDA ARS, Soybean Genomics and Improvement
Lab. BARC, Beltsville, MD, USA; (5) USDA ARS, Department of
Agronomy, Iowa State University, Ames, IA, USA; (6) USDA ARS, Soybean
Genomics and Improvement Lab. BARC, Beltsville, MD, USA
Phytopathology 100:S189
PI 399073, a plant introduction from South Korea, is the source of Rps8, one
of the genes that confers resistance to Phytophthora sojae, the causal agent of
Phytophthora root and stem rot in soybeans. Three Williams (rps8/rps8) × PI
399073 (Rps8/Rps8) BC4F2:3 populations were evaluated for the introgression
of Rps8 by association of resistance to P. sojae race 25 (1a, 1b, 1c, 1k, 7) with
72 SSR and SNP markers from a region on chromosome 13 where Rps8 was
previously mapped. A PI 399073 (Rps8/Rps8) × PI 408211B (Rps?/Rps?) F3:4
population was used to map resistance to P. sojae isolate Butmu (1a, 1b, 1k, 2,
7), and was positioned below the introgression site found in the backcrosses.
Williams (rps8/rps8) × PI 399073 (Rps8/Rps8) BC4F4:5 lines, each line having
different location and size of introgression in this region of chromosome 13,
were inoculated with the isolates of race 1, 4, 7, 17, 25, and Butmu. The
phenotypes of each line were different from each other for the same isolate, as
well as to different isolates, this could be attributed to different Rps genes
present in PI 399073 that were introgressed differentially in the lines tested.
The position and size of the introgression on chromosome 13 in a particular
BC4F4:5 line could carry one or more Rps genes, the response to a different
pathogen isolate could depend on which gene or genes were present in that
line. These results suggest that PI 399073 could have two or more resistance
to Phytophthora sojae genes on chromosome 13.
Aggressiveness of different Fusarium graminearum chemotypes on wheat
cultivars with different level of resistance to Fusarium head blight
K. D. PURI (1), S. Zhong (1)
(1) North Dakota State University, Fargo, ND, USA
Phytopathology 100:S189
Fusarium head blight (FHB), caused by F. graminearum Schw., is a
destructive disease of wheat and barley throughout the world. The disease is
responsible for both direct yield reduction and mycotoxin contamination of
grains. The major mycotoxins produced by the pathogen include
deoxynivalenol (DON) and its derivatives [3-acetyl deoxynivalenol (3ADON) and 15-acetyl deoxynivalenol (15-ADON)] as well as nivalenol
(NIV), which pose health hazards to human and animals. The relative
aggressiveness of 132 isolates collected during 1980 to 2000 in North Dakota,
43 isolates collected in 2008 from different counties of North Dakota and 59
isolates from China were evaluated after their chemotype. PCR assay
indicated that 124 (93.9%) isolates from the old collection (1980 to 2000) and
24 (55%) from the new collection (2008) were of 15-ADON chemotype, and
46 (77.9%) from China were of NIV chemotype. Fourteen isolates from each
of 15-ADON and 3-ADON chemotypes, and two from the NIV chemotype
were tested for aggressiveness on three wheat cultivars/line (Grandin, SteeleND and ND 2710), which are susceptible, moderately susceptible and
moderately resistant to FHB, respectively. Mean disease severity induced by
the isolates varied from 13.5 to 55.6% and difference in aggressiveness among
isolates were highly significant (P = 0.0001). Majority of 3-ADON producing
isolates had higher disease severity compared to 15-ADON or NIV isolates,
but no isolate/variety interaction was detected. The results indicate that the 3ADON chemotype isolates of F. graminearum have increased in the
population of North Dakota and in general were more aggressive than 15ADON and NIV isolates.
Comparison of molecular and mycelium assay for determining
benzimidazole resistance in field populations of Venturia inaequalis in
Indiana
K. L. QUELLO (1), K. Chapman (1), J. Beckerman (1)
(1) Purdue University, West Lafayette, IN, USA
Phytopathology 100:S189
Apple scab, caused by the fungus Venturia inaequalis, is the most destructive
disease on apples in the Midwest, and is controlled primarily by fungicides.
As a result, fungicide resistance has become a problem in orchards. Fungicide
resistance testing requires pure cultures of the fungus. Unfortunately, isolating
pure cultures of V. inaequalis after the end of spring is difficult due to the
microflora on the apple leaf. The use of a molecular assay in situ could avoid
this requirement. We developed a screen that utilizes PCR in situ to detect
Topsin-M (thiophanate-methyl) resistance. V. inaequalis isolates collected
from Indiana were screened with mycelium assays for thiophanate-methyl
resistance. Isolates were found to range from sensitive (no growth at 0.5 µg
active ingredient (a.i.) thiophanate-methyl /mL) to low resistance (growth at
0.5 µg a.i./mL but not 5 µg a.i./mL) to medium resistance (growth at 5 µg
a.i./mL but not at 50 µg a.i./mL) to very high resistance (rapid growth at 50 µg
a.i./mL). To test the accuracy of a molecular assay, concordance between
known mutations in the beta-tubulin gene and phenotype was determined.
DNA was extracted from pure cultures and the beta-tubulin gene was
amplified and digested. Restriction enzyme BstUI was used to verify a
restriction fragment length polymorphism (RFLP) at codon 198 that
corresponded to very high fungicide resistance. 68% of resistant isolates were
positive for the polymorphism. The remaining resistant isolates that did not
contain the RFLP were sequenced. 100% of these isolates possessed a point
mutation at codon 240 in the beta-tubulin gene. This mutation can be
differentiated by PCR-RFLP using Cac8I. All resistant isolates could be
identified with the two restriction enzyme digests. These two PCR-based
RFLP detection methods could be used to rapidly detect thiophanate-methyl
resistance isolates of V. inaequalis at any time.
Extended-duration row covers to suppress bacterial wilt on muskmelon:
Optimizing a new management strategy
E. SAALAU ROJAS (1), M. L. Gleason (1), J. C. Batzer (1)
(1) Iowa State University, Ames, IA, USA
Phytopathology 100:S189
Bacterial wilt (pathogen: Erwinia tracheiphila) causes major losses on
muskmelon in the Midwest U.S. Extending the period during which plants are
covered by spunbond row covers may shield crops from cucumber beetles,
which vector the pathogen. Experiments at two Iowa State University research
Vol. 100, No. 6 (Supplement), 2010
S189
farms (Muscatine and Gilbert, IA) in 2008 validated the ability of extendedduration row covers to suppress incidence of bacterial wilt on muskmelon.
Treatments in a latin square design were: 1) no row cover; 2) row cover
removed at the beginning of anthesis (start of bloom); 3) row covers removed
10 days after anthesis, with row cover ends opened at anthesis to allow
pollination; and 4) row covers removed 10 days after anthesis, with bumble
bee boxes inserted under row covers at anthesis to provide pollination. In both
trials, wilt incidence in the non-covered control was much higher than in the
row-covered treatments. Yield in the extended-duration row cover treatments
was similar when row ends were opened or when a bumble bee box was
inserted under the cover. At Muscatine, the extended-duration row covers
significantly reduced incidence of bacterial wilt at harvest compared to row
cover removal at anthesis. At Gilbert, where melons were transplanted 3
weeks later than at Muscatine, all row cover treatments resulted in similar
levels of wilt suppression. The results suggest that row covers can effectively
suppress cucurbit bacterial wilt, and that timing of transplanting may
determine whether extending the row-covered period provides an additional
margin of wilt protection.
Pathotype diversity of Phytophthora sojae plant isolates from Iowa
S. M. STEWART (1), A. E. Robertson (1)
(1) Department of Plant Pathology, Iowa State University, Ames, IA
Phytopathology 100:S190
Phytophthora root rot (PRR) caused by Phytophthora sojae can infect
soybeans at all growth stages, causing pre- and post-emergence damping-off
and root and stem rot. The most effective way to manage PRR is through the
use of P. sojae-resistant cultivars however, the pathogen continues to diversify
and overcome resistant genes (Rps) present in commercial cultivars. This
host-pathogen system follows Flor’s gene-for-gene hypothesis, and there are
13 known Rps genes. Pathotype diversity has been monitored in Iowa since
1966. Prior to 1975, race 1, which is capable of defeating the resistance gene
Rps7, was the only pathotype reported in Iowa however, two decades later
100% of isolates of P. sojae recovered from soybean plants were able to infect
plants with Rps7. Since only 4.6% of isolates of P. sojae in 1976 were able to
infect plants with Rps-1k, soybean cultivars with Rps-1k were marketed
commercially for PRR management, but by 2004, 73.3% of isolates of P.
sojae recovered from soybean plants could infect plants with Rps-1k. In 2008,
the pathotype diversity of 41 isolates of P. sojae recovered from 15 soybean
plants sampled from six commercial fields in Iowa was assessed using 14
differentials. The isolates belonged to six unique pathotypes. In four fields,
only one unique pathotype was recovered from the plants sampled, while in
the other two fields, two unique pathotypes were recovered. In the study,
100% of the isolates were able to infect plants with Rps-7 and 85.4% could
infect plants with Rps-1k. As expected, the endemic P. sojae population in
Iowa continues to diversify and selection pressure posed by commercial P.
sojae-resistant cultivars results in a greater number of isolates compatible on
these cultivars.
Effectiveness of Brassica short-cycle cover crops in
Phytophthora capsici and Fusarium spp. in cucurbit fields
S. THRU PPOYIL (1), M. Babadoost (1)
(1) UIUC, Urbana-Champaign, IL, USA
Phytopathology 100:S190
managing
A study was conducted in 2008 to determine effectiveness of Brassica shortcycle cover crops in managing Phytophthora capsici and Fusarium spp. in
cucurbit fields. Mustard cultivars, Florida Broadleaf (FBL) and Tilney were
seeded on 29 April in a field with a history of Phytophthora blight and
Fusarium fruit rot of pumpkins and watermelon. The mustard crops were
grown for 45 days and then incorporated into top 10-cm layer of the soil after
cutting the mustard plants with a disk cultivator. Jack-o-Lantern pumpkin
‘Magic Lantern’, processing pumpkin ‘Dickinson’, and cucumber ‘Eureka’
were grown in the mustard amended area. Incidence and severity of seedling
death, leaf spot, vine infection and fruit rot caused by P. capsici and Fusarium
spp. were assessed on a biweekly schedule starting from seedling emergence
on 14 July until harvest on 26 September. No seedling infection or leaf
spot were observed in the plots. Percentage of vines infected with P. capsici in
the plots on 19 September were 16.0, 22.5, 23.0, and 27.5% in the plots
amended with FBL, Tilney, FBL+Tilney, and control plots, respectively. Similarly, percentage of fruits infected with P. capsici on 26 September were 32.2,
33.5, 33.6, and 42.4% in plots amended with FBL, Tiney, FBL+Tilney, and
control plots, respectively. No Fusarium infection was detected in the plots.
Virulence and genetic diversity of Phakopsora pachyrhizi in Nigeria
M. TWIZEYIMANA (2), P. Ojiambo (4), J. Haudenshield (1), G. CaetanoAnollés (1), K. Pedley (5), R. Bandyopadhyay (6), G. Hartman (3)
(1) Dept. of Crop Sciences, University of Illinois, IL; (2) Dept. Crop Sciences,
University of Illinois, IL and International Institute of Tropical Agriculture
(IITA), Nigeria; (3) Dept. of Crop Sciences, University of Illinois and USDAS190
PHYTOPATHOLOGY
ARS; (4) Dept. of Plant Pathology, North Carolina State University, Raleigh,
NC; (5) Foreign Disease-Weed Science Research Unit, USDA-ARS, Fort
Detrick, MD; (6) International Institute of Tropical Agriculture (IITA),
Ibadan, Nigeria
Phytopathology 100:S190
Soybean rust, caused by Phakopsora pachyrhizi, is a major disease in many
soybean-producing areas in Nigeria. To determine the virulence and the
genetic structure of Nigerian field populations of the soybean rust pathogen, a
total of 116 purified isolates established from infected leaves randomly
collected from soybean fields in four agroecological zones in 2005 was used.
The virulence variability of the isolates was determined using a set of four
soybean accessions with Rpp1, Rpp2, Rpp3, and Rpp4 resistance genes, two
highly resistant and two highly susceptible genotypes. Principal component
and cluster analyses on the number of uredinia per cm2 of leaf tissue
separated the rust isolates into seven pathotype clusters. Isolates in cluster III
were the most virulent, while those in cluster IV were the least virulent. In a
follow-up study, 18 simple sequence repeat markers were used to study the
genetic diversity using the same116 isolates and an additional 146 isolates
collected from infected plants in two fields (73 isolates in each field) located
292 km apart. There was a high genetic variation in Nigerian P. pachyrhizi
populations. Eighty-four distinct genotypes were identified among isolates
from the three agroecological zones, while 48 distinct genotypes were
identified from 146 isolates analyzed from both fields. Nei’s average genetic
diversity across geographical regions was 0.22 while for both fields was 0.09.
Hierarchical analysis of molecular variance revealed significant (P < 0.05) and
low genetic differentiation among all populations of P. pachyrhizi. However,
the majority (> 90%) of the genetic diversity was distributed within a soybean
field, while almost 6% was distributed among fields within geographic
regions. The phylogenetic analysis showed three groups in Nigerian rust
populations with one major group comprising more than 90% of the isolates.
However there was a poor correlation between virulence and genetic
variation. This work will be useful in breeding and management of soybean
rust by facilitating the deployment of rust-resistant cultivars.
Performance of SCN-resistant soybean varieties in fields infested with
different soybean cyst nematode HG types
G. L. TYLKA (1), G. D. Gebhart (1), C. C. Marett (1)
(1) Iowa State University, Department of Plant Pathology, Ames, IA, USA
Phytopathology 100:S190
There are hundreds of soybean varieties resistant to the soybean cyst
nematode (SCN). These varieties vary in yield and the ability to control SCN
populations. The HG type test is a greenhouse test that assesses SCN
reproduction on the different sources of resistance used in breeding SCNresistant soybean varieties. Each year, we evaluate the agronomic
performance and SCN control of SCN-resistant soybean varieties in field
experiments, and results reveal how the HG type of an SCN population relates
to performance of SCN-resistant soybean varieties in the field. There are nine
experimental locations statewide annually, three each in northern, central, and
southern Iowa. Plots are four rows wide, spaced 76 cm (30 inches) apart and
5.2 meters (17 feet) long. Each variety is replicated four times per location.
Soil samples are collected from each plot at planting to verify the presence of
SCN and to determine the initial SCN population density. Also, an HG type
test is conducted on the SCN population obtained from the spring soil samples
at each location. At harvest, another soil sample is collected from each plot to
determine SCN population densities. The center two rows of each plot are
harvested, and yield and SCN population densities are averaged for each
variety at each location. The highest-yielding SCN-resistant varieties often are
those with a source of resistance on which there was low (<5 percent) SCN
reproduction in the HG type test. But in some experiments, the highest
yielding SCN-resistant soybean varieties are those with a source of resistance
on which there was relatively high (>20 percent) SCN reproduction in the HG
type test. Also, in some experiments, SCN population densities declined or did
not increase during the growing season on varieties with sources of SCN
resistance on which there was >20% reproduction in the HG type test.
Discovery of genes underlying soybean QTLs conferring partial
resistance to Phytophthora sojae
H. WANG (4), L. Waller (5), S. Tripathy (5), S. K. St. Martin (3), L. Zhou
(5), K. Krampis (5), D. M. Tucker (1), I. Hoeschele (2), S. Maroof (1), B.
Tyler (5), A. E. Dorrance (4)
(1) Department of Crop and Soil Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA; (2) Department of Statistics,
Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; (3)
The Department of Horticulture and Crop Science, The Ohio State University,
Columbus, OH, USA; (4) The Department of Plant Pathology, The Ohio State
University, Wooster, OH, USA; (5) Virginia Bioinformatics Institute, Virginia
Polytechnic Institute and State University, Blacksburg, VA, USA
Phytopathology 100:S190
Phytophthora sojae causes soybean root and stem rot, resulting in an annual
loss of 1–2 billion dollars in soybean production worldwide. Partial resistance
confers a broad-spectrum durable resistance to P. sojae and is currently
thought to be a more stable alternative than single gene mediated resistance.
Few QTLs have been mapped for soybean partial resistance to P. sojae and
little is known about the molecular mechanisms behind it. In this study, five
potential QTLs on Chromosomes 12, 13, 14, 17 and 19, each explaining 4–7%
of phenotypic variation, were identified from 186 RIL of a F4:7 population
from a cross of the partially resistant cultivar ‘Conrad’ and the susceptible
cultivar ‘Sloan’ by composite interval mapping. Global expression profiling
identified a large number of genes showing expression contrast between
‘Conrad’ and ‘Sloan’ either after inoculation or constitutively. Of these, 55
genes map to the QTL regions and include defense-related proteins such as
auxin response factors, heat shock proteins, transcription factors, membrane
transporters, NBS-LRR proteins, pyruvate decarboxylase, cytochrome P450,
cysteine protease and H(+)/calcium ATPase. Eighteen of the 55 (32.7%)
proteins are either unknown or have uncharacterized functions. Fifteen genes
under QTLs were selected and their expression was confirmed by qRT-PCR.
The results indicate the possibility of a complex QTL-mediated resistance
network and provide the clues for further functional studies of soybean partial
resistance to P. sojae. These genes could also be used as markers for breeding
and thus improving soybean production.
Field and greenhouse evaluation of fungicide seed treatment control of
sudden death syndrome of soybean
J. D. WEEMS (2), G. Zhang (2), K. A. Ames (2), J. P. Bond (1), C. A.
Bradley (2)
(1) Southern Illinois University, Carbondale, IL, USA; (2) University of
Illinois, Urbana, IL, USA
Phytopathology 100:S191
Sudden death syndrome (SDS), caused by Fusarium virguliforme, is a yield
reducing disease common in many soybean producing states. Results of recent
research indicated that infection can occur during early radicle emergence,
suggesting fungicide seed treatments may provide protection from the
pathogen during the early stages of soybean development. In 2008, a field
study across two locations and a greenhouse study were conducted to test
eleven fungicide seed treatments and an untreated control across four cultivars
for effects on F. virguliforme infection and development. The southern Illinois
location (Valmeyer) was naturally infested with F. virguliforme, the central
Illinois location (Urbana) was naturally infested with F. virguliforme and soil
was augmented with sterilized grain sorghum colonized by F. virguliforme,
and the greenhouse study was artificially infested with F. virguliforme
inoculum. Roots collected from plots were scanned and analyzed using
WinRHIZO. Foliar symptoms of SDS were rated during plant growth and
harvest data were collected to monitor disease development. At Valmeyer,
SDS was most prevalent and seed treatments had a significant (P = 0.0002)
effect on early season plant stand, with the untreated control having the lowest
stand. Furthermore, roots from untreated plots collected from Valmeyer had significant root tip reductions (P = 0.0052) and increased average root diameter
(P = 0.0451), suggesting lateral root and root hair reduction. Seed treatments
had no other significant effect on the pathogen or disease development.
The 2007 and 2008 Fusarium head blight epidemics in Nebraska
S. WEGULO (1), P. Baenziger (1), L. Nelson (1), J. Hernandez Nopsa (1), J.
Millhouse (1), N. Mengistu (1), J. Breathnach (1)
(1) University of Nebraska-Lincoln, Lincoln, NE, USA
Phytopathology 100:S191
Because of a variable climate, including drought during some years, Fusarium
head blight (FHB) occurs sporadically in Nebraska. In 2007 and 2008, FHB
epidemics occurred in the state for the first time in more than a decade.
Infection of wheat heads by Fusarium graminearum was favored by excessive
rainfall before and during flowering. The most affected areas were the south
central and eastern parts of the state. However, in 2008, FHB was observed as
far west as Imperial in the southwestern part of the state where irrigated fields
were more severely affected. Northwest, the Nebraska Panhandle was spared
in both years due to dry conditions. A shift towards reduced tillage or no-till
to conserve water and soil and inclusion of corn and wheat in crop rotation
schemes has led to buildup of FHB inoculum in Nebraska. Yields were
reduced not only by FHB but by other foliar diseases favored by wet weather.
The major foliar diseases were Septoria tritici blotch, powdery mildew, and
tan spot. In addition to reducing yield and grain quality, FHB caused
accumulation of the mycotoxin deoxynivalenol (DON) in grain. Yield losses
of up to 20% were estimated in the most severely affected areas in the south
central and eastern parts of the state. In 2007, the overall loss statewide in
grain yield was estimated at 2.0% or 1.68 million bushels valued at $9.4
million based on a June 11, 2007 wheat price of $5.57/bushel. In 2008, the
overall loss statewide was estimated at 2.3% or 1.64 million bushels valued at
$13.3 million based on an August 28, 2008 wheat price of $8.11/bushel.
Additional losses were incurred in reduced prices for the infected grain with
high levels of DON. In the most severely affected areas in both years, DON
concentrations of more than 18 ppm were recorded in the most susceptible
cultivars.
Detection of Melon necrotic spot virus in Olpidium sp. infested cucumbers
C. D. WOLTJEN (1), D. J. Lewandowski (1)
(1) The Ohio State University, Columbus, OH, USA
Phytopathology 100:S191
Melon necrotic spot virus (MNSV) is an important pathogen of cucumbers
and melons that can lead to a reduction in fruit quality and economic losses.
MNSV is vectored by zoospores of Olpidium sp. and can also be mechanically
transmitted. In 2008, we received a call from a greenhouse cucumber grower
in Ohio with a high percentage of symptomatic cucumber (Cucumus sativus)
plants exhibiting large necrotic foliar lesions. Leaf samples from symptomatic
cucumbers tested positive for MNSV by DAS-ELISA. Roots of symptomatic
plants were found to be infested with Olpidium sp. The objectives of our
research were to compare the sequence of this Ohio MNSV isolate to other
known MNSV isolates and to determine the susceptibility of several cucurbit
species. Sap from MNSV-infected cucumber leaves was rub-inoculated onto
the cotyledons of three C. melo and seven C. sativus cultivars. Necrotic local
lesions on cotyledons of all ten cultivars were confirmed to be MNSVpositive by DAS-ELISA. MNSV was also transmitted from the original
Olpidium sp. infested substrate to one C. sativus variety. cDNA was
synthesized from total RNA extracted from MNSV-infected leaves using an
MNSV primer complementary to the 3′-UTR. The coat protein (CP) ORF was
amplified with PCR using the same 3′ primer and a primer located upstream
of the CP ORF. The gel-purified PCR product was sequenced and compared
to other known isolates of MNSV. The CP of the Ohio MNSV isolate is 84–
94% and 74–97% identical to the other isolates of MNSV at the nucleotide
and amino acid levels, respectively.
Bacterial species associated with internally-discolored horseradish roots
J. YU (1), M. Babadoost (1)
(1) University of Illinois, Urbana, IL, USA
Phytopathology 100:S191
Internal discoloration of horseradish roots is a complex disease, caused by at
least three fungal pathogens, Verticillium dahliae, V. longisporum, and
Fusarium solani. In addition to the fungal species associated with internally
discolored horseradish roots, bacteria have been routinely isolated from the
affected roots. This study was conducted to identify bacterial species
associated with internally discolored horseradish roots. Horseradish root
samples were collected from major horseradish growing areas in North
America, including Illinois, Wisconsin, California, and Ontario (Canada), and
were assayed for presence of bacteria. The outer layer of the diseased roots
were peeled and surface sterilized in a 6% sodium hypochlorite solution for 1
minute, followed by a 95% ethanol concentration for 3 minutes, and then
rinsed in sterile-distilled water three times. Five segments from each root were
placed onto nutrient agar (NA) plates. The plates were incubated at 22–28°C
with 12 h light/12 h darkness. Bacterial growth were observed after 5, 10, 15
days of incubation. Single-cell colonies of each isolated bacterium were
grown on NA. Characteristics of each purified colony were recorded. Isolated
bacteria were identified using the Biolog program and polymerase chain
reaction (PCR)-restriction fragment length polymorphism (RFLP) assay
followed by analyzing 16s rDNA sequences. Pseudomonas fluorescence,
Bacillus cereus, and Erwinia spp. were the main bacterial species isolated
from horseradish root samples.
Genetic variation of Phytophthora sojae populations from Ohio and South
Dakota
L. X. ZELAYA-MOLINA (1), M. A. Draper (2), A. E. Dorrance (1)
(1) Dept. of Plant Pathology, OARDC, Wooster, OH; (2) formerly Plant
Science Department, South Dakota State University, Brookings, SD
Phytopathology 100:S191
Phytophthora sojae is an important plant pathogen of soybean and negatively
impacts yield each year in the north central region. Understanding how the
pathogen population is evolving will assist in developing effective
management strategies. Our objective was to assess the population variation
of P. sojae isolates collected from 2 states, OH and SD and 2 intensively
sampled fields within OH, Sandusky and Wood, with 21 polymorphic SSR
markers. The average number of alleles was 3.6 per loci for the OH
population, and 2 of the 32 isolates were putative heterozygotes for 2 SSRs.
Seventeen alleles were exclusive to the OH population. SD population had
fewer alleles, 2.9; while one isolate was heterozygous for one SSR, and 4
alleles were exclusive. The Sandusky and Wood populations had an average
of 3.1 and 3.3 alleles, respectively; Wood population had two heterozygous
isolates. Nei’s genetic distance and Fst analysis indicates a moderate genetic
differentiation between the populations. The results agree with previous
Vol. 100, No. 6 (Supplement), 2010
S191
reports of low level of outcrossing in these populations that could account for
the generation of different pathotypes in the field.
Mapping multiple novel resistance genes against Phytophthora sojae in
soybean PI 408211B
Z. ZHANG (2), S. A. Berry (2), R. Mian (3), S. K. St. Martin (1), A. E.
Dorrance (2)
(1) Department of Horticulture and Crop Science, The Ohio State University,
Columbus, OH, USA; (2) Department of Plant Pathology, The Ohio State
University/OARDC, Wooster, OH, USA; (3) USDA-ARS and Department of
Horticulture and Crop Science, The Ohio State University/ORADC, Wooster,
OH, USA
Phytopathology 100:S192
Fourteen Rps genes conferring resistance to Phytophthora sojae, which causes
Phytophthora root and stem rot, have been identified in different soybean
cultivars and plant introductions (PI). PI 408211B was proposed to have one
novel dominant resistance gene against P. sojae race OH17 (vir1b, 1d, 2, 3a,
3b, 3c, 4, 5, 6 and 7) and three previously documented dominant resistance
genes against race OH25 (vir1a, 1b, 1c, 1k and 7). Simple sequence repeat
S192
PHYTOPATHOLOGY
(SSR) and single-nucleotide polymorphisms (SNPs) markers tightly linked
with the resistance to OH17 were identified. Using the soybean sequence
assembly, new SSR and SNPs markers were developed to fine map this gene
on a mapping population of 79 F4:5 recombinant inbred lines from a cross,
‘Williams’ X PI408211B. The gene was mapped between Scf260-027 and
Satt530 with a map distance of 1.3cM and 4.9cM, respectively, on
chromosome 3. The result was validated in an independent population of 48
F2:4 lines from a cross ‘Sloan’ X PI408211B. A population of 360 BC4:7
lines from backcrosses of ‘Stressland’ X PI408211B which does not have a
locus for resistance to OH17 but maintained resistance to OH25 was used to
map the resistance gene against OH25. Bulk segregant analysis (BSA) was
used to screen 177 polymorphic SSR markers on twenty chromosomes with
10 to 20 cM intervals. None of the SSR markers previously linked with known
Rps genes was associated with the resistance to OH25, which suggests that
this resistance occurs at novel loci. BSA indicates that the resistance to OH25
is associated with chromosome 9 and upper part of chromosome 16. Single
marker association analysis on ‘Williams’ X PI408211B F4:5 identified a
region at lower half of chromosome 3 which was also associated with the
resistance to OH25.
2009 Northeastern Division Meeting Abstracts
Abstracts presented at the APS Northeastern Division meeting in Quebec City, Canada, October 28–30, 2009. The abstracts are arranged alphabetically, by first
author’s name.
Rust diseases of cultivated turfgrasses: Understanding an old foe
L. A. BEIRN (1), J. Crouch (2), B. B. Clarke (1)
(1) Rutgers University, New Brunswick, NJ, USA; (2) USDA-ARS, Cereal
Disease Laboratory, St. Paul, MN, USA
Phytopathology 100:S193
Rust is a common disease of cool-season turfgrasses that can decrease the
aesthetic and economic value of many cultivated species, particularly
Kentucky bluegrass (Poa pratensis L.). Chemical control of rust is costly and
sometimes ineffective; therefore the use of resistant cultivars is important for
the effective management of this disease. Over the past ten years, increased
susceptibility to rust has been observed for several Kentucky bluegrass
cultivars in the U.S., most notably the once highly resistant ‘Midnight’ types.
It has been theorized that new races or even new species of the pathogen may
be responsible for this shift in cultivar susceptibility, but the data needed to
test this hypothesis is lacking. In the current study, we are using molecular
markers to evaluate turfgrass rust populations. To date, 63 rust infested leaf
samples have been obtained from graminicolous hosts in North America, the
United Kingdom, Australia, and Chile. A reliable DNA extraction protocol
was developed and both the complete internal transcribed spacer (ITS) region
and 5.8S ribosomal DNA of the samples were amplified and sequenced.
Assembled sequences ranged from 682 to 701 base-pairs in length, including
the partial sequences of the flanking 18S and 28S rDNA. Bayesian
phylogenetic analysis identified Puccinia coronata, P. graminis, and P.
striiformis from infested samples, with P. coronata and P. graminis being
most prevalent. Sequence data generated from this study has been used to
design species-specific molecular markers to develop a real-time PCR
protocol that can be utilized by turfgrass breeders, pathologists and
diagnosticians for a quick identification of turfgrass rust species.
Compost amendment, a potential alternative to soil fumigation for the
control of strawberry verticillium wilt
V. BERNIER-ENGLISH (1), T. J. Avis (2), B. Mimee (1), H. Antoun (1), R.
J. Tweddell (1)
(1) Centre de recherche en horticulture, Pavillon de l’Envirotron, Université
Laval, Québec, QC, Canada, G1V 0A6; (2) Department of Chemistry,
Carleton University, Ottawa, ON, Canada, K1S 5B6
Phytopathology 100:S193
The application of certain composts is known to provide natural biological
control against several diseases and appears as an interesting environmentallyrespectful approach for the control of plant diseases. Verticillium wilt, caused
by Verticillium dahliae, is an important disease affecting strawberry (Fragaria
× ananassa). Currently, pre-plant soil fumigation with metham sodium
(Vapam®) is commonly used to control the disease. However, Vapam
fumigation implies serious risks for health and the environment and often
leads to the eradication of beneficial organisms and to a negative shift in the
biological equilibrium. The objective of the study was to evaluate the effect of
compost application and Vapam fumigation on verticillium wilt incidence and
on vegetative development and fruit yield of strawberry plants. Greenhouse
and field assays have been conducted with three composts produced from
either bovine manure, marine residues or forest bark residues. Composts were
applied at different rates to V. dahliae naturally-infected field plots fumigated
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
or non-fumigated with Vapam and planted with strawberries (cv. Seascape).
In greenhouse assays, composts were applied to sandy substrate inoculated or
non-inoculated with V. dahliae and planted with strawberries (cvs. Seascape
and Chambly). The results indicate that incorporation of marine residues
compost significantly increased vegetative development of strawberry plants
in greenhouse and significantly reduced verticillium wilt incidence in the
field. The results also indicate that soil fumigation significantly decreased
fruit yield and total soil microbial biomass. Although fumigation significantly
decreased soil populations of V. dahliae, it did not reduce verticillium wilt
incidence.
Glyphosate effect on DON content and Fusarium graminearum inoculum
production in wheat and barley
M. BÉRUBÉ (4), A. Vanasse (4), S. Rioux (1), N. Bourget (1), Y. Dion (2),
G. Tremblay (2), G. Bourgeois (3)
(1) Centre de recherche sur les grains, Quebec City, Quebec, Canada; (2)
Centre de recherche sur les grains, Saint-Mathieu-de-Beloeil, Quebec,
Canada; (3) Horticulture Research and Development Centre, Agriculture and
Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada; (4) Laval
University, Quebec City, Quebec, Canada
Phytopathology 100:S193
Fusarium head blight (FHB) is an important disease of wheat and barley,
particularly in the wet conditions of Eastern Canada. The principal pathogen
associated with FHB, Fusarium graminearum, produces deoxynivalenol
(DON), a mycotoxin that makes the grain unfit for food or feed. A recent
survey conducted in eastern Saskatchewan revealed that glyphosate
application in the previous 18 months within minimum-till system was
significantly associated with higher FHB levels in wheat and barley. The
objective of this study was to determine the glyphosate effect, used on
soybean as the previous crop, on DON content and F. graminearum inoculum
production in wheat and barley under three different tillage practices:
conventional-till, minimum-till and no-till. Six field experiments (two species
× three tillage practices) were conducted in Saint-Augustin-de-Desmaures and
Saint-Mathieu-de-Beloeil in 2007 and 2008. Glyphosate and check herbicide
treatments chosen according to weed species were applied as main plot
treatments on RoundUp Ready™ soybean. The following year, three wheat
and three barley cultivars of different FHB resistance levels were seeded as
subplot treatments. In each main plot, two Petri plates containing a Fusariumselective medium were placed facing the ground in order to capture spores
coming from the previous crop. In 2007, there were no significant herbicide ×
cultivar interaction nor herbicide effects on DON content and inoculum
production in any of the 12 experiments. In 2008, DON content was
significantly (P = 0.046) enhanced by glyphosate use (1.5 vs 1.0 ppm) in only
one trial of Saint-Augustin (barley, minimum-till), but there was no significant
effect of glyphosate on F. graminearum inoculum production for any of the
trials. Therefore, it seems that glyphosate used on soybean the previous year
has no or low impact on DON content and F. graminearum inoculum
production under Quebec cropping conditions, whatever the tillage practice
used.
Fifty years of breeding for disease resistance in turfgrasses: Where we’ve
been and where we’re going
S. A. BONOS (1)
(1) Rutgers University
Phytopathology 100:S193
Vol. 100, No. 6 (Supplement), 2010
S193
In the past fifty years, dramatic improvements have been made in breeding for
disease resistance in cool-season turfgrasses. Significant breeding progress
has been made for leaf spot (caused by Drechslera poae) and stem rust
(caused by Puccina graminis) resistance in Kentucky bluegrass (Poa
pratensis), gray leaf spot (caused by Pyricularia grisea) resistance in
perennial ryegrass (Lolium perenne), brown patch (caused by Rhizoctonia
solani) resistance in tall fescue (Festuca arundinacea) and colonial bentgrass
(Agrostis capillaris), and dollar spot (caused by Sclerotinia homoeocarpa)
resistance in creeping bentgrass (Agrostis stolonifera). There are some
diseases for which significant improvements have not been made including
red thread (caused by Laetisaria fuciformis) resistance in perennial ryegrass
and pythium blight (caused by P. aphanidermatum and other Pythium spp.) in
most cool-season turfgrasses. Historically, the dramatic improvements in
disease resistance of the cool-season grasses have been attributed to
traditional/conventional breeding techniques; however, it is likely that
functional genomics and molecular techniques that identify specific genes and
mechanisms involved in disease resistance will be significant in the
development of cultivated turfgrasses in the future.
Evaluation of a dynamic model for primary infections caused by
Plasmopara viticola on grapevine in Quebec
T. CAFFI (2), V. Rossi (2), O. Carisse (1)
(1) Agriculture and Agri-Food Canada, Horticultural Research Centre, 430
Gouin boul., Saint-Jean-sur-Richelieu, Québec, Canada, J3B 3E6; (2) Istituto
di Entomologia e Patologia vegetale, Università Cattolica del Sacro Cuore,
Via E. Parmense 84, I-29100 Piacenza, Italy
Phytopathology 100:S194
A dynamic model for the prediction of Plasmopara viticola primary infections
was evaluated by comparing model predictions with disease onset in 43 cases
(locations per years) in Eastern Canada in 2008 and 2009. The model
simulates the development of all oospore cohorts during the primary inoculum
season, including: oospore germination; production and survival of sporangia;
release, survival and dispersal of zoospores; infection and incubation.
Bayesian analysis was used to evaluate the sensitivity, specificity and
accuracy of the model predictions. First seasonal onset of downy mildew
symptoms ranged between 8 June and 29 June depending year and vineyard.
For each vineyard, one to 20 simulation runs were performed depending on
the number of oospore cohorts formed, for a total of 545 simulations. All
observed infections were correctly predicted by the model. A total of 313
simulations resulted in no infection and in 284 cases no disease developed.
Only one observed infection was not predicted by the model. Finally, 29 out
of 313 simulations predicted an infection that did not result in observed
disease. From this validation analysis, it was concluded that this model could
be used in Eastern Canada to predict the occurrence of the first infection and
trigger the initiation of a fungicide spray program against grape downy
mildew.
Age-related susceptibility of strawberry leaves and berries to infection by
Podosphaera aphanis
O. CARISSE (1), J. Bouchard (2)
(1) Agriculture and Agri-Food Canada, 430 Gouin, St-Jean-sur-Richelieu, Qc,
Canada, J3B 3E6; (2) Plant Science department, Laval University, Quebec
City, Quebec, Canada, G1K 7P4
Phytopathology 100:S194
Powdery mildew, caused by Podosphaera aphanis, is a major disease of
strawberry for which only few management tools are available. The
importance of the disease varies with production systems (June bearing vs day
neutral) which could be explained in part by the concurrent presence of
susceptible leaves or berries and abundant airborne inoculum. Age-related
susceptibility was studied by inoculating strawberry leaves and berries at
different age group. The experiment was conducted for the June bearing
cultivar ‘Jewel’ and the day neutral cultivar ‘seascape’. On eight occasions in
2007, five plants for each cultivar were inoculated with dry conidia using a
settling tower. They was a significant effect of leaf and berry age group on the
susceptibility which decreased exponentially as leaves or berries aged to reach
zero when the leaves were completely expanded or the berries at the pink
stage at the time of inoculation. The proportion of maximum mildew severity
as a function of leaf or berry growth stage was predicted using non-parametric
regression (R2 = 0.96 to 0.97). The prediction values were further validated
with data collected in field naturally infected by P. aphanis. There was a
linear relation between predicted and observed proportion of maximum
mildew severity (R2 = 0.95 to 0.98). The results of this study showed that
timing fungicide sprays based on periods of high leaf and berry susceptibility
should greatly improve management of strawberry powdery mildew.
First report of clubroot caused by Plasmodiophora brassicae on spring
canola in Maine
S. B. JOHNSON (1), D. H. Lambert (1), P. J. Sexton (1)
S194
PHYTOPATHOLOGY
(1) University of Maine, Orono, ME, USA
Phytopathology 100:S194
Spring canola (Brassica napus L.) was introduced into Maine as a potential
oilseed crop in 1999. Since then, acreage has increased from 120 to about
3500 acres and is rapidly becoming an important rotation crop with potato. In
August of 2008, canola plants in two fields in Aroostook County were
observed with classical symptoms of clubroot. Severely affected plants were
stunted and ripening prematurely. Many of the plants had the roots fully
involved with the disease. Infected plants were collected from the fields. The
roots were macerated and the resultant slurry allowed to settle. Abundant
spores of Plasmodiophora brassicae were observed in the slurry. Spring
canola was sown and inoculated with an excess of 50,000 spores per plant. A
like number of spring canola plants were not inoculated. All canola plants
were examined twelve weeks after inoculation. Typical root clubbing
symptoms were evident in all of the inoculated plants. The roots were
macerated and Plasmodiophora brassicae spores were observed from the
resultant slurry. No symptoms or spores were present in any of the
uninoculated plants. The pathogen is not a recent introduction to Maine.
Pathogen buildup or spread to uninfested areas is a concern in Maine.
Broccoli is susceptible to Plasmodiophora brassicae and is an important cash
crop often used in rotation with potatoes in Maine. The economic impact to
the broccoli industry has the potential to be greater than the impact to the
canola industry. To our knowledge, this is the first report Plasmodiophora
brassicae on spring canola in Maine.
White rot of garlic caused by Sclerotium cepivorum -- a new disease in
Maine
S. B. JOHNSON (1), D. H. Lambert (1), B. Watt (1), R. Kersbergen (1)
(1) University of Maine, Orono, ME, USA
Phytopathology 100:S194
There are over 70 commercial garlic (Allium sativum var ophioscorodon)
growers in Maine representing all 16 counties. Most garlic producers in Maine
are market gardeners producing many crops. However, the contribution to
farm income from garlic is disproportionably large when compared to the area
planted. Garlic grown in Maine is distributed within Maine and to other states.
In July of 2008, garlic plants were observed with symptoms which appeared
to consistent with white rot. Severely affected plants were stunted with
yellowing and wilting of the leaves. Bulb decay was present as were sclerotia.
Infected plants were collected from the field. Sclerotium cepivorum Berk was
isolated from the diseased bulbs. Established chives (Allium schoenoprasum)
were used as susceptible host. These were inoculated with a sclerotia/mycelial
mixture. Uninoculated chives plants were used as controls. All chives plants
were examined twenty weeks after inoculation. Typical symptoms were
evident in all of the inoculated plants. The pathogen, Sclerotium cepivorum,
was isolated from the inoculated and symptomatic plants. No symptoms were
present in any of the uninoculated plants. The pathogen is a recent
introduction to Maine. While reputedly present a year or two previously,
disease has now been confirmed. Pathogen buildup and spread to uninfested
areas is a concern in Maine. The current practice of importation of seed stock
and exchange of live plant material may contribute to new appearances and
further spread of the disease. At present, there is no current knowledge on the
distribution and prevalence of garlic white rot.
Solutions to the imbroglio over the nomenclature of Gremmeniella
abietina
G. LAFLAMME (1)
(1) Natural Resources Canada, Canadian Forest Service, Laurentian Forestry
Centre, Quebec, QC, Canada G1V 4C7
Phytopathology 100:S194
Scleroderris canker is caused by the fungus Gremmeniella abietina. In North
America, the disease was first noticed in red pine plantations in Michigan,
USA, in 1951 and was then referred to as the X disease. In 1954, similar
damage was reported in Ontario, Canada. The causal agent was first identified
in Maple, Ontario, in 1962, as Scleroderris lagerbergii. Taxonomists changed
the name of the genus, in 1969, to Ascocalyx and Gremmeniella. Two years
later, a third genus name appeared in the literature: Lagerbergia. Within the
accepted species Gremmeniella abietina, three races were created in 1975:
North American, European and Asian. In 1989, two varieties were recognized,
one representing the three previous races and the second for G. abietina found
on spruce and fir. In the 1990s, based on the host affected by the disease, a
new vocabulary appeared: small tree type and large tree type, or, according to
pathogen traits, types A and B. This was followed later by amplitypes
(European, Northern, Alpine) which evolved later into biotypes (Alpine,
Fennoscandian and European). A new biotype from Spain will soon be added
to this list. Race and type are not valid ranks in fungal taxonomy. After
molecular analyses, it now seems more evident that G. abietina is a European
species, known in North America under the name European race. Fungi found
on pine, fir and spruce in North America and on Todo-fir in Japan (the latter is
currently referred to as the Asian race) are four different species. The case of
G. laricina being one or two species in Europe and North America remains to
be clarified.
Integration of biofungicides and conventional fungicides for management
of peach brown rot
N. LALANCETTE (1), K. McFarland (1)
(1) Rutgers University, Agricultural Research & Extension Center, Bridgeton,
NJ, USA
Phytopathology 100:S195
The biorational fungicides Serenade MAX (Bacillus subtilis QST 713),
Kaligreen (potassium bicarbonate), and Trilogy (hydrophobic extract of neem
oil) were examined in integrated programs with conventional fungicides
during the 2009 growing season for management of brown rot blossom blight
and fruit rot on ‘Encore’ peach. Experimental programs consisted of low and
high rates of each biofungicide applied during bloom (pink, full bloom, and
petal fall timings) for blossom blight control and as the middle of three
applications at 18-, 9-, and 1-day preharvest for rot control during the fruit
ripening period. Treatments were applied using an airblast sprayer (935 L/ha)
to single trees arranged in a randomized complete block design with four
blocks; non-treated buffer trees surrounded each treatment tree. A standard
commercial program and non-treated control (NTC) were included for
comparison. Results of analyses of variance of both the blossom blight and
fruit rot dependent variables showed significant model and treatment effects.
Blossom blight canker incidences (% shoots with canker) for Trilogy-low
(2.5%), Serenade-low (1.3%), Kaligreen-high (1.3%), and Trilogy-high (0%)
were significantly less than the NTC (10.0%) and not significantly different
from standard (0%). These integrated treatments provided 75–100% control of
blossom blight canker development. At harvest, all six integrated programs
had significantly less brown rotted fruit (22.0–35.3% fruit rot) than the NTC
(90.4%) and statistically equivalent incidence of rot to the standard (16.2%).
Similar results were obtained for a postharvest evaluation of fruit after 3-days
incubation; integrated programs had 14.7 to 29.3% fruit with brown rot versus
26.3% for the standard. These results demonstrated that biorational fungicides
can be integrated with conventional fungicides to provide effective brown rot
control programs. Furthermore, the results suggest that early season blossom
blight control may be possible with biofungicides alone. Data from additional
seasons are required to substantiate these findings.
Evaluation of strawberry breeding lines for tolerance to black root rot
and black vine weevil feeding
J. A. LAMONDIA (1), R. S. Cowles (1)
(1) Connecticut Agricultural Experiment Station, Windsor, CT, USA
Phytopathology 100:S195
Perennial strawberries are an important high value crop in the northeast U.S.
Root diseases and root-feeding insects reduce yield and strawberry planting
longevity. The most important root disease is strawberry black root rot, caused
by Rhizoctonia fragariae. Feeding by root weevil larvae, especially black vine
weevil (BVW), also reduces root mass and damages or kills plants. We
conducted field assessment of strawberry cultivars over three years for yield,
vigor, and root health to identify tolerance to black root rot as well as leaf
feeding preference bioassays to identify tolerance to BVW. Several cultivars
were identified as having characteristics desirable as parents for crosses.
Primetime and Lester exhibited resistance or tolerance to black root rot and
non-preference to BVW in feeding preference trials; the cultivar Idea was
moderately susceptible to root rot, but produced a large, vigorous root system.
Progeny of crosses made between Primetime, Lester, Allstar, Delmarvel, and
Idea were carefully selected for resistance or tolerance to black root rot in
greenhouse pots and in the field in infested soils as well as low preference in
BVW feeding trials. Progeny were also screened for fruit yield, size, and
flavor. Selection over three years reduced the progeny population from >4,000
genotypes to a few elite clones with promising horticultural characteristics,
tolerance to black root rot, and low feeding preference by BVW. Our results
demonstrated that sufficient variation exists in certain octoploid parents to
develop effective resistant/tolerant lines. Because there were differences in
disease reactions between greenhouse evaluations of juvenile plants and field
evaluations on mature plants, evaluations in the field are essential in selecting
for black root rot tolerance.
Changing climate, changing forests: Two decades of tree dieback and
decline in Maine
W. H. LIVINGSTON (1)
(1) University of Maine, Orono, ME, USA
Phytopathology 100:S195
A number of tree dieback and decline episodes have occurred in Maine forests
over the past 25 years: Ash dieback 1985–95 (Fraxinus nigra), island spruce
decline 1995–2000 (Picea glauca), white pine decline 1995–2001 (Pinus
strobes), beech decline 1999–2004 (Fagus grandifolia), and balsam fir decline
1999–2004 (Abies balsamea). In all studies, dendrochronology was the key
analytical approach for establishing consistencies between likely inciting
stresses and diebacks or declines. Historical abandonment of agricultural
fields created conditions where native tree species regenerated on sites where
they were not well adapted for long-term survival. In white pine decline, trees
regenerated in high densities on sites where white pine rooting patterns would
not penetrate deeply into the soil. A drought in 1995 incited a decline in these
stands. On Maine coastal islands, abandonment of sheep pastures around 1900
allowed white spruce to regenerate in pure stands not historically found on the
islands. As the stands matured, eastern dwarf mistletoe (Arceuthobium
pusillum) and spruce beetle (Dendroctonus rufipennis) built-up populations
and killed stands. Droughts are common inciting events in other diebacks and
declines (ash, beech, balsam fir) in Maine where typical years do not have a
dry season. Finally, warmer winter temperatures are allowing buildup of
insect populations of beech scale (Cryptococcus fagisuga) and balsam woolly
adelgid (Adelges piceae) that predispose trees to declines incited by drought.
The combination of land use histories altering normal disturbance and
successional patters, years of drought, warmer winters, and build-up of
invasive pests have been adversely affecting survival of trees in Maine.
Sensitivity of Phytophthora capsici isolates from cucurbits in the
northeastern U.S. to dimethomorph, cymoxanil, and mefenoxam
A. M. MADEIRAS (1), R. L. Wick (1), D. R. Cooley (1)
(1) University of Massachusetts, Amherst, MA, USA
Phytopathology 100:S195
Phytophthora capsici has become an important pathogen of cucurbit crops in
the northeastern United States over the last few years. Monitoring fungicide
resistance development in this pathogen is crucial to the success of disease
control programs. The purpose of this study was to determine sensitivities of
local P. capsici isolates to dimethomorph, cymoxanil, and mefenoxam. Single
spore isolates from each of ten cucurbit fields in New York and Massachusetts
were randomly selected and assayed. The 37 isolates tested for sensitivity to
dimethomorph demonstrated a range of 50% effective concentration (EC50)
values from 0.21 to 0.63 mg/L. Of 37 isolates tested for sensitivity to cymoxanil, 21 (56.8%) had EC50 values >50 mg/L and 16 (43.2%) had EC50 values
<50 mg/L. EC50 values for cymoxanil ranged from 1.04 to 132.8 mg/L. Of 39
isolates tested, 35 (89.7%) were sensitive (relative colony diameter [RCD]
<30% of nonamended control) and 4 (10.3%) were intermediately sensitive
(RCD 30–90%) to mefenoxam. The results of this study parallel those of
investigators in other locations and provide information which can be used to
monitor changes in dimethomorph, cymoxanil, and mefenoxam sensitivity in
populations of P. capsici on cucurbits in the northeastern U.S.
Elucidation and management of bacterial diseases on sweet onion in
Pennsylvania
M. A. Mansfield (1), B. K. GUGINO (1)
(1) The Pennsylvania State University, University Park, PA, USA
Phytopathology 100:S195
Sweet onions are an emerging crop in Pennsylvania; however, they are
susceptible to a number of bacterial diseases that cause leaf and bulb decay. In
2008, it was estimated that 50% of the sweet onion crop in Lancaster County
was culled in the packing shed as a result of bacterial diseases. In addition,
recent studies reported an association between disease incidence and severity
on onions grown using plastic mulches typical of production in PA. Our
objectives were to identify and characterize the bacteria associated with
symptomatic onions in commercial fields and evaluate the effect of bare soil,
straw and different types/colors of plastic mulches with and without thrips
control on bacterial disease incidence and severity in a replicated field trial.
Based on preliminary morphological identification and pathogenicity tests
using an onion slice bioassay, Burkholderia cepacia, B. gladioli, Erwinia
caratovora, E. chrysanthemi, Pantoea ananatis, P. agglomerans, Pseudomonas syringae, P. viridiflora, Xanthomonas axonopodis, and X. campestris
were identified from symptomatic onions collected from 15 commercial fields
across PA in 2009. This is the first report of P. ananatis and P. agglomerans
causing disease on onion in PA. Further molecular characterization of these
isolates is ongoing. In the field trial, bacterial diseases were most prevalent on
onions grown on bare soil followed by those on white plastic mulch without
thrips control. Additional data will be presented on the effects of mulch type
and thrips control on onion bacterial disease incidence and severity and its
implication for disease management.
Proposing a new species of Fusarium: F. ‘aestuarinus,’ a pathogen of
Spartina alterniflora associated with wetland dieback in eastern marshes
R. E. MARRA (1), W. H. Elmer (1)
(1) Connecticut Agricultural Experiment Station, New Haven, CT, USA
Phytopathology 100:S195
Sudden Vegetation Dieback (SVD) is a syndrome characterized by rapid loss
of vegetation, particularly smooth cordgrass (Spartina alterniflora). The
Vol. 100, No. 6 (Supplement), 2010
S195
phenomenon has been observed in salt marshes of the eastern seaboard
extending from Louisiana north to Maine. Morphological assessments of fungi
associated with S. alterniflora in SVD sites have revealed a preponderance of
isolates in the genus Fusarium that could not be assigned to known species.
Based on morphology and greenhouse pathogenicity studies, the isolates
separated into two groups, pathogens and nonpathogens. Phylogenetic
analyses of three nuclear genes – beta-tubulin, calmodulin, and translation
elongation factor 1-alpha – corroborated morphological and pathogenicity
studies. Phylogenies were constructed using 20 pathogenic isolates, 18
nonpathogenic isolates, and nine previously described Fusarium species.
Maximum Parsimony and Maximum Likelihood analyses, using individualgene and combined-gene datasets, produced concordant topologies that
strongly support the hypothesis that the pathogenic and nonpathogenic isolates
constitute two phylogenetically distinct clades. From these data, we conclude
that the pathogens represent a single species in the Fusarium section
Sporotrichiella, for which we propose the name Fusarium ‘aestuarinus.’
Isolates in the nonpathogenic group further cluster into two distinct clades,
both clearly belonging to the F. incarnatum-equiseti species complex.
Additional analyses reveal that beta-tubulin sequences from F. langsethiae
and F. equiseti share strongest similarities to that from a more distantly related
ascomycete, Arthrinium sp., highly suggestive of horizontal gene transfer, and
warranting further study.
Occurrence of basil downy mildew in the eastern U.S. in 2009
M. T. MCGRATH (3), C. A. Wyenandt (2), R. N. Raid (5), M. Babadoost (1),
R. L. Wick (4)
(1) Department of Crop Sciences, University of Illinois, Urbana, IL, USA; (2)
Department of Plant Biology and Plant Pathology, Rutgers University,
Bridgeton, NJ, USA; (3) Department of Plant Pathology and Plant-Microbe
Biology, Cornell University, LIHREC, Riverhead, NY, USA; (4) Department
of Plant Soil and Insect Sciences, University of Massachusetts, Amherst, MA,
USA; (5) University of Florida, Everglades Research & Educ Ctr, Belle
Glade, FL, USA
Phytopathology 100:S196
Downy mildew (caused by Peronospora belbahrii) is a new disease of basil in
the USA. It was first detected in FL in Oct 2007. There were several reports in
the eastern USA in 2008. Occurrence in 2009 was monitored through sentinel
plots planted with the cucurbit downy mildew project plots throughout the
eastern USA and a publicly-accessible spreadsheet on the web. Downy
mildew was reported in 2009 on basil grown in greenhouses and outdoors, in
both commercial crops and gardens. The first reported observation was 14 Jan
in GA. Downy mildew was also reported in FL, SC, NC, TN, VA, DE, NJ,
PA, NY, CT, MA, VT, IL, IN, ND, and CA. Not all reports were made by
plant pathologists or confirmed by microscopic examination. Entries in the
spreadsheet include the reporter’s name and method of diagnosis. Downy
mildew was reported widespread with large impact in some areas. In NJ,
symptoms were first observed in June and progressed during the season
throughout the production area which covers more than 600 A. Downy
mildew was also widespread in FL, and in IL where commercial basil field
production is estimated at 550 A. Entire crops have been lost because of this
disease. Downy mildew is now recognized to be established in the USA and is
anticipated to continue occurring every year. Until host plant resistance
becomes available, it appears that downy mildew may force growers to make
drastic changes in production practices, principally applying fungicides to a
crop that rarely needed pesticide applications previously.
Control of foliar diseases in organically-produced tomato with
biopesticides
M. T. MCGRATH (1), G. M. Fox (1)
(1) Department of Plant Pathology and Plant-Microbe Biology, Cornell
University, LIHREC, Riverhead, NY, USA
Phytopathology 100:S196
Tomato is an important crop for organic vegetable growers. Foliar diseases
occur commonly in the northeastern U.S. and can reduce fruit quality and
quantity. EPA-defined biopesticides compliant with U.S. National Organic
Program were evaluated in 2008 and 2009 for naturally-occurring diseases in
trellised fresh-market tomato. Main diseases were powdery mildew and
Septoria leaf spot. Applications were made weekly with a back-pack sprayer
and hand-held boom beginning before symptoms were seen. In 2008, degree
of control calculated from canopy severity on 6 Oct was 75% for powdery
mildew and 58% for Septoria leaf spot with Actinovate SP (0.0371%
Streptomyces lydicus). It was 98% and 68% with Companion (0.03% Bacillus
subtilis GB03), 100% and 61% with Regalia (5% extract of Reynoutria
sachalinensis), 99% and 54% with Organocide (5% sesame oil), and 86% and
0% for Sporatec AG (18% rosemary oil, 10% clove oil, and 10% thyme oil).
Degree of control of both diseases obtained with Regalia alternated with
Kocide 3000 (46% copper hydroxide), Organocide plus Kocide (both at low
rates), and Sporatec plus Saf-T-Side was not significantly different from
S196
PHYTOPATHOLOGY
Kocide alone applied weekly or the conventional fungicide program (control
of 99–100% and 94–100%). Copper fungicide is considered a standard being
the main product used currently by organic growers. Another biopesticide,
Taegro (24.5% Bacillus subtilis var. amyloliquefaciens strain FZB24), was
only effective for powdery mildew (56%). In 2009, Septoria leaf spot was the
dominant disease. The most effective biopesticides were Companion and
Actinovate. They were at least as effective as Kocide. Similar control was
obtained with low rates of Organocide plus Kocide. The most effective
treatments in 2009 included conventional fungicides used alone or combined
with biopesticides.
Identification and characterization of silicon transporters in wheat
(Triticum aestivum)
J. MONTPETIT (1), J. Vivancos (1), W. Rémus-Borel (1), C. Grégoire (1), F.
Belzile (1), R. R. Bélanger (1)
(1) Université Laval, Quebec, Quebec, Canada
Phytopathology 100:S196
Silicon (Si) is not considered as an essential element for plant growth but its
supply is known to be beneficial, namely in preventing biotic and abiotic
stresses. However, its positive effects are variable since in planta
accumulation differs among plant species and a direct correlation between
benefits and absorption has been shown. Some species of the Gramineae
family can accumulate up to 10% on a dry weight basis while others only
accumulate less than 0.1%. Recently, studies with rice have shown that Si
transport is mainly regulated by two genes, Lsi1 and Lsi2. Lsi1-2 like genes
have also been reported in barley and corn. The objective of this project was
to identify and characterize orthologous Si-transport genes in wheat, given the
ability of this plant to accumulate high Si concentrations. With the design of
degenerate primers, two genes presenting high homology (>80%) to Lsi1 and
Lsi2 in rice were identified. Following their isolation, the Si-transport activity
of Lsi1 was verified in Xenopus laevis oocytes, a heterologous system.
Functional characterization is in progress in the model plant Nicotiana
tabacum by the intra-cellular localization of these transporters. Preliminary
results suggest that Lsi1 transporter in wheat is localized across the entire
plasma membrane, unlike OsLsi1 located in specific distal parts of the cell.
This localization of Si transporters could explain the difference in Si
absorption between wheat and rice.
Breeding hybrid poplar in Québec to improve their resistance to Septoria
musiva
M.-J. MOTTET (1), P. Périnet (1)
(1) Ministère des Ressources naturelles et de la Faune, Direction de la
recherche forestière, 2700, rue Einstein, Québec, Qué., Canada G1P 3W8
Phytopathology 100:S196
The poplar (Populus spp.) breeding program in Québec is based on multi-trait
selection including disease resistance. Infection by the native fungus Septoria
musiva Peck causes severe stem deformation and breakage leading to top
dieback or death of the trees. In Québec, bioclimatic conditions have a
significant impact on canker incidence and severity; damage generally
decreases following a south-north gradient. For about 20 years the breeders
have been selecting clones to improve Septoria canker resistance. Interspecific
hybridization aims to capture growth vigour, cold hardiness, and site
adaptability from the species P. trichocarpa (T) and P. maximowiczii (M)
while retaining Septoria resistance from P. deltoides (D), and to some extent
P. nigra (N) and P. balsamifera (B). Despite the high susceptibility of T and
M species and considering that Septoria resistance is apparently recessive,
improvement in resistance was accomplished in some species and hybrids (T,
N, TD, DN x M, NM, MB) through selection and testing. The objective is to
incorporate resistance or tolerance genes originating from many sources in
order to achieve durable resistance. Progenies or clonal populations are first
screened in nursery or early tests where artificial inoculations with different
isolates contribute to accelerate the screening. Then, superior clones are tested
in several locations including Septoria prevalent sites. More than 5000 clones
have been evaluated since 1969. For now, about 40 clones belonging to
different hybrid types and families are used for planting in Québec including
18 resistant/tolerant clones in Septoria zones. Some of those clones are
periodically replaced to improve traits of interest and increase genetic
diversity. Even if Septoria canker expands in geographic range since year
2000, selection for Septoria resistance has not been defeated by evolution of
new races or other pest.
The potential for post-harvest foliar urea sprays to reduce ascospore
production by Venturia inaequalis
R. NORTON (1), C. A. Smith (1), W. E. MacHardy (1), W. Lord (1)
(1) University of New Hampshire, Durham, NH, USA
Phytopathology 100:S196
Apple scab, a disease caused by the ascomycete Venturia inaequalis, is an
important disease of apples in the Northeast. In the Northeast, apple scab is
typically controlled by frequent applications of chemical fungicides in the
spring. Consumer concerns about the potential harmful effects of pesticides on
human health and the environment has created a need for alternative
management options. We are examining the use of urea, as an alternative to
chemical fungicides for controlling apple scab. Although previous studies
have shown that urea reduces the production of ascospores, the primary
inoculum for apple scab, the studies did not compare application rate, timing
of application, and winter hardiness in a single comprehensive study. Cortland
leaves, infected with V. inaequalis, were treated immediately post-harvest, at
the start of leaf fall, and at 95% leaf fall. A 5% urea application was made
either as a single spray or in split applications. Our data suggests that postharvest foliar urea applications significantly reduce the production of
ascospores in V. inaequalis. In conjunction with the ascospore study, we are
also examining the effects of post-harvest foliar urea sprays on winter
hardiness, fruit set, and foliar nitrogen content. Results indicate that postharvest foliar urea applications have no adverse effects on tree health.
The effect of demethylation inhibitor fungicides on Sclerotinia
homoeocarpa population structures
J. T. POPKO (2), C. Ok (2), W. R. Autio (2), M. T. McGrath (1), G. Jung (2)
(1) Department of Plant Pathology and Plant-Microbe Biology, Cornell
University; (2) Department of Plant, Soil and Insect Sciences, University of
Massachusetts-Amherst
Phytopathology 100:S197
Dollar spot (Sclerotinia homoeocarpa F.T. Bennett) is the most economically
important turfgrass disease in North America. Dollar spot is primarily
controlled by fungicide application on golf courses; however, fungicide
resistance has been confirmed in three of the five fungicide classes used to
control dollar spot. Among the confirmed classes, the sterol demethylation
inhibitor (DMI) fungicide class is the most widely used. The objective of this
project was to investigate the effect of propiconazole (DMI) rates on changes
in dollar spot population structure using in-vitro fungicide assays and field
efficacy results. Two sites (Hickory Ridge Country Club, HRCC and South
Deerfield Turf Research Center, SDTRC) were selected for the experiment.
Dollar spot was sampled prior to fungicide application and at the end of the
experiment to examine change in population structure. Samples were also
taken 7 and 14 days after fungicide application from infection centers that
displayed actively growing mycelia to determine the sensitivity of isolates
causing reduced DMI efficacy. All samples were subjected to an in vitro
fungicide assay using a single discriminatory concentration (0.1 µg a.i./ml) of
propiconazole to determine relative mycelia growth percentage (RMG%).
Propiconazole (0.44, 0.88, 1.28 and 1.72 kg a.i. ha–1) and the industry
standard chlorothalonil (non-DMI/12.67 kg a.i. ha–1) fungicides were applied
to both sites to test field efficacy. The initial sampling from the HRCC (n =
433 isolates) revealed the pre-existence of DMI sensitive and insensitive subpopulations. All isolates from SDRC (n = 458) were DMI sensitive. All
samples from propiconazole treated plots 7 and 14 days after application
contained only isolates from the insensitive sub-population regardless of rate
applied at HRCC. Non-DMI treated plots (untreated or chlorothalonil)
sampled 7 and 14 days after application contained isolates of both subpopulations. Reduced field efficacy using propiconazole was observed at
HRCC, whereas complete control was observed at the SDRC.
Structural defense mechanisms in trees: What’s new?
D. RIOUX (1)
(1) Natural Resources Canada
Phytopathology 100:S197
As is the case for many herbaceous plants, preexisting defense structures are
essential in helping trees resist pathogens or mechanical damage. For instance,
by insulating the inner living tissues from heat damage, the thick bark of
sequoia trees is considered an important factor in their tolerance to fire.
Another example is the wax that covers the foliage that often has an influence
on the germinating rate of some pathogen spores or can even help prevent the
penetration of stomata. When such preformed defense elements fail to impede
pathogen ingress, trees respond by forming different structures to limit or stop
pathogen invasion. Compartmentalization processes are certainly among the
most important induced mechanisms that explain tree resistance to various
stresses. Basically, it involves the formation of barriers that bound infected
tissues and thus limit the extent of such lesions in trees. Lignin and suberin
often impregnate the walls of cells involved in compartmentalization whereas
phenols are usually a major component of their cytoplasm. Lately, resistance
of eucalyptus trees to a leaf pathogen has been attributed to these types of
compartmentalization responses. Likewise, in a recent study, it was clearly
shown that such reactions occur in elm calli inoculated with a wilt pathogen.
Genes and substances potentially involved in metabolic pathways leading to
compartmentalization barriers have been reported in recent years. In
particular, methyl jasmonate can induce the formation of traumatic resin
canals in conifers, and these canals are regularly found in compartmentali-
zation xylem barriers. Interestingly, embolism seems to be a significant trigger
of compartmentalization and this could result in practical applications, e.g.
when sugar maple trees are tapped to collect their sweet sap. Finally,
even though compartmentalization structures are composed of antifungal
compounds, some fungi have developed strategies to breach these defensive
tissues.
Summer N-fertilization effects on annual bluegrass putting green turf
J. A. ROBERTS (1), J. A. Murphy (1), B. B. Clarke (1)
(1) Rutgers University, New Brunswick, NJ, USA
Phytopathology 100:S197
Anthracnose, caused by Colletotrichum cereale Manns, is a devastating
disease of putting green turf. Increased N fertility has been reported to reduce
anthracnose severity on annual bluegrass [Poa annua L. f. reptans (Hausskn)
T. Koyama] turf. In 2007, a 3-yr field study was initiated in North Brunswick,
NJ to determine the effect of rate and frequency of soluble-N fertility during
mid-season on anthracnose severity of annual bluegrass turf maintained at 3.2
mm. The date of initiating N fertilization (mid-May vs. mid-June) was also
evaluated during 2008 and 2009. Nitrogen was applied at 4.9 kg ha–1 every 1,
2, 4 and 8 wk and 9.8 kg ha–1 every 2 and 4 wk as a solution of NH4NO3.
Anthracnose severity, assessed as area under the disease progress curve, was
reduced linearly with increasing total N rate (9.8 to 58.8 kg ha–1). Nitrogen
applied at 58.8 kg ha–1 total (4.9 kg ha–1 wk–1 or 9.8 kg ha–1 2 wk–1) had the
greatest reduction in anthracnose severity throughout the study. Nitrogen
applied at 29.9 kg ha–1 over the season (4.9 kg ha–1 2 wk–1) was the lowest N
rate to significantly reduce disease severity, and anthracnose was most severe
on turf receiving N at 19.6 and 9.8 kg ha–1 (4.9 kg ha–1 4 wk–1 or 8 wk–1) over
the season. Initiating N fertilization before symptom expression (mid-May)
reduced anthracnose severity compared to fertilization initiated at the onset of
disease (mid-June) in 2008. Thus, fertilization techniques that increased midseason N fertility, within the range of 29.9 to 58.8 kg ha–1, were effective at
reducing anthracnose severity on annual bluegrass turf.
Using lime-sulfur to control sooty blotch and flyspeck in organic apple
production in southeastern New York State
D. A. ROSENBERGER (1), F. W. Meyer (1), A. L. Rugh (1)
(1) Cornell University’s Hudson Valley Lab, Highland, NY, USA
Phytopathology 100:S197
Lime-sulfur (LS) was applied to control sooty blotch and flyspeck (SBFS) on
apple fruit in five trials in which replicated plots were sprayed to drip using a
handgun. In 2005, Golden Delicious apples receiving two sprays of LS at 10
ml/L during July followed by one spray of thiophanate-methyl plus captan
(210 and 600 mg/L of active ingredient) in Aug had no more SBFS than trees
that received thiophanate-methyl plus captan (TM-C) in all three sprays. In
2006, four summer applications of LS at either 5 or 10 ml/L on a 20-day
interval or six sprays at 2.5 ml/L on a 10-day interval controlled flyspeck just
as well as four sprays of TM-C, but a four-spray program of LS at 2.5 ml/L
was less effective. In 2007, LS at 2.5 ml/L was applied alone on 7 and 30 June
and in a tank mix with 300 mg/L of Cuprofix Disperss (71% basic copper
sulfate) on 24 July and 14 Aug. This treatment reduced flyspeck by only 77–
78% on Empire and Golden Delicious fruit compared to unsprayed controls
whereas TM-C provided 94–96% control. During the very wet summer of
2009, 98% of unsprayed Royal Court fruit failed to meet the U.S. Fancy grade
at harvest due to SBFS whereas eight summer sprays of LS at 2.5 ml/L
resulted in 65% out-of-grade fruit. When the eight-spray LS program was
modified by applying LS alone in June and Sep but adding 318 mg/L of
Nordox (56% copper oxide) to LS in the four July-Aug sprays, only 32% of
fruit were down-graded for SBFS but 26% showed copper injury. LS can be
used to control SBFS in organic apple production, but additional research is
needed to optimize rates and spray timings and to determine if LS applied
during summer reduces fruit size.
Stem susceptibility of six eastern Canadian tree species to Phytophthora
ramorum
M. SIMARD (2), S. C. Brière (1), A. K. Watson (3), D. Rioux (2)
(1) Canadian Food Inspection Agency; (2) Canadian Forest Service; (3)
McGill University
Phytopathology 100:S197
Phytophthora ramorum (Pr) is an emerging pathogen that causes diseases
known as sudden oak death, ramorum leaf blight and ramorum shoot dieback.
Even though Pr has been reported to naturally infect around 120 species, it has
not been detected in the wild in eastern North America. However, there is real
concern that Pr could be introduced and spread into this area. To better
estimate this risk, seedlings of the following six eastern Canadian forest
species were stem-inoculated with Pr: Abies balsamea (Ab), Acer saccharum
(As), Betula alleghaniensis (Ba), Fraxinus americana (Fa), Larix laricina
(Ll), and Quercus rubra (Qr). Bark necrosis, colonization by Pr as well as host
defense reactions were evaluated. Two months after inoculation, nearly 25%
Vol. 100, No. 6 (Supplement), 2010
S197
of Ll and Ab seedlings died. Necrotic areas on the bark were larger in Ll, Ab,
and Qr than in Fa, As, and Ba. Chlamydospores were observed close to the
inoculation point in the phloem of Ll, Ab, and Qr. Pr hyphae were abundant in
the phloem and cambium but also in the xylem of the two coniferous species
where the colonization is facilitated mainly through the invasion of ray cells.
In broadleaf species, hyphae were observed in a few xylem vessels and fibers
close to the inoculation point, except for Qr where Pr was abundant in xylem
vessels and still present up to 5 cm above the inoculation wound. However,
among the six species, Qr was the only one where defense reactions were
clearly apparent, especially when the inoculation occurred later in the growing
season. Overall, Ab, Ll, and to a certain extent Qr appeared susceptible to Pr
and they could be killed should Pr be introduced during conditions conducive
to disease development.
Molecular characterization of the biocontrol activity of Pseudozyma
flocculosa
B. TEICHMANN (1), F. Lefebvre (1), C. Labbé (1), R. Bélanger (1)
(1) Biocontrol Laboratory, Pavillon de l’Envirotron, Université Laval, 2480,
boul Hochelaga, Québec, G1V 0A6, Canada
Phytopathology 100:S198
The basidiomycetous fungus P. flocculosa is a natural inhabitant of the
phyllosphere and has been isolated as a biocontrol agent (BCA) against
powdery mildews. It secretes large amounts of an antifungal cellobiose lipid,
flocculosin, presumably involved in its biocontrol activity. However, the
molecular and genetic basis of glycolipid production and secretion is largely
unknown in P. flocculosa. The related fungus Ustilago maydis secretes a
highly similar glycolipid, ustilagic acid (UA), which also displays
antibacterial and antifungal activity. Recently, a biosynthetic gene cluster was
characterized in U. maydis and found to contain all genes required for the
efficient production and secretion of UA. By analyzing the database of the
recently sequenced genome of P. flocculosa, we hypothesized that a
homologous gene cluster regulating flocculosin synthesis could be found in P.
flocculosa. Comparison of the sequences of all 12 genes against the genome
of P. flocculosa revealed that they were also present within a specific cluster
with the exception of one gene encoding the alpha-hydroxylase Ahd1,
necessary for alpha-hydroxylation of the fatty acid. On the other hand, the
flocculosin gene cluster contained an additional gene encoding an acetyltransferase, probably involved in the acetylation of a further acetyl-group at
the cellobiose moiety. It has already been shown that the presence of powdery
mildew on a plant leaf triggers strong growth of P. flocculosa thereby
affecting the pathogen. It remains to be elucidated which role flocculosin
plays in this biocontrol activity. One hypothesis is that the release of
flocculosin leads to formation of lesions in the membrane of the pathogen
cells followed by the release of nutrients stimulating growth of the BCA. To
validate this hypothesis, we are currently trying to generate a mutant strain
deficient in its ability to produce flocculosin in order to analyze the biocontrol
potential of the resulting phenotype.
Evaluation of basil (Ocimum spp.) cultivars and breeding lines for
susceptibility to downy mildew
A. WYENANDT (2), J. Simon (1), C. Park (3)
(1) Director, New Use Agriculture and Natural Plant Products Program
(NUANPP), Department of Plant Biology and Plant Pathology, Rutgers
University, New Brunswick, NJ, USA; (2) Extension Specialist in Vegetable
Pathology, Department of Plant Biology and Plant Pathology, Rutgers
S198
PHYTOPATHOLOGY
University, Bridgeton, NJ, USA; (3) Research Associate, New Use
Agriculture and Natural Plant Products Program (NUANPP), Department of
Plant Biology and Plant Pathology, Rutgers University, New Brunswick, NJ,
USA
Phytopathology 100:S198
Since 2007, downy mildew (Peronospora belbahrii) on sweet basil (Ocimum
basilicum) has caused significant losses in New Jersey and other basil
production areas of the eastern U.S. No known resistance in basil to downy
mildew has been reported. In 2008, different basil species, cultivars, and
Rutgers University breeding lines (30 in total) were evaluated for
susceptibility to downy mildew in a field trial in southern New Jersey. On 27
Jul, all basil was hand transplanted in a randomized complete block design
with four replications. The field was artificially-infested with downy mildew
by transplanting infected sweet basil plants into rows on 31 Jul. On 20 Aug
and 21 Sep basil plants were rated for downy mildew infection using a plus
scale rating system. Ocimum basilicum was the most susceptible among all O.
species and varieties evaluated. While sporulation ratings varied among the
sweet basil varieties, popular commercial cultivars such as ‘Martina’, ‘Nufar’
and ‘Poppy Joe’s’ were among the most susceptible. Symptoms and
sporulation on Ocimum × citriodorum and O. americanum cultivars were
present, but far less than on O. basilicum cultivars. ‘Spice’, ‘Blue Spice’, and
‘Blue Spice Fil’ were the least susceptible to basil downy mildew with no
visible symptoms developing on the leaves. Similar findings were observed on
a second but non-inoculated basil cultivar trial in northern New Jersey. This is
the first report of potential resistance in Ocimum spp. to downy mildew.
Observations from this study show that genetic resistance is possible.
Selection criteria such as foliar morphology, plant architecture, as well as, the
presence of secondary metabolites are being examined as potential avenues
for developing downy mildew resistance basil cultivars.
Wetwood – An ignored corner in forest pathology
D. YANG (1)
(1) FPInnovations-Forintek Division, 319 rue Franquet, Quebec, QC, Canada,
G1P 4R4
Phytopathology 100:S198
Wetwood, or water pocket, is caused by anaerobic bacteria and occurs in
many softwood and hardwood species. The bacteria enter trees through
wounds or roots and produce pectinolytic enzymes to destroy the vessel and
ray pit membranes of wood. The reproduction and metabolites of these
bacteria form a foetid liquid in wood, which results in a high moisture content
(MC) of the wetwood. Because the MC of wetwood is much higher than
average, wetwood usually requires relatively long periods for adequate drying
in sawmill. Degradation of pectic substances of the middle lamella causes
weakness in the chemical bonds between wood cells. Consequently, weak
bonding increases the risk of warping and checking in lumber during drying
process. Wetwood also has a lower permeability than normal wood; this, in
turn, affects the wood’s treatability with preservatives. The economic losses
resulting from wetwood for wood production and utilization are enormous.
Many studies have been conducted in sawmill on drying wetwood using
various physical, chemical, biological or mechanical methods, but the problem
has yet to be solved. Studies on wetwood infection mechanisms and its control
measurements in forest are limited. More attention should be given to
wetwood problem, and wetwood-free trees are required for lumber
manufacturing and wood utilization.
2010 Southern Division Meeting Abstracts
Abstracts presented at the APS Southern Division meeting in Orlando, Florida, February 7–8, 2010. The abstracts are arranged alphabetically, by first author’s
name.
Implications of fungicide application timings and irrigation on disease
control and peanut yield
J. AUGUSTO (1), T. Brenneman (1)
(1) Dept. Plant Pathology, UGA, Tifton, GA, USA
Phytopathology 100:S199
Night application and/or fungicide redistribution with irrigation may improve
control of stem rot (Sclerotium rolfsii) and increase peanut (Arachis hypogaea
L.) yield by enhancing fungicide penetration to the lower canopy. Four
applications of chlorothalonil (1.26 kg a.i./ha), prothioconazole plus
tebuconazole (0.23 kg a.i./ha), tebuconazole (0.21 kg a.i./ha), flutolanil plus
propiconazole (0.45 kg a.i./ha) or pyraclostrobin (0.21 kg a.i./ha), and two
applications of fluoxastrobin (0.17 kg a.i./ha) or azoxystrobin (0.31 kg a.i./ha)
were applied either early morning (AM = 3 - 5 a.m., with folded leaves) or
during daylight (PM = 10 a.m. - 12 p.m., with unfolded leaves) in irrigated
and nonirrigated plots to evaluate disease control and peanut yield in 2008
(dry year) and 2009 (wet year). In 2008 leaf spot control was similar
regardless of spray timings, fungicides, or irrigation. The AM application of
all systemic fungicides except fluoxastrobin decreased stem rot in nonirrigated
plots, but only azoxystrobin and prothioconazole plus tebuconazole decreased
stem rot more in AM than in PM sprays in irrigated plots. Yields were higher
with AM sprays of tebuconazole and prothioconazole plus tebuconazole in
nonirrigated plots, and with flutolanil plus propiconazole, pyraclostrobin,
tebuconazole and prothioconazole plus tebuconazole in irrigated plots than
with PM sprays. In 2009, leaf spot was severe and spray timings with
systemic fungicides gave similar control regardless of irrigation;
pyraclostrobin had the lowest ratings. The AM sprays of pyraclostrobin,
flutolanil plus propiconazole and prothioconazole plus tebuconazole had
lower stem rot and higher yields than PM sprays, irrespective of irrigation.
The effects of spray timings and irrigation on fungicide efficacy are not the
same for all products.
Bacterial leaf scorch of blueberries: A new threat to the southeastern
industry
P. M. BRANNEN (2), L. Nissen (2), T. Denny (2), C. Chang (3), M.
Tertuliano (1)
(1) Entomology Dept., Univ. of Georgia, Tifton, GA; (2) Plant Pathology
Dept., Univ. of Georgia, Athens, GA; (3) Plant Pathology Dept., Univ. of
Georgia, Griffin, GA
Phytopathology 100:S199
The Xylella fastidiosa bacterium is the causal agent of bacterial leaf scorch
(BLS) of blueberry, predominantly a problem on southern highbush cultivars
(Vaccinium corymbosum interspecific hybrids), but also more recently
confirmed to be present in rabbiteye (Vaccinium virgatum) cultivars.
Symptoms include marginal leaf scorch, leaf drop, yellowing of stems, and
eventual plant mortality. Initial typing of the blueberry strain places it in an A-
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
type category, not closely related to Pierce’s disease (G-type) strains. The
glassy-winged sharpshooter, Homalodisca vitripennis, a known insect vector
of other X. fastidiosa diseases, has been established as a potential vector of the
bacterium in Georgia, since it is the most prevalent sharpshooter found in
commercial blueberry plantings. A 2008 survey determined the prevalence of
BLS in Georgia, and 71.1% of farms were positive for BLS in at least one
field. Field resistance or tolerance was observed among some cultivars.
However, highly susceptible cultivars are predicted to incur complete loss
within 10 years of planting.
Efficacy of fungicides applied in furrow for peanut disease control
T. B. BRENNEMAN (1), J. Augusto (1)
(1) Department of Plant Pathology, University of Georgia, Tifton, GA
Phytopathology 100:S199
Prothioconazole (0.20 kg/ha), azoxystrobin (0.11 kg/ha), or penthiopyrad
(0.35 kg/ha) were applied to Tifguard peanut (Arachis hypogaea) in furrow
(spray volume 35 L/ha) at planting in two replicated trials in 2009. All plots
received only chlorothalonil during the season for foliar diseases. Data for the
trials could be combined, and none of the treatments affected plant stands.
Expanding leaves were bioassayed with Sclerotium rolfsii after emergence but
no residues were detectable. However, prothioconazole reduced leaf spot at
harvest whereas other treatments did not. Prothioconazole and penthiopyrad
each reduced stem rot at both midseason and harvest, but only penthiopyrad
significantly increased yield (512 kg/ha greater than the nontreated control).
In furrow fungicides are known to reduce diseases of seedlings and roots, but
can also reduce foliar diseases of peanut.
Evaluation of Pasteuria usgae as a biological control of sting nematode
(Belonalaimus longicaudatus)
J. H. CAMPBELL (1), J. L. Starr (1), K. L. Ong (1)
(1) Dept. of Plant Pathology & Microbiology, Texas A&M University,
College Station, TX, USA
Phytopathology 100:S199
The plant-parasitic sting nematode (Belonalaimus longicaudatus) is one of the
most devastating nematode pests on turfgrass. In recent years, the turfgrass
industry has seen a number of chemical control measures taken off the market
leaving no effective alternative. The effectiveness of a commercial
formulation of Econem (Pasteuria usgae, an obligate bacterial parasite
specific to sting nematodes) was tested on a bermudagrass putting green in
Texas. A complete-block design with five replicates of each treatment was
used. Granular applications of 100 thousand (30 g product formulation) or 200
thousand spores (60 g product formulation) per 16 square foot plot were
applied monthly from April through July 2009. Effects of treatments on
nematode population densities, root health and length, turf color and turf
density were evaluated over time. There was no effect of treatments on sting
nematode populations, root health or turf density. Turf color was significantly
greater at both the 100k and 200k levels from the untreated controls at the
0.05 level. Average root length was statically greater at the 200k level than the
other treatments. Less than two percent of nematodes in bacterial treated plots
were encumbered by P. usgae endospores at the termination of the
experiment.
Vol. 100, No. 6 (Supplement), 2010
S199
Components of resistance to Cercospora arachidicola in medium maturity
peanut varieties with moderate early leaf spot resistance in the field
E. G. CANTONWINE (2), A. K. Culbreath (1), D. Baskin (2), D. Murphy (2)
(1) University of Georgia, Tifton, GA, USA; (2) Valdosta State University,
Valdosta, GA, USA
Phytopathology 100:S200
Until recently, resistance to early leaf spot, caused by Cercospora
arachidicola, has been linked to late-maturity in peanut (Arachis hypogaea).
An experiment was conducted to evaluate the components of resistance to C.
arachidicola in two medium maturity peanut cultivars with moderate field
resistance, Georgia-03L and Tifguard. Their responses to inoculation were
compared to those of Georgia Green, a susceptible medium maturity cultivar,
and two resistant late-maturing cultivars, Georganic and York. Leaves taken
from the second position of flowering plants were detached, placed in beakers
of saturated sand, and inoculated with spores of C. arachidicola. Leaves were
maintained in a dew chamber at 24°C, 100% relative humidity, and 12-hr
photoperiod for 32 days. Infection frequency, lesion diameter, incubation
period, latent period, and the number of spores per lesion area were compared
for the genotypes. The only resistance component observed for Georgia-03L
was a reduced infection frequency, 0.35 lesions/cm compared to 0.53
lesions/cm for Georgia Green. Tifguard had a lower infection frequency (0.29
lesions/cm), smaller lesion diameter, longer latent period, and fewer spores
per lesion area than Georgia Green. Infection frequency was also lower for
York (0.23 lesions/cm) and Georganic (0.40 lesions/cm) than Georgia Green,
and York and Georganic had smaller lesions than Georgia Green. York had
fewer spores per lesion area than all cultivars tested.
Effect of cotton cultivar selection on soil populations of Fusarium
oxysporum f. sp. vasinfectum
S. CHAWLA (1), J. E. Woodward (3), T. Wheeler (2)
(1) Department of Plant and Soil Science, Texas Tech University, Lubbock,
TX, USA; (2) Texas AgriLife Research, Texas A&M System, Lubbock, TX,
USA; (3) Texas Tech University, Lubbock, TX, USA & Texas AgriLife
Extension Service, Texas A&M System, Lubbock, TX, USA
Phytopathology 100:S200
Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f. sp.
vasinfectum (Fov), is an important disease of cotton (Gossypium hirsutum L.)
in portions of West Texas. A microplot study was conducted over the 2008
and 2009 growing seasons to investigate the influence of planting susceptible
and/or resistant cotton cultivars, FiberMax (FM) 9058F and Stoneville (ST)
4554B2F, respectively on soil population of Fov. Fibermax cultivars, when
planted 2 consecutive years resulted in large increase of Fusarium wilt. The
hypothesis was that cultivars can affect population density of Fov in the soil.
Microplots (75 cm diameter × 45 cm deep) were augmented with field soil
naturally infested with Fov and Meloidogyne incognita. Treatments consisting
of rotation schemes containing ST 4554B2F and FM 9058F were arranged in
a randomized complete block with nine replications. Baseline soil populations
(46.2 cfu/g soil) were enumerated for each microplot via soil dilution plating
on a semi-selective medium. It was observed that FM 9058F planted in
sequential seasons increased Fov populations (79.4 cfu/g soil); however,
populations in microplots planted to ST 4554B2F over two seasons remained
constant (45.8 cfu/g soil). Soil populations in microplots initially planted to
FM 9058F followed by ST 4554B2F remained unchanged (44.2 cfu/g soil);
whereas, Fov populations in microplots initially planted with ST 4554B2F
followed by FM 9058F were not different from those planted to FM 9058F for
two seasons. Results from this study may be useful in developing long term
management strategies that can be implemented into integrated programs for
sustaining the production of cotton in fields infested with Fov.
Effects of chlorothalonil and dodine applied alone and in combination
with systemic fungicides on late leaf spot of peanut
A. K. CULBREATH (1), T. B. Brenneman (1), R. C. Kemerait (1)
(1) University of Georgia, Tifton, GA
Phytopathology 100:S200
In the southeastern U.S., control of late leaf spot, caused by Cercosporidium
personatum, of peanut (Arachis hypogaea) requires multiple applications of
fungicides. Recent renewed interest in the fungicide dodine for leaf spot
control prompted comparison of this fungicide to chlorothalonil alone and in
combination with four systemic fungicides in a randomized complete block
field experiment at Tifton, GA in 2009. All fungicides were applied seven
times at ca.14 day intervals starting at 37 days after planting. Leaf spot
epidemics were severe, with final leaf spot severity ratings (Florida 1-10
scale) of 9.3 (> 95% defoliation) for the nontreated control. The dodine (0.45
kg a.i./ha) treatment had final leaf spot severity ratings of 8.1 compared to 5.1
for 1.26 kg a.i./ha of chlorothalonil (LSD = 0.7). Final leaf spot ratings for
mixtures of dodine (0.3 kg a.i./ha) with propiconazole (0.063 kg a.i./ha),
thiophanate methyl (0.2 kg a.i./ha), tetraconazole (0.063 kg a.i./ha) and
cyproconazole (0.03 kg a.i./ha) were 7.2, 7.5, 6.1 and 6.6, respectively,
S200
PHYTOPATHOLOGY
compared to 4.8, 5.0, 5.1, and 5.2 (LSD = 0.7) for chlorothalonil (0.84 kg
a.i./ha) for those respective treatments.
Comparative evaluation of the survivability of Acidovorax avenae subsp.
citrulli in stored seeds
B. DUTTA (1), R. Walcott (1)
(1) University of Georgia, Athens, GA, USA
Phytopathology 100:S200
Bacterial fruit blotch (BFB), one of the most economically important bacterial
diseases of cucurbits worldwide, is caused by Acidovorax avenae subsp.
citrulli (Aac). Infested seeds are the primary source of inoculum and under
favorable environmental conditions, up to 100% yield loss can occur. Recent
reports indicating that Aac can be transmitted to seedlings from infested seeds
stored for more than 38 years, suggest that the bacterium can withstand
desiccation during storage. However, no detailed studies have been conducted
to dissect the mechanisms of long term bacterial survival in seeds. Hence, the
objective of this work was to compare the ability of Aac to survive on host
and non-host seeds with Xanthomonas campestris pv. campestris (Xcc),
Pantoea stewartii subsp. stewartii (Pnss) and Ralstonia solanacearum (Rs).
Watermelon, tomato, cabbage, and corn seeds (n = 100 g) were artificially
inoculated (separately) with suspensions containing 108 CFU/ml of each of
the four bacteria. Inoculated seeds were air-dried overnight and stored at 4°C
and 50% R.H. The bacterial populations on five replicated samples (n = 1 g of
seed) from each treatment were estimated weekly for 3 months on semiselective media. In two independent trials, the Pnss was undetectable on all
seed types at the end of 3 months. In contrast, the populations of Aac and Xcc
declined to 102 to 103 CFU/g of seeds whereas Rs populations declined to 104
to 105 CFU/g of seeds, irrespective of seed type. Seed type was not a
significant factor in bacterial survival. These data suggest that Aac, Xcc, and
Rs are more tolerant to desiccation than Pnss. The data also suggest that it is
likely that the ability of Aac to survive for 38 yrs in stored seed is due to the
location of the bacterium in the seed rather than some unique characteristic of
the bacterium.
Current status and future of HLB
T. GOTTWALD (1), M. Irey (2), A. Bergamin-Filho (3), R. Bassanezi (4)
(1) USDA-ARS, Fort Pierce, FL; (2) Southern Gardens Citrus, US Sugar
Corp., Clewiston, FL; (3) ESALQ, Universidade de São Paulo, Brazil; (4)
Fundecitrus, Araraquara, Brazil
Phytopathology 100:S200
Results from studies on the increase in HLB incidence and spread in China
and Reunion Island indicate a rate of disease increase leading to a multi-year
epidemic requiring 7 to 10 years for infection to approach an asymptote of
100%. In contrast, more recent studies in Brazil, Vietnam, and Florida suggest
a much more rapid rate of disease increase and spread. An HLB epidemic was
examined in a plantation of over 4,800 ha in South Florida where no new
citrus had been introduced for 10 y and thus spread was entirely dependent on
psyllid transmission. The level of psyllid infestation was unprecedented
compared to previously recorded psyllid infestations. The psyllid vector was
relatively newly introduced to Florida and thus lacks the biological and
environmental constraints found in its native range. Consequently the HLB
epidemic in Florida is undoubtedly one of the worst on record. Stochastic
Markov-Chain Monte Carlo models indicated a prevalence of secondary
spread with occasional primary spread from outside the plots. Interpretations
of the stochastic models combined with survival analyses show spread over
multiple scales from local to regional are occurring simultaneously and
continually in Florida. Edge effects analyses indicate a prevalence of
infections that accumulate at the transition of plantings and areas devoid of
citrus such as the plantation perimeter, internal roads, canals, ponds, etc. This
edge effect diminishes rapidly toward the interior of the planting and is
generally well described by an inverse power function.
MeloCon WG® and SoilGard 12G® used in a program as a methyl
bromide alternative to control nematodes and soil borne diseases in
fruiting vegetables
H. B. HIGHLAND (1)
(1) Certis USA, Nokomis, FL
Phytopathology 100:S200
With the advent of the Montreal Accord of 2007 on restricting ozone
depleting gases, and as a result of further state led restrictions, the use of
methyl bromide and other fumigants in agriculture has been on a steady
decline. As such effective and safe alternative treatments are being
investigated, labeled and used in commercial production. The loss of
fumigants is especially deleterious to the production of fruiting vegetables,
primarily tomatoes and peppers, in the southeastern US, where soil borne
diseases and nematodes can be of particular concern. A program of MeloCon®
WG and SoilGard® 12 G, marketed by Certis USA, have been shown to be
very effective when used alone or in combination to control nematodes and
soil pathogens in field trials in the US. MeloCon® WG is a naturally occurring
and beneficial soil fungus (Paecilomyces lilacinus strain 251) that controls a
wide range of plant parasitic nematodes. MeloCon® WG has been shown in
replicated field trials to control both southern root knot nematodes
(Meloidogyne incognita) and stubby root nematodes (Trichodorus spp. and
Paratrichodorus spp.), as well as many others. SoilGard® 12G is also a
naturally occurring and beneficial soil fungus (Gliocladium (Trichoderma)
virens strain GL-21) that controls a wide range of soil borne pathogens,
including southern blight (Sclerotium rolfsii), Fusarium crown rot, and pepper
blight (Phytophthora capsici). Replicated field trials using tomatoes with
these products in conjunction with soil applied herbicides resulted in
improved plant growth, increased survival, and increased yields, similar to
methyl bromide and other chemical standards.
Yield loss associated with sheath blight disease of rice
C. A. HOLLIER (1), D. E. Groth (2)
(1) Department of Plant Pathology and Crop Physiology, LSU Agricultural
Center, Baton Rouge, LA, USA; (2) Rice Research Station, LSU Agricultural
Center, Crowley, LA, USA
Phytopathology 100:S201
Sheath blight is one of the most important rice diseases in the southern USA
rice-producing area. Yield loss estimates are made annually but accurate
measurements need to be taken. Fungicides were used a tool to influence
sheath blight development in small plots. Applications were made with the aid
of CO2– pressurized sprayers delivering 93L/ha of solution at various times to
halt or delay disease development to determine the affect of disease
development on yield at different growth stages. Early disease development
on enclosed canopy rice reduced yield greater than epidemics that were halted
until late stages of crop development. Yield losses ranged from 7.83% for
heading stage development (late developing disease), 16.65% for boot stage
development (intermediate developing disease) to 28.63% for green ring stage
development (early developing disease).
Management strategies for Pierce’s disease: An increasing threat to grape
production in the southern US
D. HOPKINS (1)
(1) University of Florida, Mid-Florida REC, Apopka, FL
Phytopathology 100:S201
Pierce’s disease (PD) of grapevine, caused by Xylella fastidiosa, affects grape
production across the southern U.S. and is especially damaging in the
Southeast, where it is the primary factor limiting the development of a grape
industry based on the high-quality Vitis vinifera grape. PD is increasing in
severity in the southeastern U.S. as a result of warmer winter temperatures,
increasing the risk of PD in the Piedmont region. Currently, the only feasible
control for PD in most of the southeastern U.S. is genetic plant resistance.
Management strategies currently being used or tested include vector control,
removal of reservoir hosts, various transgenes in grape cultivars or rootstocks,
and biological control with benign strains of X. fastidiosa. In Temecula CA,
an area wide leafhopper vector management program has been credited with
saving the grape industry from a 100% loss to PD. Several transgenic grape
lines are ready for field trials to evaluate resistance to PD, including lines
containing transgenes for anti-microbial proteins, for programmed cell death,
and for diffusible signal factor. In Florida, injection of a benign strain (EB921) of X. fastidiosa into transplants has controlled PD in a Vitis vinifera cv.
Cabernet Sauvignon planting for 13 years. This control could be available for
commercial use in 2–3 years.
Occurrence of boscalid-insensitive isolates of Didymella bryoniae in
commercial watermelon fields in South Carolina
A. P. KEINATH (1), E. Fillippeli (1)
(1) Clemson Coastal REC, Charleston, SC, USA
Phytopathology 100:S201
Insensitivity to boscalid in Didymella bryoniae, causal agent of gummy stem
blight on cucurbits, was found in Georgia in 2007. In 2009, isolates were
collected in South Carolina from watermelon leaves with symptoms of
gummy stem blight in four commercial fields in three counties and one
research plot. All five sites had been sprayed with a boscalid-pyraclostrobin
premixture (Pristine) in 2009 and in prior years. Sensitivity of these isolates to
boscalid was compared to sensitivities of isolates that had never been exposed
to boscalid collected from watermelon in 1998 or from muskmelon in 2002
and isolates previously exposed to Pristine collected from watermelon in
2005. When possible, the 2009 isolates were collected from sites sampled in
2005 and 1998. Suspensions of conidia and ascospores of 57 isolates were
placed on water agar amended with 0, 0.01, 0.10, 1.0, or 10.0 mg/l technical
grade boscalid. On each plate, spore germination was counted after 24 h.
Relative percentage germination (germination on amended media/germination
on nonamended medium) was regressed against the logarithm of fungicide
concentration to calculate EC50 values. Insensitive isolates were found at all
five sites sampled in 2009. Of 30 isolates collected in 2009, 13 had EC50
values >10 mg/l, 11 had EC50 values >1 mg/l, and 6 had EC50 values <1 mg/l.
EC50 values for all 27 isolates collected in 1998 to 2005 were <1 mg/l. Spores
of 27 of the 30 isolates collected in 2009 germinated on agar amended with
10.0 mg boscalid per liter compared to only 1 of the 27 isolates collected in
1998 to 2005. Ten isolates collected in 2009 were insensitive to 10 mg/l,
based on germination of >50% of conidia and ascospores on amended
medium. Isolates of D. bryoniae from South Carolina are now insensitive to
both pyraclostrobin and boscalid.
Baseline sensitivity to fluopicolide in Phytophthora capsici isolates from
the eastern United States
A. P. KEINATH (1), E. Fillippeli (1), M. K. Hausbeck (2), C. Kousik (3)
(1) Clemson Coastal REC, Charleston, SC, USA; (2) Michigan State
University, East Lansing, MI, USA; (3) USDA, ARS, Charleston, SC, USA
Phytopathology 100:S201
Fluopicolide was registered in 2007 to control diseases caused by Oomycete
pathogens such as Phytophthora capsici on cucurbits and peppers. In this
study, 69 isolates of P. capsici from Michigan (24 isolates), South Carolina
(17), Georgia (14), Florida (11), and North Carolina (3) recovered from
watermelon (22), pepper (11), bean (10), squash (9), cucumber (6), or
unknown hosts (11) were tested to determine their sensitivities to fluopicolide.
In three assays, isolates were grown on V8 agar amended with technical grade
fluopicolide dissolved in DMSO. For the mycelial growth assay,
concentrations were 0, 0.03, 0.10, 0.30 and 1.0 mg/l. For the sporangia
production assay, concentrations were 0, 0.03, 0.10, and 0.30 mg/l with a few
isolates also tested at 0.01 mg/l. For the zoospore germination assay, isolates
were initially tested at 0.10, 1.0, and 10.0 mg/l; some isolates then were tested
at 0.03 or 31.6 mg/l. Percentage colony diameter, zoospore germination, and
sporangia production relative to the nonamended control was regressed
against the logarithm of fungicide concentration to calculate EC50 values. All
isolates of P. capsici tested were sensitive to fluopicolide in all three assays.
EC50 values for each assay were non-normally distributed. The median
concentration was 0.28 (range 0.11 to 1.56), 0.04 (<0.01 to 0.14), and 2.08
(0.14 to 13.74) mg/l in the mycelial growth, sporangia production, and
zoospore germination assays, respectively. The ratio between the least and
most sensitive isolates was 14 for mycelial growth and sporangia production.
For zoospore germination, the ratio was 98 across all isolates but ranged from
3 to 44 for isolates within states. For mycelial growth and zoospore
germination, isolates from Michigan had a higher mean EC50 value than
isolates from other states (P < 0.05). Zoospore germination was much less
sensitive and sporangia production was much more sensitive to fluopicolide
than mycelial growth was.
Redbud yellow ringspot disease: Thirty years of research
A. G. LANEY (1), R. C. Gergerich (1), I. E. Tzanetakis (1)
(1) University of Arkansas
Phytopathology 100:S201
In the 1970s a disease was found infecting eastern redbud, Cercis canadensis.
Symptoms include chlorotic ringspots, oak-leaf, and vein chlorosis in mature
leaves and are usually expressed early in the season. Previous work revealed
the presence of virus-like double membrane-bound bodies in diseased plants.
Similar bodies have been found associated with several diseases including
rose rosette, high plains disease, fig mosaic, European mountain ash ringspot,
and thistle mosaic. Recently, the genomes of the viruses associated with Fig
mosaic (FMV) and European mountain ash ringspot (EMARaV) were
sequenced, and found to be negative sense ssRNA viruses related to
tospoviruses. We have obtained sequence information of a virus found in
yellow ringspot diseased plants, provisionally named Redbud yellow ringspotassociated virus (RYRaV). Detection protocols have been developed and used
in a survey of symptomatic redbud trees. RYRaV was found closely
associated with diseased trees as more than 90% of tested material was
infected with the virus. Potential field alternative hosts were surveyed and a
several herbaceous hosts were inoculated mechanically and by grafting.
Transmission studies using eriophyid mites are under way.
Genetic diversity of the Sclerotinia homoeocarpa population in Florida
D. LIBERTI (1), G. T. Cooper (1), J. A. Rollins (1), P. F. Harmon (1)
(1) University of Florida, Plant Pathology Department, Gainesville, FL
Phytopathology 100:S201
Dollar spot disease of turfgrass, caused by the fungus Sclerotinia
homoeocarpa, is the most important turfgrass disease occurring world-wide
on all cool and warm season turfgrass species. The ribosomal DNA (rDNA)
sequences were obtained from twenty-six isolates collected from Floridian
warm season turfgrass species including bermudagrass (Cynodon dactylon
(L.) Pers.), seashore paspalum (Paspalum vaginatum Sw.), St. Augustinegrass
(Stenotaphrum secundatum (Walter) Kuntze) and zoysiagrass (Zoysia
japonica Steud.) and from the Floridian cool season turfgrasses, rough
Vol. 100, No. 6 (Supplement), 2010
S201
bluegrass (Poa trivialis L.) and creeping bentgrass (Agrostis palustris Huds).
Isolates were collected from 26 distinct golf courses and other turfgrass
swards in 12 Florida counties between 2004 and 2009. These data plus 14
other S. homoeocarpa rDNA sequences from GenBank were subjected to
phylogenetic analysis using the neighborg-joining method and choosing
Sclerotinia sclerotiorum (Lib.) De Bary and Poculum henningsianum (Plottn.)
T. Schumach. as outgroups. Phylogenetic reconstructions based on sequences
of internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2)
indicated that twenty out of twenty-six Floridian isolates clustered in a group
that represents a newly identified biotype of S. homoeocarpa. Further
characterization of this Floridian biotype is in progress.
Effect of southern root-knot nematode (Meloidogyne incognita) on cotton
growth, yield and fiber quality
P. LU (2), R. C. Kemerait (2), C. D. Perry (2), R. F. Davis (1)
(1) USDA-ARS, Tifton, GA, USA; (2) University of Georgia, Tifton, GA,
USA
Phytopathology 100:S202
Southern root-knot nematode (SRKN) (Meloidogyne incognita) is a major
pest of cotton worldwide. Much research has been devoted to impact of
SRKN on yield; less information is available regarding impact of SRKN on
fiber quality. Objectives were to assess impact of SRKN on growth, yield, and
fiber quality at five sites planted to cotton in Georgia in 2008 and 2009. Three
sites were planted to DPL 555B/RR and two were planted to Fiber Max
9063B2F and Stoneville 4554B2RF. A randomized complete block design
with 4-6 replications was used at each site. Risk management zones for SRKN
were established at three locations based upon characteristics to include
elevation, slope, soil electroconductivty and NDVI from bare soil reflectance.
Nematicides (aldicarb, 3-6 lb/A), 1,3-dichloropropene (3-6 gal/A) and two
seed treatment nematicides were used to create differential populations of
SRN. Growth of the crop, soil populations of root-knot nematodes, and
damage to the plants were assessed. Cotton growth and yields were often
significantly and negatively correlated to root gall ratings, populations of
nematode juveniles and the number of SRKN eggs extracted from root
samples. However, most fiber quality parameters were not correlated to soil
nematode populations or damage to the cotton plants. However, in fields with
higher populations of SRKN, fiber quality properties tended to be more
strongly correlated to nematode populations and subsequent root damage than
in fields with lower populations. Often, plant growth, yield and fiber quality
were significantly different between the high and low risk management zones;
however the impact of SRKN populations was not always clear.
Quantitative modeling of the effects of temperature and wetness duration
on germination and infection of cantaloupe by Pseudoperonospora
cubensis
K. N. NEUFELD (1), P. S. Ojiambo (1)
(1) Department of Plant Pathology, North Carolina State University, Raleigh,
NC
Phytopathology 100:S202
Cucurbit downy mildew caused by Pseudoperonospora cubensis is considered
the most damaging disease of cucurbitaceous crops worldwide. Three
response surface models were developed based on independent experiments in
which cantaloupe plants were inoculated with P. cubensis and exposed to a
range of leaf wetness durations (2–24 h) and fixed temperatures (5–30°C) in
growth chambers. Germination was assessed at the end of each wetness period
and infection was recorded 5 days after inoculation as percent leaf area with
chlorotic and necrotic symptoms. Models were evaluated for their ability to
predict germination and infection of P. cubensis. Optimum germination and
infection was observed at 16.5 and 20.6°C, respectively, while little
germination or infection occurred at 5 or 30°C. Optimum infection for
wetness periods 4–8 h was observed at t = 20°C, but wetness periods > 8 h
had broader optimum curves. Model 1 of the form f(w,t) = f(t)•(1-exp
{-[Bw]D}) resulted in smaller asymptotic standard errors and yielded higher
correlations between observed and predicted germination and infection data
than either model 2 of the form: f(w,t) = A{1-exp[-f(t)•(w-C]D} or model 3:
f(w,t) = [1-exp(-Bw)2] / cosh[(t-F)G/2]. Models 1 and 2 had non-significant
lack-of-fit statistics while a lack-of-fit test was significant for model 3 for both
germination and infection data. These models accounted for up to 98% of the
total variation in the data. Risk threshold charts were developed to estimate
the potential risk of cucurbit downy mildew epidemics in the field based on
temperature and duration of leaf wetness.
Efficacy of strobilurin fungicides and host resistance for control of gray
leaf spot of corn
M. A. NEWMAN (1)
(1) University of Tennessee, Jackson, TN, USA
Phytopathology 100:S202
S202
PHYTOPATHOLOGY
Gray leaf spot (GLS) of corn, caused by Cercospora zeae-maydis is a
common foliar disease that reduces corn yields in Tennessee and many other
states. Two strobilurin fungicides (azoxystrobin and pyraclostrobin) have
shown a high degree of control of GLS in tests conducted over the last three
years (2006–2008) at the Research and Education Center at Milan, TN. Each
fungicide was sprayed at (01.1 kg/ha a.i.) with Penetrator Plus @ 0.125% v/v
as an adjuvant. Four-row plots 30’ long were randomized and replicated four
times. Rows were on 30” centers and planted no-till in a field infested with
GLS. The following three Pioneer corn hybrids with different levels of
resistance to GLS were used: susceptible P 32T22, moderately susceptible P
33R76 and tolerant P 33V14. Each fungicide was sprayed once over the top at
the VT growth stage (tassel) in 20 gallons of water per acre. Yield increases
over the untreated control for the three-year period were significantly greatest
for the susceptible hybrid for both fungicides. For the susceptible hybrid, the
average three-year yield increase over the untreated was 1613 kg/ha with
azoxystrobin and 1545 kg/ha with pyraclostrobin. The average three-year
yield increase using the moderately susceptible hybrid was 1210 kg/ha with
azoxystrobin and 470 kg/ha with pyraclostrobin. For the tolerant hybrid, the
average three-year increase in yield was 538 kg/ha with azoxystrobin and 403
kg/ha with pyraclostrobin respectively. These results indicate that spraying
strobilurin fungicides can increase corn yields, especially on the more GLS
susceptible hybrids.
Screening Gulf Coast forest species for susceptibility to Phytophthora
ramorum
J. A. PREUETT (2), D. J. Collins (2), T. L. Widmer (1), D. G. Luster (1)
(1) USDA/ARS Foreign Disease-Weed Science Research Unit, Fort Detrick,
MD, USA; (2) Urban Forestry Program, Southern University and A&M
College, Baton Rouge, LA, USA
Phytopathology 100:S202
Phytophthora ramorum, the causal agent of sudden oak death, is an emerging
pathogen of California oak woodlands. This pathogen poses a threat to woody
plants in many areas of North America, due to the broad host range of the
pathogen and the wide distribution of hosts. The US Gulf Coast area is
considered a high risk due to the suitable climate, but the question remains
whether Gulf Coast woody understory species represent possible hosts for the
pathogen. The following woody plant species, native to the Gulf Coast forest:
yaupon (Ilex vomitoria), spice bush (Lindera benzoin), southern magnolia
(Magnolia grandiflora), and eastern baccharis (Baccharis halmifolia) were
tested for their reaction to P. ramorum. This study was conducted at the
USDA/ARS plant pathogen containment greenhouse facility at Ft. Detrick,
MD. Foliage of four test plants was inoculated with 50,000 zoospores per ml
until the foliage was completely wet. The test was repeated three times for
each plant species. Inoculated plants were placed in a dew chamber at 20°C
for 4 days. After this incubation period, the leaves were detached, scanned on
a flatbed scanner, and the leaf lesion areas were assessed for disease using
ASSESS 2.0 software. Yaupon and southern magnolia appeared to be
susceptible to P. ramorum. The average percentage of lesion leaf area was 4.9,
0.2, 28.1 and 32.1% for inoculated spice bush, eastern baccharis, yaupon and
southern magnolia plants, respectively. This is compared to 1.2, 0.4, 0.1 and
0.6%, respectively, for the non-inoculated controls. We plan to continue this
research to analyze additional Gulf Coast forest plant species for reaction to P.
ramorum.
Characterization of rice blast resistance gene Pi-z(t) in rice germplasm
using DNA markers and pathogenicity assays
M. ROY-CHOWDHURY (1), Y. Jia (2), A. Jackson (2), M. Jia (2), R.
Fjellstrom (2), R. Cartwright (1)
(1) University of Arkansas, Cell and Molecular Biology Program,
Fayetteville, AR, USA; (2) USDA-ARS, Dale Bumpers National Rice
Research Center, Stuttgart, AR, USA
Phytopathology 100:S202
The Pi-z(t) gene in rice confers resistance to a wide range of races of the rice
blast fungus, Magnaporthe oryzae. The objective of the present study was to
identify Pi-z(t) in 131 worldwide rice germplasm using DNA markers and
pathogenicity assays. Four simple sequence repeat (SSR) markers (RM527,
AP4791, AP5659-1, AP5659-5) closely linked to Pi-z(t) were first used to
predict the existence of Pi-z(t) in rice germplasm and results were verified
using pathogenicity assays with an avirulent IE1k / two virulent races, IB33
and IB49. A total of 98 germplasm containing one to four SSR alleles for the
Pi-z(t) gene was found to be resistant to IE1k and susceptible to IB33 and
IB49, suggesting these germplasm contain different Pi-z(t) haplotypes.
Eighteen germplasm containing one to four SSR alleles were found to be
resistant to all three races, suggesting the presence of other R gene(s) in
addition to Pi-z(t). Five germplasm containing three to four SSR alleles were
found to be susceptible to all races, indicating the absence of the R gene(s) or
presence of non functional components of Pi-z(t) in these germplasm. Six
germplasm containing one to four SSR alleles, with one having novel alleles,
were found to be resistant to IB49 and IE1k but susceptible to IB33,
suggesting that other R gene in these germplasm confer resistance to IB49.
Three germplasm containing two to three SSR alleles were found to be
resistant to IB33 and IE1k and susceptible to IB49, suggesting the presence of
additional R gene(s) to IB33 in these germplasm. Finally, one germplasm with
novel SSR alleles was found to be resistant to all races, suggesting the
presence of Pi-z(t) independent R gene(s) in this germplasm. These
characterized germplasm should be useful for genetic studies and marker
assisted breeding for improving blast resistance worldwide.
Zebra chip of potato: Current status and future outlook
C. M. RUSH (1), D. C. Henne (1), F. Workneh (1) and N. Gudmestad (2)
(1) Texas AgriLife Research, Amarillo, TX; (2) North Dakota State
University, Fargo, ND
Phytopathology 100:S203
In Texas, potatoes are grown in the Rio Grande Valley, the Winter Garden
area near San Antonio, and the Panhandle. In 2000, potatoes from the lower
Rio Grande Valley displayed brown necrotic flecks and streaking of the
medullary rays. In fry tests, chips from these tubers exhibited dark brown
blotches and stripes, which were initially referred to as Texas Defect, but later
renamed Zebra Chip (ZC). Zebra Chip affects all market classes of potatoes
by reducing yield and quality, and now has been identified in California,
Colorado, Kansas, Nebraska, New Mexico and Wyoming. In areas where ZC
has become established, it is the most economically important impediment to
profitable potato production. Recently, a phloem-restricted proteobacterium,
Candidatus Liberibacter, has been associated with ZC. Two newly named
species, Ca. Liberibacter psyllaurous and Ca. Liberibacter solanacearum, have
been reported as etiological agents of the disease. Both are transmitted by the
potato psyllid Bactericera cockerelli, but sequence analysis suggests that the
two are likely the same species. However, slight differences between the two
suggest the possibility of strains. No genetic resistance to ZC has been
identified, insecticides for psyllid control are often ineffective, and factors
which impact disease epidemiology are largely unknown. In response to this
national threat to the potato industry, a multidisciplinary, multistate team of
researchers and extension specialists initiated a program with the goal of
reducing losses from ZC to economically sustainable levels by development
of a comprehensive, environmentally responsible disease management
program. To accomplish this, an advisory board of farmers and representatives
from ag-industry, together with the participating scientists, identified seven
priority focus areas (Disease Etiology and Vector/Pathogen Diversity,
Epidemiology, Pest Management, Breeding, Economics, Risk Assessment and
Technology Transfer), each with a number of specific objectives, which
together constitute an integrated systems approach to resolving the ZC
problem.
Etiology of zoysiagrass diseases in northwest Arkansas
T. N. SPURLOCK (1), E. A. Milus (1)
(1) University of Arkansas, Fayetteville, AR, USA
Phytopathology 100:S203
The increased use of zoysiagrass in Northwest Arkansas has raised awareness
of its susceptibility to a range of pathogens. The most destructive and
widespread disease is large patch caused by Rhizoctonia solani AG 2-2 (LP).
The disease occurs on warm season turfgrasses in the transition zone and is
very destructive to zoysiagrass in Northwest Arkansas. Symptoms include
irregular patches of dying turf up to several meters in diameter with distinct
lesions on the leaf sheaths and stems. Management of large patch with
fungicides is expensive and control is variable, possibly indicating that other
microorganisms are associated with the disease. The objectives of this study
were to determine if other microorganisms isolated from large patch areas
contribute to disease severity. Fungi and oomycetes were isolated from leaf
sheaths, stems, and rhizomes of zoysiagrass with large patch symptoms from
fairways of three golf courses in Northwest Arkansas. Isolates were grouped
by morphological characteristics and frequency of isolation recorded. R.
solani AG 2-2 (LP) was the most frequently isolated fungus from all sampling
locations. Isolates representative of other morphological groups were tested
for pathogenicity on Zoysia japonica cv. Meyer and Zoysia matrella cv.
Cavalier. Individually, R. solani and Gaeumannomyces graminis var. graminis
increased the proportion of shoots with lesions (DS) and decreased biomass
(B) over the non-inoculated control in both cultivars. When zoysiagrass was
inoculated with another isolate in addition to R. solani, a Fusarium isolate and
a sterile white basidiomycete caused significantly less disease (DS and B) in
Cavalier than R. solani alone. However, the addition of a Pythium isolate with
R. solani decreased B over R. solani alone in both cultivars and DS in Meyer.
Disease severity and growth for the combination of R. solani and G. graminis
var. graminis was not significantly different from these pathogens inoculated
singularly.
Comparison of seed treatments for control of soybean seedling diseases in
field soil at three temperatures
K. E. URREA (1), J. C. Rupe (1), C. S. Rothrock (1)
(1) University of Arkansas
Phytopathology 100:S203
Seedling diseases frequently reduce seed germination, seedling emergence,
stand, vigor, and yield, and sometimes require replanting. Since seedling
disease severity depends on the pathogens present in the soil and the
environmental conditions, evaluation of seed treatments and cultivars in the
field can be very erratic. The objectives of this research were to evaluate
selective and broad spectrum fungicide seed treatments and cultivars in field
soil under controlled environmental conditions. Three cultivars were treated
with six fungicide treatments or not treated and planted in soil collected from
two fields in April, May and June in 2008 and 2009. Tests were conducted in
growth chambers at 21°C (April soil), 25°C (May soil) or 28°C (June soil) and
soils were watered when matric potentials reached –30 J/kg. After two weeks,
the tests were rated for stand, root rot and plant growth and isolations were
made from the roots. In 2008, seed treatments resulted in greater stands than
the control at all three temperatures only for the cultivar Archer. In 2009, seed
treatments resulted in a significant increase in stands for low and high quality
seed of the cultivar Hutcheson, but not for HBK4924. Allegiance® was the
most effective fungicide tested across all the temperatures in increasing plant
stands. For all temperatures and soils, Pythium sylvaticum followed by
Fusarium oxysporum were the most frequently isolated pathogens from roots.
These results indicate that by controlling environmental conditions seed
treatment fungicides and the importance of different seedling pathogens can
be efficiently evaluated.
Cellular mechanisms that indicate needle health of seedlings of loblolly
pines
C. WALKINSHAW (1)
(1) USDA Forest Service, Pineville, LA
Phytopathology 100:S203
Health of needles on seedlings is readily apparent in pines. Symptoms of
disease are easy to recognize by observing the whole leaf or thin sections of
needle tissue. In this study histology techniques were used on both healthy
and diseased needles that were fixed, sectioned and examined for tannin and
other variables, including necrosis of resin ducts. The largest variation in
number of starch grains and cells with excess tannin occurred in the resin
ducts. These were lined with epithelial cells and had an outer layer of
parenchyma cells that were often torn during normal growth. Sporadic healing
occurred in areas near phenol cells. Energy for repair of these cells appeared
to originate from the collenchyma. Bands of phloem within the needle traces
had high starch content. Phenol oxidase, acid phosphatase and peroxidase
enzymes as measured by histochemical techniques, combined to hydrolyze
cell contents. These observations describe the biology of a number of cellular
changes that are associated with susceptibility of loblolly pine needles to
decline in the greenhouse and field.
Development of a screening protocol for assessing baseline sensitivity to
fungicides for Phakopsora pachyrhizi, the soybean rust pathogen
N. A. Ward (1), R. W. SCHNEIDER (1), C. G. Giles (1), C. L. Robertson (1)
(1) Department of Plant Pathology & Crop Physiology, Louisiana State
University Agricultural Center, Baton Rouge, LA
Phytopathology 100:S203
Soybean rust, caused by Phakopsora pachyrhizi, was first discovered in the
continental United States in November 2004. Since then it has been detected
throughout the U.S. and Ontario, Canada. The disease has been particularly
severe in the Southeast where producers are forced to apply fungicides. These
continual applications have the potential to select for fungicide resistant
strains of the rust pathogen, and these strains could easily overwinter along
the Gulf Coast on kudzu and other alternative hosts. The purpose of this study
was to develop a sensitive and repeatable assay for establishing baseline
sensitivity concentrations for two classes of fungicide chemistry, namely
triazoles and strobilurins. The following fungicides were included in this
study: tetraconazole (Domark®), flutriafol (Topguard®), azoxystrobin
(Quadris®), and pyraclostrobin (Headline®). Freshly produced urediniospores
were collected by brushing and discarding existing spores from infected
leaves with an artist’s paintbrush. These leaves were then incubated in a moist
chamber (25°C) for 2 days after which hyaline urediniospores were produced
in abundance on the lower leaf surface. These leaves were placed (lower
surface down) on a grid suspended over a plastic petri dish, and the upper leaf
surface was gently tapped. This dislodged the urediniospores into the dish,
and the spores were then dabbed with the brush to break apart clumps. Water
agar plates amended with various concentrations of the fungicides were
inoculated by touching spores in the spore collection plates with the tip of
the brush and then touching the surface of the amended agar plates with
Vol. 100, No. 6 (Supplement), 2010
S203
the brush. Spore germination was assessed after incubation for 4 hours in the
dark at 25°C.
Pod yield of peanut breeding lines from fields infested with Sclerotinia
minor or Verticillium dahliae
J. E. WOODWARD (1), M. R. Baring (3), C. E. Simpson (4), T. A.
Baughman (2)
(1) Texas AgriLife Extension Service, Lubbock, TX, USA; (2) Texas
AgriLife Extension Service, Vernon, TX, USA; (3) Texas AgriLife Research,
College Station, TX, USA; (4) Texas AgriLife Research, Stephenville, TX,
USA
Phytopathology 100:S204
Diseases such as Sclerotinia blight (Sclerotinia minor Jagger) and Verticillium
wilt (Verticillium dahliae Kleb.) can drastically reduce peanut (Arachis
hypogaea L.) yields in Texas. Field trials were conducted to evaluate the
performance of advanced peanut breeding lines in a field naturally infested
with S. minor. Pod yields were increased by 2457, 1391, 1226 and 981 kg ha–1
for breeding lines TX-3, TX-2, TX-1 and TX-4, respectively, when compared
to the commercial standard ‘Flavor Runner 458’. Yield for these breeding
lines were equivalent to or greater than that of the partially resistant cultivar
‘Tamrun OL07’. Separate trials were conducted on the Southern High Plains
to evaluate the performance of breeding lines TX-3, TX-4, TX-5 and TX-6 in
fields infested with V. dahliae. Pod yields were greatest for breeding line TX3 and ‘Flavor Runner 458’, 5038 and 4960 kg ha–1, respectively, whereas
yield was lowest for breeding line TX-5 (3966 kg ha–1). Pod yields for the
cultivars ‘McCloud’, TX-6, ‘Tamrun OL02’ and ‘Tamrun OL07’ where
intermediate ranging from 4476 to 4254 kg ha–1. Results from these studies
indicate that there are varying levels of resistance to S. minor and V. dahliae
among the breeding lines evaluated. Breeding line TX-3 may be suitable for
fields co-infested with both pathogens.
Characterization of interacting genes with the rice blast fungus
avirulence gene AVR-Pita
J. Xing (1), S. Lee (2), Y. JIA (2), L. Yuan (1)
(1) Central South University, Changsha, P.R. China; (2) Dale Bumpers
National Rice Research Center, Stuttgart, AR, USA
Phytopathology 100:S204
The AVR-Pita gene in Magnaporthe oryzae determines the efficacy of the Pita blast resistance gene. AVR-Pita encodes a predicted metalloprotease with
223 amino acids. AVR-Pita176 with deletion of 57 amino acids at the amino
terminus was previously shown to be involved in Pi-ta mediated blast
resistance as a putative effector protein. In order to study the role of AVR-Pita
in fungal pathogenicity and blast resistance, AVR-Pita176 was used as bait to
identify interacting genes from a yeast two-hybrid library constructed using
mRNAs isolated from a U.S. tropical japonica cultivar Katy leaves at different
time points after inoculation with M. oryzae. Identified AVR-Pita interacting
proteins will be verified using in Vitro binding techniques. In addition, three
predicted proteins, AVR-Pita223, AVR-Pita176, and AVR-Pita166 will be used
to examine interaction specificity in the yeast two-hybrid assays. The roles of
AVR-Pita interacting proteins in fungal pathogenicity and blast resistance will
be investigated and progress will be presented.
Histopathology of ‘rapid blight’, a disease caused Labyrinthula terrestris
on cool-season turfgrasses
K. YADAGIRI (1), J. L. Kerrigan (1)
(1) Entomology, Soils, and Plant Sciences, Clemson University, Clemson, SC,
USA
Phytopathology 100:S204
Rapid blight is a disease on cool-season turfgrasses, caused by a
microorganism known as Labyrinthula terrestris. Symptoms of rapid blight
include water-soaked lesions and browning or bronzing of foliage that lead to
S204
PHYTOPATHOLOGY
yellowing and death of the infected turf. So far, eleven states in the US on
both coasts have reported rapid blight, in addition to other countries including
the United Kingdom, Spain and Argentina. Saline irrigation water and soil are
favorable for disease causation. Rapid blight is more severe on salt-sensitive
varieties of turfgrasses that are mostly cool-season turfs, such as rough
bluegrass (Poa trivialis), perennial ryegrass (Lolium perenne), annual
bluegrass (Poa annua) and colonial bentgrass (Agrostis tenuis). Labyrinthula
terrestris is an unusual pathogen on turf; it belongs to a group of organisms
commonly referred to as marine net-slime molds, which have been primarily
known to cause diseases on sea grasses. Details of the host-pathogen
interactions of Labyrinthula terrestris on turfgrasses have not been
investigated. We are, therefore, documenting the infection processes and life
cycle using light and electron microscopy to better understand the
pathogenicity of this organism and, ultimately, apply these finding to provide
better means of controlling rapid blight disease.
Epidemiology of soybean rust in soybean sentinel plots in Florida
H. M. YOUNG (2), J. J. Marois (2), D. L. Wright (2), D. F. Narváez (1), G. K.
O’Brien (2)
(1) Monsanto Co., St. Louis, MI, USA; (2) University of Florida, NFREC,
Quincy, FL, USA
Phytopathology 100:S204
Since its discovery in 2004 in the Southeastern United States, soybean rust
(SBR) severity has been variable from year to year. It is important to
understand the epidemiology of the pathogen in Florida as it may serve as an
inoculum source for other areas of the country. This study examined the
incidence and severity of SBR in relation to prevailing weather data, growth
stage, and maturity group (MGIII, MGV, MGVII) in soybean plots (15 m
square) across north Florida that were part of the national sentinel plot
network from 2005 through 2008. On average, plots became infected 30 days
earlier in 2008 than 2005. Precipitation was the principle factor affecting
disease progress, where disease increased rapidly after rain events and was
suppressed during dry periods. In 2008, there was a significant increase in
disease incidence and severity as reflected in the area under the disease
progress curve. This was associated with the occurrence of Tropical Storm
Fay, which deposited up to 290 mm of water in the plot locations. Results
from this study may lead to a better understanding of the impact of weather on
the epidemiology of this pathosystem.
Effect of temperature on latent period of Stagonospora nodorum blotch
on winter wheat under field conditions
A. D. ZEARFOSS (1), C. Cowger (2), P. S. Ojiambo (1)
(1) Department of Plant Pathology, North Carolina State University, Raleigh,
NC, USA; (2) USDA-ARS, North Carolina State University, Raleigh, NC,
USA
Phytopathology 100:S204
Stagonospora nodorum is the causal agent of Stagonospora nodorum blotch
(SNB) and yield losses from severe disease epidemics can be as high as 50%.
To establish a model for SNB development based on the effects of
temperature on pathogen latent period and life cycle relative to the host,
batches of two winter wheat cultivars (AGS 2000 and USG 3209) were
inoculated with pycnidiospores of S. nodorum at weekly intervals over a 1
year period. After an incubation period of 72 h, plants were exposed to field
conditions where prevailing temperatures ranged from 5°C to 28°C with a
mean batch temperature of 9°C to 24°C. Latent period until the first visible
symptoms ranged from 8 to 34 days. The relationship between development of
lesions with pycnidia and accumulated thermal time will be described using a
shifted cumulative gamma distribution model using estimated base
temperatures. These results will provide valuable data that link crop growth
models with the progress of SNB and facilitate the establishment of disease
development models for use in timing fungicide applications.
2010 Potomac Division Meeting Abstracts
Abstracts presented at the APS Potomac Division meeting in Ocean City, Maryland, March 24–26, 2010. The abstracts are arranged alphabetically, by first
author’s name.
Host range determination of Colletotrichum gloeosporioides f. sp. salsolae,
a biological control agent of tumbleweed: From BLUPs to biomass loss
D. K. Berner (1), C. A. CAVIN (1)
(1) USDA, ARS, FDWSRU
Phytopathology 100:S205
Host range tests were conducted with Colletotrichum gloeosporioides f. sp.
salsolae (CGS) in quarantine to determine whether the fungus is safe to
release in N. America for biological control of tumbleweed (Salsola tragus L.,
Chenopodiaceae). Ninety two accessions were analyzed from 19 families and
10 tribes within the family Chenopodiaceae. These included 62 genera and
120 species. Disease reaction data were combined with a relationship matrix
derived from internal transcribed spacer DNA sequences and analyzed with
mixed model equations to produce Best Linear Unbiased Predictors (BLUPs)
for each species. Twenty nine species from 7 closely-related Chenopodiaceae
tribes had significant levels of disease severity as indicated by BLUPs. Most
species in the genus Salsola, which are all introduced and weedy, were very
susceptible and damaged by CGS. Of the 29 susceptible species, 10 native or
commercially important species in N. America were identified as needing
additional tests to determine the extent of any damage caused by disease.
These additional tests were done by inoculating the non-target species of
concern with CGS and weighing oven-dried shoots and roots of noninoculated and inoculated plants one month after inoculation. The shoots and
roots of each plant were scanned and the surface areas determined with image
analysis software. The damage to the shoots and roots of each plant were
standardized by dividing surface area by the corresponding dry weights to
arrive at area per unit weight. Average differences in standardized plant
damage between inoculated and controls for each plant species were
combined with corresponding disease ratings and analyzed by principal
component analysis. Results showed that most of the non-target species
clustered as not-damaged while the target and several related weedy species
were heavily damaged. Three non-target species were moderately damaged,
but these species were either perennial or not ecologically sympatric with
tumbleweed.
Extract of the brown seaweed Asophyllum nodosum and silicon reduce
plant death due to Fusarium spp. of cucurbits
G. E. Brust (3), R. E. ROSS (1), J. Jayaraj (2)
(1) Acadian Seaplants LLC, Dartmouth, NS, Canada; (2) Department of Life
Sciences, The University of the West Indies, St. Augustine, Trinidad; (3)
University of Maryland, Upper Marlboro, MD, USA
Phytopathology 100:S205
Crop losses due to Fusarium spp. are important to cucurbit growers along
with an increasing interest in natural ways to improve disease resistance.
Extracts of the brown seaweed, Ascophyllum nodosum, and products
containing silicon have both been shown to promote disease resistance in
many crops. In a 2008 watermelon trial located in Upper Marlboro, MD,
Fusarium solani symptoms were suppressed by extracts of A. nodosum. At the
final rating, 30% of the watermelon plants were dead from this pathogen in
the control plots vs. 10% in A. nodosum extract treatments. A second study
The abstracts are published as submitted. They were formatted but not
edited at the APS headquarters office.
was implemented in 2009 on Gladiator Pumpkins. Calcium silicate and A.
nodosum extract were applied to pumpkins grown in a field known to have
Fusarium spp. infected squash three years prior. At the final rating, 24.6% of
the pumpkin plants were dead in the control plots vs. 19.2% in the silicon
plots, 13.6% in the A. nodosum extract treatment, and just 6.1% in the plots
with both calcium silicate and A. nodosum extract. These field trials were
further supported by two greenhouse studies where applications A. nodosum
extract applied to cucumber plants reduced incidence of Fusarium oxysporum
and enhanced the activities of plant defense-related enzymes including
chitinase, beta-1,3-glucanase, peroxidase, polyphenol oxidase, phenylalanine
ammonia lyase and lipoxigenase as well as elevated levels of total phenols
compared to the control. The jasmonic acid pathway has been found to be
very important in plant defense responses elicited by A. nodosum. Pathogens
that are inhibited by the jasmonic acid pathway are often necrotrophs such as
Fusarium spp. A. nodosum extract may offer a valuable tool to improve the
health and productivity of cucurbits.
Detection and distribution of Bean pod mottle virus in soybean and beetle
vectors in eastern Virginia
M. E. CASSELL (1), T. P. Kuhar (2), P. B. Schultz (3), S. A. Tolin (1)
(1) Virginia Tech, Blacksburg, VA; (2) Virginia Tech-ESAREC, Painter, VA;
(3) Virginia Tech-HRAREC, Virginia Beach, VA
Phytopathology 100:S205
Bean pod mottle virus (BPMV) (genus Comovirus; family Comoviridae) is a
reemerging disease of soybeans. Vectored by the Bean leaf beetle (Cerotoma
trifurcata), this virus has become prevalent on the Eastern Shore of Virginia.
In tissue blot immunoassays (TBIA) of soybean sentinel plots for the Legume
IPM-PIPE in 2007, BPMV was detected at a high incidence at the Eastern
Shore station, but not at the Tidewater station. In 2008, beetles collected at the
Eastern Shore station were 80% positive for BPMV by TBIA, but all nonsoybean legumes tested were TBIA-negative. In a systemic survey in 2009,
BPMV was detected by TBIA in 16 of 42 soybean fields from the southern tip
of the Eastern Shore to southern Maryland. Up to 100% of the beetles
collected from 24 of 38 Virginia fields were positive for BPMV by ELISA of
individual beetles. Infectious virus was recovered from beetle extracts
prepared for ELISA. In 2009, an outbreak of BPMV was also detected in two
counties in the Northern Neck of Virginia. The primary inoculum of BPMV
remains unknown. Sampling is being conducted on the Eastern Shore to locate
plants that might serve as an early season source of BPMV for acquisition by
overwintering or first generation beetles.
Impact of mowing and fertility practices on weed species and brown
patch dynamics in rhizomatous tall fescue
M. A. CUTULLE (3), D. McCall (2), B. Horvath (1), J. Derr (3)
(1) University of Tennessee, Knoxville, TN; (2) Virginia Tech, Blacksburg,
VA; (3) Virginia Tech, Virginia Beach, VA
Phytopathology 100:S205
Tall Fescue (Festuca arundinacea) is a commonly utilized turfgrass in the
temperate and transition zone areas of the United States. It establishes quickly,
requires moderate amounts of nitrogen, and is resistant to most diseases.
However, during hot humid summers, tall fescue is under stress and is
susceptible to Rhizoctonia solani infection. The resulting disease, referred to
as brown patch, causes turf thinning, leading to encroachment from weeds
Vol. 100, No. 6 (Supplement), 2010
S205
such as bermudagrass (Cynadon dactylon). Cultural practices such as fertility
and mowing height may impact bermudagrass encroachment and brown patch
disease in tall fescue. Improved brown patch control may result in lower weed
infestations. Two mowing heights (5 and 10 cm), three levels of fertility (49,
171, and 220 kg of nitrogen annually per hectare), and preemerge herbicide
application (ronstar or no herbicide applied in 2009 only) were evaluated in an
established stand of ‘RTF’ tall fescue. Three plugs of common bermudagrass
were planted in each plot in May 2008. Data collected monthly included weed
composition and density, bermudagrass diameter, brown patch severity, and
turf quality. The experiment was repeated in May of 2009. Mowing height had
a significant effect on bermudagrass in year one and year two. A higher
mowing height resulted in less bermudagrass encroachment. Fertility did not
have an effect on bermudagrass diameter. In July and August, southern
crabgrass (Digitaria ciliaris) density was much greater in the 5 cm mowing
height plots. Tall fescue cover was significantly reduced in the 5 cm mowing
treatment due to weed competition but was acceptable at the 10 cm height.
Higher fertility resulted in increased brown patch severity. However, these
plots recovered quickly when weather was cooler and dryer. The same trends
were observed in year two, though incidence of brown patch was greater in
year two due to the increased precipitation.
A new phytoplasma lineage is associated with diseased juniper (Juniperus
occidentalis)
R. E. DAVIS (1), E. L. Dally (1), Y. Zhao (1), I.-M. Lee (1), R. Jomantiene
(2), A. J. Detweiler (3), M. L. Putnam (4)
(1) USDA-Agricultural Research Service, Beltsville, MD, USA; (2) Nature
Research Center, Vilnius LT-08406, Lithuania; (3) Oregon State University
Deschutes County Extension, Redmond, OR, USA; (4) Oregon State
University Plant Clinic, Corvallis, OR, USA
Phytopathology 100:S206
Phytoplasmas are wall-less, prokaryotic plant pathogens that are spread from
plant-to-plant by insects and are the cause of diseases in a wide range of plant
species that include angiosperms and gymnosperms. Worldwide, work is
underway to determine the possible association of phytoplasmas with plant
diseases of unsolved cause, in order to devise disease control and quarantine
measures based on knowledge of the pathogens involved. The present work
focused on a disease (juniper witches’ broom, JunWB) of Juniperus
occidentalis, a native tree indigenous to parts of western USA. Amplification
of ribosomal RNA gene sequences (rDNA) in polymerase chain reactions
(PCRs) primed by phytoplasma-universal primers indicated that a
phytoplasma was associated with the disease. Nucleotide sequences of the
rDNA were analyzed using a computer-based interface, iPhyClassifier, to
obtain virtual RFLP patterns of 16S rDNA; the results indicated that JunWB
phytoplasma represented a new lineage in the pigeon pea witches’ broom
phytoplasma group (16SrIX). The findings expand the known biodiversity of
phytoplasmas infecting conifers and raise the question of whether J.
occidentalis, previously undescribed as a phytoplasma host, could play a role
in the spread of phytoplasmal disease potentially damaging to forest and/or
landscape conifers in North America.
Effects of exogenous indole-3-acetic acid on transcriptional
reprogramming of hormone signaling and metabolism genes in potato
purple top phytoplasma-infected tomato plants
Y. DING (1), W. Wei (1), W. Wu (1), Y. Jiang (1), R. E. Davis (1), Y. Zhao
(1)
(1) Molecular Plant Pathology Laboratory, ARS-USDA, Beltsville, MD
Phytopathology 100:S206
Phytoplasmas are plant pathogenic bacteria that lack a cell wall. Plants
infected by phytoplasmas exhibit various symptoms indicative of disrupted
hormonal balance. Observations that exogenous application of auxins on aster
yellows phytoplasma-infected periwinkle plants could induce symptom
remission or even phytoplasma elimination further point to crucial roles of
plant hormones in phytoplasma pathogenesis. The present study was designed
to gain an insight into expression profiles of plant hormone signaling and
metabolism genes in healthy vs phytoplasma-infected plants, and to examine
whether exogenously applied indole-3-acetic acid (IAA, a naturally occurring
auxin) would modify the expression patterns of these genes. Columbia Basin
potato purple top (PPT) phytoplasma (a member of subgroup 16SrVI-A) and
its alternate host Rutgers tomato were used as a model pathogen-host pair.
Our preliminary data revealed that, following graft inoculation of plants with
PPT phytoplasma, expression patterns of a putative IAA biosynthesis gene
and an F-box protein-encoding gene responsible for IAA signaling were
altered. Exogenously applied IAA was able to reverse the course, bringing
expression of the two genes to the levels comparable to those in healthy and
mock-inoculated tomato plants. The findings provide a clue to understanding
mechanisms of phytoplasma pathogenesis and exogenous auxin-induced
phytoplasmal disease remission.
S206
PHYTOPATHOLOGY
The science and art of photography for art and science
J. D. EISENBACK (1)
(1) Department of Plant Pathology, Physiology, and Weed Science, Virginia
Tech, Blacksburg, VA
Phytopathology 100:S206
Several art historians have noted that paintings suddenly improved in detail
and realistic representation sometime around 1420 AD. This remarkable
enhancement of drawings and paintings was credited to use of a convex mirror
to project a scene onto a canvas. As the technology of glass production
improved, conventional lenses were made for projecting images onto a canvas
inside a large dark room, called a camera obscura. In the early 1800s light
sensitive paper replaced the canvas and pigments, and the photograph was
born. The camera obscura was downsized to the more mobile camera, a small
box with a lens and a holder for light sensitive film. Cameras and photography
became incorporated into nearly every facet of human activity, including
scientific documentation. The discovery of the photovoltaic effect by Albert
Einstein initiated the development of digital cameras in the late 1900s. Digital
photography allows images to be readily manipulated in several ways,
including high dynamic range (HDR) photography, multiple focus
photography, and the production of megapixel mosaic photographs that can be
utilized for art and science.
Effect of aging on survival and heat tolerance of anhydrobiotic seed-gall
nematode, Anguina agrostis
J. D. EISENBACK (1), C. W. Roane (1), W. Ma (1)
(1) Department of Plant Pathology, Physiology, and Weed Science, Virginia
Tech, Blacksburg, VA
Phytopathology 100:S206
A seed gall nematode, Anguina agrostis, was found parasitizing redtop
creeping bentgrass (Agrostis stolonifera), that over winters in an
anhydrobiotic state. Infested grass seed was collected on Aug. 24, 2003 and
Aug. 16, 2009 from a naturally infested site on Butt Mountain Lookout, near
the fire watchtower, in Giles County, Virginia. Samples that were stored in an
open plastic bag in a laboratory cabinet for more than 5 years were compared
to freshly collected specimens. Seed galls were soaked in tap water for 24 hr.
to evaluate the survival of juvenile nematodes. Additional seed galls were
exposed to high temperatures in a glass test tube immersed in hot water for 5,
10, 15, and 30 min. at 80, 90, and 100°C each. The nematodes were freed
from the gall with sharply pointed forceps after the gall was placed into water
for 24 hr. All treatments were replicated 6 times. Fresh galls contained an
average of 630 nematodes, of which 82% were alive; five-year-old galls had
694 nematodes, of which 72% survived. The survival of nematodes in galls
that were heat-treated at 80°C for 30 min. was 90% in fresh galls and reduced
to 68% in 5 year old galls. Fresh galls exposed to 90°C for 30 min. survival
was 68% and only 10% in five year old galls. All of the individuals were
killed in five year old galls that were treated at 100°C for 15 min.; however,
there was 35% survival in fresh galls. These experiments demonstrate that
Anguina agrostis has remarkable heat tolerance that gradually declines as it
ages.
Host range determination of Synchytrium solstitiale: Issues as a candidate
for biological control of yellow starthistle
F. M. ESKANDARI (1), W. L. Bruckart (1), T. L. Widmer (1)
(1) USDA, ARS, FDWSRU, Ft. Detrick, MD, USA
Phytopathology 100:S206
Synchytrium solstitiale, a chytrid recently evaluated for biological control of
yellow starthistle (YST, Centaurea solstitialis), was found to cause infections
on seedlings of commercially-important safflower (Carthamus tinctorius) and
two North American natives, Centaurea americana and C. rothrockii (10%,
10%, and 25% incidence, respectively) when cotyledons were exposed to
zoospores released in water. This compared to >90% incidence for inoculated
YST seedlings in that same study. The object of the present study was to
confirm susceptibility of safflower to S. solstitiale. Surface-sterilized leaves of
YST (as susceptible control), safflower, Russian knapweed (Rhaponticum
[Acroptilon] repens), and common crupina (Crupina vulgaris), were floated
on sterile water in large (15 cm) glass Petri dishes containing zoospores. Five
pieces of surface-sterilized galled leaf tissue were placed at four locations
around the perimeter and at the center of each dish to give uniform
distribution of zoospores in each test. Leaves of each test species were paired
with YST leaves in each dish, thus providing uniform inoculum for each test
species and YST within a dish. At least three dishes were set up for tests.
Results confirm that both YST and safflower are susceptible to S. solstitiale
and that Russian knapweed and common crupina are not. Data from both
studies suggest differential susceptibility to S. solstitiale occurs among plants
within the Asteraceae. Before proposal is made to introduce S. solstitiale for
biological control of YST in the U.S., additional data will be needed
concerning potential risk associated with such action, particularly relating to
safflower and safflower culture in California.
Phytophthora pini Leonian, a valid and distinct species
M. GALLEGLY (2), C. Hong (1), P. Richardson (1), P. Kong (1)
(1) Virginia Tech, Virginia Beach, VA; (2) West Virginia University,
Morgantown, WV
Phytopathology 100:S207
Leonian described Phytophthora pini in 1925. His one culture, isolated from
roots of red pine growing in Minnesota, was essentially ignored as a species
until 1956 when Waterhouse included it in her compilation of original
descriptions. Later, in 1963, Waterhouse considered P. pini to be invalid
because it was morphologically identical to P. citricola described by Sawada
in 1927. We secured the ex-type and ex-authentic cultures of P. citricola, P.
pini, and Chester’s P. cactorum var. applanata and compared them
morphologically and molecularly with each other and with isolates of P.
plurivora Jung and Burgess (same as Gallegly and Hong’s P. citricola II), P.
citricola I and P. citricola III. The results show that P. pini is identical to P.
citricola I in all molecular and morphological characters and different from P.
plurivora, the ex-type P. citricola, and P. citricola III. Incidentally, the extype culture of P. cactorum var. applanata is identical to isolates of P.
plurivora. Thus, Phytophthora pini is resurrected to distinct species status.
Characterization of Pythium species causing Pythium blight on snap
beans in the eastern US
L. HARRISON (1), S. L. Rideout (1)
(1) Virginia Tech, Painter, VA, USA
Phytopathology 100:S207
The Eastern Shore of Virginia (ESV) is an important snap bean (Phaseolus
vulgaris L.) growing region, but profitable yields are threatened by Pythium
blight, one of the most severe snap bean diseases in the US. Although this
disease is well documented, the species of Pythium causing this disease have
not been well characterized. This information is important for determining
management strategies. Isolates were collected to establish the causal agent(s)
of Pythium blight on snap beans. Because of the pathogen’s wide host range
and distribution, isolates were recovered from different hosts, including other
legumes, cucurbits and solanaceous crops, and from multiple snap beangrowing areas in the ESV, Georgia, and New Jersey. Isolates were collected
from soil by baiting and from plant tissue showing water-soaking and/or
white, cottony growth. For each isolate, pathogenicity on snaps beans was
verified, and each isolate was characterized by morphology and sequence
analysis of the rDNA-internal transcribed spacer (ITS) regions. All ESV
isolates were identified as Pythium aphanidermatum, except for one Pythium
myriotylum and one Pythium ultimum isolate. Both P. aphanidermatum and P.
ultimum were recovered from New Jersey crops. P. aphanidermatum was also
isolated from symptomatic plants in Georgia; however, multiple Georgia
isolates had 99-100% ITS sequence similarity with Pythium deliense Meurs
accessions in GenBank. These isolates also had P. deliense-characteristic
morphology, producing oospores measuring 16.5 µm diameter and similar, but
less inflated, sporangia than P. aphanidermatum. Putative P. deliense isolates
will be further characterized by sequence analysis of the cytochrome oxidase
II gene. P. deliense has not yet been reported on common bean. This research
verifies that multiple Pythium spp. are responsible for Pythium blight
symptoms on snap beans.
Does a Vicia villosa cover crop induce general suppression of Fusarium
wilt of watermelon?
J. HIMMELSTEIN (2), K. Everts (2), J. Maul (1)
(1) Sustainable Agriculture Systems Laboratory, USDA-ARS, Beltsville, MD,
USA; (2) University of Maryland, College Park, MD, USA
Phytopathology 100:S207
Current triploid watermelon cultivars have little resistance to Fusarium wilt,
caused by Fusarium oxysporum f. sp. niveum (FON) and yield losses are
increasing in the eastern U.S. A Vicia villosa (hairy vetch) green manure,
suppressed watermelon Fusarium wilt in previous trials, but the mechanism of
this suppression is unknown. The objective of this experiment was to
determine if the V. villosa cover crop suppresses Fusarium wilt via general
suppression by looking at the rate of soil respiration and microbial activity in
the presence of three cover crops at two locations in Maryland. Fall planted V.
villosa, Trifolium incarnatum (crimson clover), and Secale cereale (rye) were
grown and incorporated as green manures into the soil prior to planting the
watermelon. FON was applied three days after transplanting at the Salisbury
location and five days after transplanting in Beltsville. No visible wilt
symptoms were observed at either location. Hairy vetch and crimson clover
caused a significant increase in total fruit yield at Beltsville. The respiration
data revealed that microbial activity increased significantly directly after
cover crop incorporation, then dropped down to pre incorporation levels for 3
months. As the watermelon matured the respiration rate increased once again.
Crimson clover and hairy vetch plots had similar respiration rates and we are
testing for correlations between cover crop induced changes in soil respiration
and disease suppressiveness.
Multianalyte immunohistochemical investigation of relative hormone
levels in potato purple top phytoplasma-infected tomato plants
Y. JIANG (1), W. Wei (1), Y. Ding (1), W. Wu (1), R. E. Davis (1), R. W.
Hammond (1), Y. Zhao (1)
(1) Molecular Plant Pathology Laboratory, ARS-USDA, Beltsville, MD
Phytopathology 100:S207
Phytoplasmas are small, cell wall-less bacteria responsible for numerous
diseases in agriculturally and environmentally important plant species
worldwide. Phytoplasma infections of plants induce symptoms including
excessive shoot proliferation, witches’-broom growths, general stunting, rapid
senescence (yellowing), and abnormal floral development (virescence and
phyllody). These symptoms indicate that hormonal balance may be disrupted
in affected plants. The lack of plant hormone biosynthesis genes in all
completely-sequenced phytoplasma genomes implies that the presumed
hormonal imbalance in phytoplasma-infected plants may be caused either by
changes in endogenous hormone levels or by alterations in sensitivity to
hormones. The present study was aimed at understanding the mechanism
underlying phytoplasma-induced host hormonal imbalance. Tissue sections
prepared from Columbia Basin potato purple top (PPT) phytoplasma-infected
and
healthy
tomato
plants
were
subjected
to
comparative
immunohistochemical analyses using antibodies against auxin (IAA),
cytokinins (6-BA, trans-zeatin riboside, and cis-zeatin riboside), abscisic acid
(ABA), and gibberellic acid (GA3). The results revealed notable changes in
levels of plant hormones in PPT-infected vs healthy plants. Findings from the
study will aid understanding of the roles of plant hormones in phytoplasma
pathogenesis and disease symptom expression.
Phytophthora phaseoli; destroyer of lima bean production
S. G. KUNJETI (1), N. M. Donofrio (1), A. G. Marsh (1), B. C. Meyers (1),
T. A. Evans (1)
(1) University of Delaware
Phytopathology 100:S207
Phytophthora phaseoli Thaxt., an oomycete plant pathogen and close relative
to Phytophthora infestans (Blair et al. 2008), causes downy mildew of lima
bean (Phaseolus lunatus L.) during cool and humid weather conditions. Since
its first report in 1889, the vegetable processing industry of the humid eastern
US has been negatively affected by this disease. Since 1889 P. phaseoli has
evolved six races A,B,C,D,E and currently prevalent race F in lima bean
fields. Developing lima bean cultivars with durable resistance to this pathogen
is a more environmentally friendly and cost-efficient method of disease
management than pesticide application. To develop such a cultivar, it is
necessary to understand the underlying mechanism of how the pathogen
breaks down the plant’s defenses. To date, nothing is known about the
molecular interactions that occur during this plant-pathogen interaction.
Towards a better understanding of these mechanisms, we used next generation
sequencing technology (Illumina) to compare global gene expression of plategrown and plant-grown P. phaseoli. Our computational analysis of the
transcripts showed that most of the effector genes that were over-expressed in
P. infestans while infecting potato leaf tissue were also over-expressed in P.
phaseoli while infecting lima bean hypocotyls. Some of the well-characterized
effectors like INF1, and INF4 were confirmed by performing RT-PCR using
plate-grown, plant-grown mycelium and lima bean pods infected with P.
phaseoli as template. Effector genes that were expressed in P. phaseoli when
infecting lima bean pods were consistent with the genes expressed when
infecting lima bean hypocotyls. Our results suggest that like in P. infestans
infection of potato, P. phaseoli requires the same effector genes for the
infection of lima bean pods and hypocotyls.
Comparison of the detection of Xanthomonas campestris pv. campestris in
Brassica seeds using agar plating and real-time PCR
A. MARQUES (2), S. Abramova (1), A. Ignatov (1), A. Sechler (3), N.
Schaad (3)
(1) All-Russian Institute of Phytopathology; (2) Embrapa Recursos Genéticos
e Biotecnologia; (3) FDWSRU, USDA-ARS
Phytopathology 100:S207
Black rot of Crucifers, caused by Xanthomonas campestris pv. campestris
(Xcc), is a serious seedborne disease worldwide. Although several assays are
available for detection of Xcc from seed, seeds remain the major source of
inoculum. We compared the recovery of Xcc on three media, mFS, NSCAA,
and mCS20ABN from spiked and naturally infested seed lots using the
industry standard seed testing protocol. The highest recovery of Xcc was
found to be equal in spiked seed extracts on mFS and NSCAA. Recovery of
Xcc from naturally infested seed was found to be higher on mFS (100
cells/ml) than NSCAA (30 cells/ml). Media mCS20ABN (Chang et al., 1991)
was found to be frequently overgrown by non-target bacteria making
identification difficult. For PCR we adapted the hrpF PCR primers of Berg et
al. (2005) for RT-PCR by designing a probe sequence between primers
DLH120 and DLH125. The resulting primer and probe set was optimized,
Vol. 100, No. 6 (Supplement), 2010
S207
tested, and used for the confirmation of suspect Xcc colonies from agar media,
and detection of Xcc from the seed soak. This adapted RT-PCR primer and
probe set was found to react with Xcc and other Xanthomonas crucifer
pathogens, including X. raphani, X. armoraciae, X. barbarae, and X.
aberrans. The sensitivity of the primer and probe set was 500 cells per
milliliter. Direct RT-PCR of the spiked seed soak was not reliable; detection
was possible only from DNA extractions of the spiked seed soak.
Use of isoparaffin-based oil for controlling dollar spot and gray leaf spot
in turfgrasses
D. S. MCCALL (1), A. E. Nichols (1), M. A. Cutulle (1)
(1) Virginia Tech, Blacksburg, VA, USA
Phytopathology 100:S208
Civitas fungicide is an isoparaffin-based oil, reported to trigger Induced
Systemic Resistance (ISR) within plants. Little is known about how
isoparaffin-based oils control turf diseases. Field trials were conducted on
perennial ryegrass (Lolium perenne) and creeping bentgrass (Agrostis
stolonifera) for control of gray leaf spot and dollar spot, respectively. Civitas
(45.2 liters mineral oil per ha) plus Civitas Harmonizer (2.86 liters proprietary
pigment dispersion per ha) was applied alone and in mixture with common
fungicides for controlling each disease. Disease was assessed by visually
estimating percentage of plots showing symptoms. The isoparaffin oil plus
pigment has consistently reduced each disease tested when compared with
untreated checks. Tank mixture of the oil plus pigment combination with
reduced rates of thiophanate methyl (3336 Plus) controlled gray leaf spot as
well as full rates of thiophanate-methyl. These results indicate that this
isoparaffin oil plus pigment product suppressed disease during mild outbreaks
of dollar spot and gray leaf spot, and can be mixed with reduced rates of
several common fungicides during more severe outbreaks.
Clusters of defense-related genes in the genome of Arabidopsis thaliana
O. A. POSTNIKOVA (2), L. G. Nemchinov (2), A. M. Boutanaev (1)
(1) Institute of Basic Biological Problems, Russian Academy of Sciences,
Pushchino, Moscow Region, Russia; (2) USDA/ARS, Plant Sciences Institute,
Beltsville, MD, USA
Phytopathology 100:S208
Functional and physical clustering of unrelated genes known as operons is a
characteristic of prokaryotic genomes. A concept and consequences of gene
clusters in eukaryotic genomes are largely unexplored. In this work, we
performed computer-generated analysis of the chromosomal distribution of
genes associated with defense response in Arabidopsis thaliana. This analysis
revealed numerous clustered genes whose co-regulation may be related to the
defense responses. The genes were distributed among all chromosomes of A.
thaliana. To support computer data, we arbitrarily selected two clusters and
analyzed expression levels of their gene-members in Arabidopsis ecotypes
Col-0 and C24 during infection with yellow strain of Cucumber mosaic virus
(CMV(Y). Ecotype Col-0 is susceptible to CMV(Y), whereas C24 contains a
dominant resistance gene RCY1. Our data showed that genes compiling two
clusters were activated only in resistant ecotype C24. This indicated that coregulation of neighboring, defense-related genes in the genome of Arabidopsis
is strongly affected not only by their chromosomal location, but also by the
basic mechanisms of genetic resistance to pathogens.
Isolates of Fusarium graminearum collected 40 to 300 meters above
ground level cause Fusarium head blight in wheat and produce
trichothecene mycotoxins
D. G. SCHMALE (1), T. Fetters (1), S. Ross (2), P. Tallapragada (2), B.
Dingus (1)
(1) Department of Plant Pathology, Physiology, and Weed Science, Virginia
Tech, Blacksburg, VA; (2) Department of Engineering Science and
Mechanics, Virginia Tech, Blacksburg, VA
Phytopathology 100:S208
The genus Fusarium contains important plant and animal pathogens, some of
which produce dangerous secondary metabolites (mycotoxins). Many fusaria
use the atmosphere to travel from one habitat to another, yet their atmospheric
transport is poorly understood. We used autonomous (self-controlling)
unmanned aerial vehicles (UAVs) to collect fusaria tens to hundreds of meters
above ground level (AGL) at Virginia Tech’s Kentland Farm in Blacksburg,
VA. Eleven single-spored isolates of Fusarium graminearum collected with
UAVs 40 to 300 meters AGL during fall, winter, spring, and summer months
were able to cause Fusarium head blight on a susceptible cultivar of spring
wheat and produce trichothecene mycotoxins. Nine of these isolates produced
the mycotoxins deoxynivalenol (DON)/15-acetyl-DON, one isolate produced
DON/3-acteyl-ADON, and one isolate produced nivalenol (NIV). To our
knowledge, this is the first report of a NIV-producing isolate of F.
graminearum in Virginia, and isolates producing DON/3-acetyl-DON are rare
in populations of the fungus recovered from infected wheat plants in the
eastern U.S. A new framework for understanding punctuated changes in the
S208
PHYTOPATHOLOGY
population structure of atmospheric fusaria based on the concepts of
atmospheric transport barriers (ATBs) and Langrangian coherent structures
(LCS) is being developed and tested at both local and regional scales. This
work aims to transform our knowledge of the atmospheric transport of
microorganisms and develop new paradigms that link field and atmospheric
populations of toxigenic fusaria.
Detection of Phytophthora ramorum chlamydospores in soil by baiting
and dilution plating
P. W. TOOLEY (1), M. M. Carras (1)
(1) USDA-ARS, Ft. Detrick, MD
Phytopathology 100:S208
Chlamydospores of P. ramorum produced by mixing 20 percent V8 juice
broth cultures with sand and incubating over a 1 month period were used to
infest field soil at densities ranging from 0.2 to 42 chlamydospores/cc soil.
Chlamydospore recovery was determined by baiting with rhododendron leaf
discs and dilution plating both when soil infestation was performed (time 0)
and following 30 days storage at 4°C, as recommended in the soil and
growing medium sampling protocol on the APHIS website
(http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/protocols.shtm
l). Baiting was slightly more sensitive than dilution plating at time 0, allowing
detection of P. ramorum down to 0.2 chlamydospores/cc soil compared with 1
chlamydospore/cc for dilution plating. Following 30 days of infested soil
storage at 4°C, P. ramorum was detected using both methods at significantly
(P = 0.05) higher levels than at time 0. The results indicate that storage of P.
ramorum-infested soil at 4°C for 30 days can enhance recovery of the
pathogen.
Increasing atmospheric carbon dioxide amplifies Alternaria alternata
sporulation and antigen production, but does not impact sporulation of
Cladosporium phlei
J. WOLF (3), N. R. O’Neill (2), C. A. Rogers (4), M. M. Muilenberg (4), L.
H. Ziska (1)
(1) USDA-ARS, Crop Systems and Global Change Lab, Beltsville, MD, USA;
(2) USDA-ARS, Systematic Mycology and Microbiology Lab, Beltsville,
MD, USA; (3) University of Maryland, College Park, MD, USA; (4)
University of Massachusetts School of Public Health, Amherst, MA, USA
Phytopathology 100:S208
Although the positive impact of elevated atmospheric carbon dioxide on
pollen production has been established, impacts on fungal sporulation and
antigen production have not been elucidated. This study examines the effects
of rising atmospheric carbon dioxide on the quantity and quality of spores
produced by fungi growing on timothy hay. Timothy grass (Phleum pratense)
was grown at recent and projected future levels of carbon dioxide (300, 400,
500 and 600 µmol mol–1). Leaves were used as substrate for the growth of
Alternaria alternata and Cladosporium phlei. The abundance of spores
produced by both fungi, as well as the size (microscopy) and antigenic protein
content (ELISA) for A. alternata, were quantified. Timothy grass leaf dry
weight and carbon-to-nitrogen ratio both increased at higher carbon dioxide
levels. Leaf carbon-to-nitrogen ratio was positively correlated with the log of
A. alternata spores produced per gram of leaf, but negatively correlated with
antigenic protein content per spore. At the two highest levels of carbon
dioxide, A. alternata produced nearly three-fold more spores and more than
twice the total antigen per plant. C. phlei spore abundance increased with leaf
carbon-to-nitrogen ratio, but overall spore numbers were much lower and perplant production did not vary with carbon dioxide level. Elevated carbon
dioxide often increases the biomass and carbon-to-nitrogen ratio of plant
leaves. This study demonstrates that leaf changes induced by increasing
carbon dioxide greatly enhance spore production by A. alternata, a ubiquitous
allergenic fungus. This response may contribute to the increasing prevalence
of allergies and asthma. Sporulation of C. phlei, a specialized pathogen of
timothy grass, did not respond to increasing carbon dioxide in this study,
suggesting that specialized and generalist fungal species may respond
differently to rising atmospheric carbon dioxide. More study is needed to
predict responses of different fungal groups to global changes.
Salicylic acid preconditioning increases tomato resistance to infection by
potato purple top phytoplasma
W. WU (1), W. Wei (1), R. E. Davis (1), I. Lee (1), Y. Zhao (1)
(1) Molecular Plant Pathology Laboratory, ARS-USDA, Beltsville, MD
Phytopathology 100:S208
Columbia Basin potato purple top (PPT) phytoplasma is a newly discovered
pathogen that causes serious diseases in potato and has the potential to affect
other vegetable crops. Since the insect vector of PPT phytoplasma, the beet
leafhopper, is a polyphagous species and has a wide geographic distribution,
diseases associated with PPT phytoplasma infections may spread rapidly. The
current study was aimed at investigating strategies to increase natural
resistance of crops to PPT phytoplasma infections. The expression profiles of
a set of defense/pathogenesis-related genes were examined in PPT
phytoplasma-infected tomato plants. Results indicated that a delayed onset
and a lack of sustained expression of a subset of defense-related genes may be
key factors involved in PPT phytoplasmal disease development. Pretreatment
of plants with SA prior to graft inoculation with PPT phytoplasma
significantly altered the expression patterns of the same subset of genes and
resulted in partial resistance of tomato to PPT infection. The findings shed
new light on molecular mechanisms of phytoplasma pathogenesis and should
aid in devising new strategies to mitigate phytoplasmal diseases.
Analysis of visual symptomatology in peach and plum inoculated with
U.S. Plum pox virus isolates
A. Younkins (1), A. L. Stone (3), D. J. SHERMAN (3), W. L. Schneider (3),
R. Scorza (2), V. D. Damsteegt (3)
(1) Dickinson College, Carlisle, PA, USA; (2) USDA-ARS-AFRS,
Kearneysville, WV, USA; (3) USDA-ARS-FDWSRU, Frederick, MD, USA
Phytopathology 100:S209
Plum pox potyvirus (PPV) is an economically devastating potyvirus that
affects Prunus species. Discovered in the United States in 1999, the
Pennsylvania PPV isolates were primarily found in peaches (Prunus persica).
When several of these original Pennsylvania isolates were inoculated onto
plums (Prunus domestica), the isolates either did not transmit or showed few
symptoms. This suggests that Pennsylvania PPV isolates were more adapted
to peach as a host. An expanded experiment was designed using a greater
number of Pennsylvania and New York PPV isolates to identify a U.S. PPV
isolate with severe visual symptoms in plums, and to determine if symptom
severity correlated with PPV titer. Two plum varieties (Bluebyrd and Stanley)
were inoculated with fourteen PPV isolates from New York and Pennsylvania
by aphid (Myzus persicae) or by grafting. Visual symptom severity was
determined using a standardized symptom rating system. PPV titers were
measured using Enzyme Linked Immunosorbent Assay (ELISA) and Realtime one step reverse transcription-PCR (RT-PCR). In contrast to PPV
infection in peach, there was little correlation between average symptom
rating and average ELISA titer or average Real-time RT-PCR Ct value in
plum. One PPV isolate had been maintained for 10 years in both hosts: peach
and plum. When this isolate was inoculated onto plum from both peach and
plum sources, differences in titer and symptoms showed a possible host
adaptation between PPV maintained in plum or in peach tissue.
Vol. 100, No. 6 (Supplement), 2010
S209
Author Index
Abad, J. A., S1, S173
Abad, Z., S150, S153
Abad, Z. G., S150
Abbas, H. K., S1
Abbasi, P. A., S1
Abd-Elgawad, M. M., S1
Abdelkarim, M. M., S2
Abel, C. A., S1
Ablova, I. B., S65
Abonyi, F., S51, S51
Abou Ghanem-Sabanadzovic, N.,
S2, S112, S112, S112
Abou Tabl, A., S176
Abouzeid, M., S34
Abrahamsen, U., S36
Abrameit, A., S54
Abramova, S., S207
Abril, M., S134
Accinelli, C., S1
Acevedo, M., S45
Achar, P., S2
Achenbach, U. C., S15
Acosta, T., S96
Acosta-Leal, R., S2, S120, S149
Adams, I., S154
Adaskaveg, J., S2, S2, S163,
S166
Adaskaveg, J. E., S141
Adelfinskaya, Y., S63
Adhikari, T. B., S3, S45, S45,
S76
Adkins, S., S70, S134, S134
Adkison, H., S129
Agindotan, B. O., S3
Agra, O., S33
Agyemang, P. A., S3
Ahn, K., S97
Ahonsi, M. O., S3
Aime, M. C., S113
Akridge, J. R., S46
Al Rwahnih, M., S3, S3
Alabi, O. J., S3, S101
Alananbeh, K. M., S182
Aldrich-Wolfe, L., S4
Alexandrova, A. V., S64
Alezones, J. M., S4
Al-Hamdany, M., S4
Ali, A., S4
Ali, M. B., S38
Ali, S., S4
Alishiri, A., S4
Alkharouf, N. W., S127
Allen, C., S22, S55, S55, S66,
S115, S173
Allen, P., S30
Allen, R., S110
Allen, T. W., S5, S49, S112
Almeida, R., S62
Almeyda, C. V., S5
Alrwahnih, M., S5
Altland, J. E., S74
Alvarado-Hernandez, M., S5
Alvarez, A., S86
Alvarez, A. M., S66, S78, S78,
S96, S98
Alvarez, E., S5, S5
Alvarez-Medina, A., S6
Alves, E., S36
Amaradasa, B. S., S6, S152
Ames, K. A., S191
Amini, N., S6
Ammar, E., S6, S27, S184
Amorim, L., S41, S118, S119, S119
Amsden, B. F., S6
Amyotte, S., S64
Anchieta, A., S64
Anco, D. J., S6, S6
Ancona, V., S7
Anderson, A. J., S158
S210
PHYTOPATHOLOGY
Anderson, C., S177
Anderson, G., S11
Anderson, G. M., S66
Anderson, R., S158
Andreote, F. D., S35
Andrews, D. L., S98
Andrews, K., S9
Antigliani, V., S158
Antolínez, C. A., S7, S29
Antoun, H., S193
Anu, A. E., S117
Appel, D. N., S7
Araujo, L., S122
Araújo, W. L., S35, S77
Arellano, C., S114
Arevalo, H. A., S77
Arevalo Zelada, J., S125
Arif, M., S7, S7, S7, S19, S30
Aritua, V., S7
Arredondo, F., S158
Arriaga, F. J., S16
Arrieta, M. L., S8
Arul, J., S65
Asalf, B., S8
Ashton, P., S154
Atallah, Z. K., S8, S24, S64, S79
Atehnkeng, J., S8
Athinuwat, D., S8
Atiri, G. I., S113, S126
Auge, R. M., S44
Augusto, J., S8, S199, S199
Autio, W. R., S197
Avanzato, M., S111
Avenot, H. F., S9
Avila, L. L., S9, S9
Avis, T. J., S193
Awurum, A. N., S9
Ayodele, M., S117, S117
Ayres, A. J., S74
Azevedo, J., S35
Babadoost, M., S9, S108, S126,
S190, S191, S196
Babujee, L., S55, S55
Baccari, G. V., S60
Backup, P., S17
Backus, E. A., S9
Bacon, C., S10, S147
Bacon, C. W., S10
Badebo, A., S93
Baenziger, P., S134, S191
Baeza-Montañez, L., S98
Bag, S., S10
Bagewadi, B., S10, S154
Bai, Y., S72
Baier, K., S10
Bajwa, S., S31
Bakker, P., S165
Bala, K., S150, S153
Balakrishnan, N., S101
Balci, Y., S151
Baldwin, T., S147
Baldwin, T. T., S10
Balint-Kurti, P., S11, S114
Balkcom, K. S., S16
Balla, B., S51
Bandyopadhyay, A., S11
Bandyopadhyay, R., S8, S117,
S128, S162, S190
Banihashemi, Z., S85, S85
Banu, S. P., S11
Barak, J., S47, S155, S168
Barash, I., S22
Barbara, D., S64
Barbetti, M., S35, S39, S60
Barbosa, J. C., S11, S87
Bardsley, S. J., S11
Barham, J. D., S153
Baring, M. R., S204
Barnes, J. S., S131
Barney, W., S126
Barooti, S., S83
Barouti, S., S84, S124
Barphagha, I., S47
Barrera, W., S12
Barros, N., S121
Barry, K. C., S160
Barthe, G. A., S86
Bartz, J., S33
Bartz, J. A., S12
Basart, J. P., S188
Bascur, G., S99
Baskin, D., S200
Bass, J., S103
Bassanezi, R., S200
Bassanezi, R. B., S74
Bastas, K., S12
Basu Thakur, P., S12
Bates, A. A., S53
Batson, W. E., S153
Batuman, O., S12
Batzer, J. C., S15, S54, S112,
S112, S189
Baudoin, A. B., S12
Baughman, T. A., S204
Baum, T., S58, S141, S187
Baum, T. J., S116, S132, S179,
S186, S187
Bautista, N., S89
Bayon, C., S12
Baysal-Gurel, F., S13, S13, S20,
S124
Beattie, A. C., S74
Beattie, G. A., S136
Becker, J. O., S141
Becker, K., S158
Beckerman, J., S23, S69, S189
Beckerman, J. L., S168, S183
Bednarek, P., S158
Beer, S. V., S144
Behlau, F., S13, S172
Behn, J. L., S182
Beirn, L. A., S13, S193
Bejarano, J. C., S13
Beladi, S., S13
Bélanger, R., S198
Bélanger, R. R., S196
Belasque, Jr., J., S11
Belcher, A., S114
Belcher, A. R., S14
Belknap, W. R., S101
Beltrán, C., S42
Belzile, F., S196
Ben Kalifa, H., S33
Bennett, A., S167
Bennett, M. H., S70
Bennett, P. J., S33
Bennetzen, J., S40
Benson, M., S94, S102
Benson, M. M., S90
Bent, A. F., S26
Bentley, C., S94
Berestecky, J. M., S98
Bergamin Filho, A., S11, S78,
S200
Berger, P. H., S156
Bernal, A., S8, S127
Bernal, A. J., S7, S21, S22, S29,
S29, S39, S86
Berner, D. K., S64, S205
Bernier-English, V., S193
Berry, S. A., S189, S192
Bérubé, M., S193
Bestor, N. R., S14, S182
Bextine, B. R., S115, S116
Beyer, D., S164
Bhat, R. G., S115
Bhat, S., S142
Bhattacharyya, M. K., S80, S183
Bi, J., S118
Bian, W., S60
Bienapfl, J. C., S14
Biggs, A. R., S56
Bilodeau, G., S14, S14
Bindschedler, L., S56
Bird, G., S185
Birren, B., S64
Bittner, R. J., S14
Blaser, J. M., S15
Blazheva, D., S30
Block, C. C., S188
Blomquist, C. L., S14
Bluhm, B., S142
Bluhm, B. H., S35, S52, S109,
S120, S148
Bock, C. H., S15, S15
Bockelman, H., S65, S104
Bockus, W. W., S45
Bocsanczy, A., S15
Boehm, M. J., S33
Boerma, H. R., S61
Bogdanove, A., S56
Bogdanove, A. J., S159
Bohannon, R. C., S15, S143
Bohlmann, H., S117
Boland, G. J., S17, S17
Bolton, M. D., S102, S111
Bonasera, J. M., S144
Bond, J. P., S191
Bondalapati, K. D., S15
Bonde, M. R., S16
Bonman, J. M., S3, S45
Bonn, G., S116, S172
Bonos, S. A., S193
Boonham, N., S154
Boquet, D. J., S115
Bordovsky, J. P., S138
Borenshtein, M., S33
Borges, B., S4
Borneman, J., S141
Borth, W. B., S82
Bost, S. C., S16
Bostock, R. M., S25, S48, S53,
S104, S141
Botermans, M., S81
Bottner-Parker, K. D., S69
Bouchard, J., S194
Bourgeois, G., S193
Bourget, N., S193
Bourland, F. M., S153
Boutanaev, A. M., S208
Bowden, R. L., S41
Bowen, K. L., S16, S20, S128
Boyetchko, S. M., S99
Boyette, D., S162
Bradley, C., S137, S185
Bradley, C. A., S3, S104, S145,
S182, S191
Bradley, J., S38
Brady, J., S16
Brady, J. A., S88
Brancher, N., S99
Brannen, P. M., S16, S199
Bransby, D., S16
Brantner, J. R., S16
Brar, D. S., S11
Brar, H. K., S183
Brauer, K., S183
Braverman, M., S162
Breakspear, A., S75
Breathnach, J., S191
Bremer, D., S91
Brennan, J., S17
Brennan, J. M., S17
Brenneman, T., S199
Brenneman, T. B., S8, S199, S200
Brewbaker, J. L., S98
Brewe, C., S108
Brewster, W. K., S63
Brian, B. K., S2
Bricault, C., S99
Brière, S. C., S197
Britt, R., S58, S58
Brlansky, R. H., S42, S61, S107,
S111, S143, S143, S172
Brodal, G., S36
Broders, K. D., S17, S17, S33
Bronstein, P. A., S160
Broome, J. C., S17
Brose, I. E., S188
Brown, D., S18
Brown, D. W., S18
Brown, J. K., S50, S53, S110,
S138, S172
Brown, R. W., S82
Brown, T., S18
Browne, G. T., S115
Bruce, M., S157
Bruckart, W. L., S34, S206
Brueggeman, R., S43
Brust, G. E., S18, S205
Buck, J., S116
Buell, C., S153
Buell, C. R., S127
Bull, C. T., S79
Burbank, L., S85
Burelle, N., S19
Burkett-Cadena, M., S18
Burlakoti, P., S18
Burnham, A. M., S18
Burnquist, W. L., S78
Burr, T. J., S8
Burskey, C., S39
Busman, M., S18
Butchko, R., S18
Butler, D. M., S19
Butler, D. N., S90
Byamukama, E., S19, S183
Bylemans, D., S163
Caasi, D., S7
Caasi, D. J., S19
Cabezas, L. F., S129
Cacciola, S. O., S76
Caesar, A. J., S68
Caesar-TonThat, T., S68
Caetano-Anollés, G., S190
Caffi, T., S19, S194
Cahalane, G., S17
Cai, X., S19
Calderon, C., S19
Caligari, P. D., S110
Calla, B., S106
Calle, J., S171
Callow, P. W., S83
Calvo-Velez, P., S20
Camacho, F., S29
Camargo, L. E., S21, S74
Camp, A. R., S20, S20
Campbell, H. L., S20
Campbell, J. H., S199
Campbell-Nelson, K., S92
Canteros, B. I., S13
Cantonwine, E. G., S200
Cao, C., S20
Cao, M., S105
Capelluto, D. G., S158
Caprara, M., S21
Caragea, P., S188
Carbone, I., S85, S92, S94, S157
Cárdenas, M. E., S21, S22
Cardona, N., S171
Carisse, O., S194, S194
Carlson, B., S21
Carnes, M. E., S21, S106
Carpenter, D., S126
Carr, J., S70
Carr, J. P., S147
Carras, M. M., S208
Carson, M., S21
Cartwright, K., S162
Cartwright, R., S145, S202
Cartwright, R. D., S22, S114
Caruano-Yzermans, A., S38
Carvalho, G., S21
Casassa, L. F., S45
Cassell, M. E., S205
Castillo, J. D., S21
Castlebury, L. A., S173
Castro, A., S110
Castroagudin, V. L., S22
Caswell-Chen, E. P., S27, S113
Cating, R. A., S22, S174
Cattaneo, M. G., S136
Cavin, C. A., S205
Center, T. D., S108
Cepero de García, M. C., S42
Céspedes, M. C., S21, S22
Cha, J., S25
Chaikam, V., S11
Chakrabarti, A., S75
Chalupowicz, L., S22
Chaluvadi, S., S40
Chambers, A. Y., S153
Champoiseau, P. G., S22, S173
Chanda, A. K., S22
Chandra-Shekara, A., S56
Chang, C., S199
Chang, P. S., S173
Chao, S., S104
Chapara, V., S183
Chapman, K., S189
Chapman, K. S., S23, S183
Charkowski, A., S19, S78
Charudattan, R., S176
Chatnaparat, T., S103
Chaudhary, P., S185
Chaudhury, A., S23
Chaves, A., S23
Chaves, G., S176
Chawla, S., S200
Chellemi, D. O., S57, S173
Chen, B., S24
Chen, C., S23, S30, S41, S53,
S105, S146, S152
Chen, H., S90
Chen, J., S24, S24, S73, S119
Chen, K., S44
Chen, P., S70
Chen, R., S47
Chen, X., S53, S132
Chen, Y., S23, S46
Chen, Z., S22, S37, S39, S47,
S64, S96, S164
Cheng, D. M., S72
Cheng, Y., S53
Cheong, E., S56
Cheong, K., S97
Cheung, H. K., S24
Chi, M., S97
Chiampiriyakul, P., S121
Childers, A. C., S138
Chintamanani, S., S11
Chitrakar, R., S24
Chitrampalam, P., S24
Chittem, K., S25, S42
Cho, J., S25
Cho, Y., S25
Choi, D., S97
Choi, J., S25, S97
Choi, Y., S25, S25
Choiseul, J., S17
Chou, C., S25
Choudhary, N., S42, S111
Chuaboon, W., S103
Chung, K., S24
Chunxue, C., S159
Cibils Stewart, X., S26
Cilia, M., S100
Cisneros, F. M., S26, S184
Ciuffetti, L. M., S160
Civerolo, E., S24
Civerolo, E. L., S30, S72, S72
Clancey, M. S., S188
Clardy, J., S54
Clark, W. D., S28
Clarke, B., S90
Clarke, B. B., S13, S109, S193,
S197
Clinckemaillie, A., S26
Clough, S. J., S106
Cloutier, S., S151
Cobine, P. A., S29
Cochran, A., S93, S93, S163
Cochran, K. A., S26
Cock, P. J., S80
Coffey, M. D., S14, S76, S150
Cohen-Kandli, M., S22
Collins, D. J., S202
Colyer, P. D., S153
Comstock, J., S78
Comstock, J. C., S106, S173,
S175
Comstock, J. P., S26
Conley, S., S67, S67
Conley, S. P., S186
Consonni, C., S158
Conway, W., S58
Conway, W. S., S56
Cook, D. E., S26
Cook, D. R., S105
Cooksey, D. A., S118
Cooley, D. R., S195
Cools, H. J., S12, S49
Coop, L., S179
Cooper, G. T., S201
Coplin, D. L., S27, S184
Coram, T., S53
Corcuff, R., S65
Corley, J., S126
Correa, V. R., S27, S184
Correll, J., S53, S142, S145
Correll, J. C., S22, S35, S135
Cortez, A., S128
Costa, C. R., S87
Cotes, A. M., S42
Cotes, J. M., S87
Cotton, J. A., S27
Cotty, P. J., S8, S39, S43, S55,
S81, S103, S162
Covert, S., S59
Cowger, C., S144, S144, S204
Cowles, R. S., S195
Cox, K., S27, S50
Craven, K., S40
Cregan, P., S189
Cregan, P. B., S61
Creswell, T., S69
Crouch, J., S13, S27, S27, S101,
S156, S193
Crow, J., S116
Crutcher, F. K., S27
Cruz, L., S28
Cruz, L. C., S29
Cruz, L. F., S21
Csinos, A., S28, S55
Csinos, A. S., S57, S142
Cuellar, A., S5
Culbreath, A., S105
Culbreath, A. K., S105, S105,
S200, S200
Cummins, D., S17
Cuomo, C., S64, S75, S156
Curry, A., S134
Curry, K. J., S83, S134
Curtis, M., S104
Cusicanqui, J., S41
Cutulle, M. A., S28, S80, S152,
S205, S208
D’Amico, K. M., S29
da Graça, J. V., S66
da Silva, M. P., S28, S184
da Silva, R., S11
Daane, K., S119
Dabala, R. R., S28
Dai, W. D., S18
Dalchau, N., S70
Dally, E. L., S29, S206
Damann, K. E., S28, S124
Damayanti, T. A., S101
Damsteegt, V. D., S42, S209, S111
Danchok, R. S., S120
Dangott, L. J., S27
Danies, G., S7, S29
Daros, E., S111
Datnoff, L., S52
Datnoff, L. E., S36, S174
Daub, M., S136
Daub, M. E., S121
Daubert, S., S3, S5
Daughtrey, M. L., S90
Davidson, R. M., S157, S164
Davis, E., S141
Davis, E. L., S116, S132, S186
Davis, M., S164
Davis, R. E., S29, S69, S135,
S206, S206, S207, S208
Davis, R. F., S202
Davis, R. M., S53, S153
Davis, T. M., S77
Dawson, W. O., S56, S154
de Afonseca Lourenço, S., S119
De Boer, S. H., S70
de Cara, M., S29, S39, S95, S95
de Cock, A. W., S150, S153
de Faria, R. S., S21
de Figueiredo, P., S7
de Jonge, R., S64
de la Cerda, K. A., S152
De La Fuente, L., S28, S29, S97
de Silva, A., S98
de Silva, A. S., S78, S96
de Souza, S. R., S111
Deah, K. L., S52
Deahl, K., S161
Deb, D., S158
Dedeurwaerder, G., S26
Dee, M. M., S131
Degaetano, A. T., S26
Del Castillo, J. M., S29
Del Ponte, E. M., S121
del Rio Mendoza, L. E., S18, S89,
S89
del Rio, L. E., S77, S184, S189
del Río, M., S86
Delaney, M. A., S30
Delfosse, P., S32, S32, S76
Delic, D., S81
Delvaux, B., S58
Deng, X., S24, S73
Deng, Y., S49
Denny, T., S199
Denny, T. P., S45
DeRobertis, C., S28
Derr, J., S28, S205
Detweiler, A. J., S206
Devorshak, C., S156
Dewdney, M. M., S85, S86, S130,
S174
Dewey, R. L., S37
Dhawan, R., S11
Dhingra, A., S142
Diaz Arias, M. M., S30
Dicklow, B., S32
Dickman, M., S58, S58, S160
DiLeo, M. V., S104
Dillard, H. R., S20
Ding, Y., S206, S207
Dingus, B., S208
Dion, Y., S193
Divo de Sesar, M., S99
Dobhal, S., S30
Dobinson, K. F., S64
Doddapaneni, H., S30
Dodds, P., S75
Dodge, S., S136
Dolezal, W. E., S98, S181
Domier, L. L., S71
Vol. 100, No. 6 (Supplement), 2010
S211
Donahoo, R., S52
Donahoo, R. S., S30
Donald, P., S30
Dong, W., S31
Donofrio, N. M., S66, S207
Doraiswamy, V., S54
Dorel, M., S58
Dorrance, A., S185
Dorrance, A. E., S33, S33, S39,
S123, S184, S189, S190, S191,
S192
dos Santos, J., S121
Dossey, S., S53
Dou, D., S158
Doubledee, M., S31
Douhan, G. W., S152
Doustseddigh, H., S31
Dowell, F., S134
Drake, J., S77
Draper, M. A., S161, S191
Dreaden, T. J., S176
Drennan, J. L., S147
Driver, J. G., S31
Dror, O., S22
Druffel, K. L., S5, S130, S130
du Toit, L., S53, S142
du Toit, L. J., S169
Duan, Y., S30, S41, S145
Duarte, I., S31
DuBose, V. B., S60
Duffy, B., S54, S59, S120
Duffy, S., S149
Dugan, D., S39
Dumenyo, C. K., S61
Dumonceaux, T., S69
Dung, J. K., S31
Dunkle, R. L., S155
Dutt, M., S86
Dutta, B., S148, S200
Duveiller, E., S156
Duvivier, M., S26
Dyer, A. T., S87
Eastwell, K., S130
Eckstein, B., S11
Egel, D. S., S32, S32
Eggenberger, A., S187
Eggertson, Q. A., S153
Eghbalian, F., S32
Eichenlaub, R., S22
Eisenback, J. D., S32, S206, S206
El Jarroudi, M., S32, S32, S76
Elad, Y., S33
Elafifi, S., S33
Elateek, S. Y., S33
Elen, O., S36
Elena, S. F., S149
El-Habbak, M., S107, S119
Elkahky, M., S12, S33
Elliot, M. L., S175
Ellis, J., S75
Ellis, M. A., S6, S6
Ellis, M. L., S33, S33, S184
Ellis, S. D., S33
Ellor, T., S164
Elmazaty, M., S33
Elmer, W. H., S195
El-Sheshtawi, M., S33
El-Tarabily, K. A., S34, S34
Emery, R., S87
Engels, R., S64
English, J. T., S43
Ensley, S. M., S109
Enzenbacher, T. B., S34
Eskandari, F. M., S34, S206
Esker, P., S67, S67, S185
Esker, P. D., S34, S79, S119,
S186, S188
Eskridge, K., S147
Esquivel, J., S127
Estevez De Jensen, C., S34
Eujayl, I. A., S123
Evans, T. A., S66, S207
S212
PHYTOPATHOLOGY
Eversole, K., S167
Everts, K., S32, S51, S99, S207
Everts, K. L., S146
Exner, C., S184
Fulton, J., S86
Funnell-Harris, D. L., S37, S37,
S185
Furman, B. J., S109
Faghihi, J., S185
Fajolu, O. L., S131
Fan, J., S61
Fang, X. L., S35
Faris, J. D., S151, S151
Farley, L., S39
Farnsworth, J. L., S40
Farrell, J. D., S184
Farrokhi Nejad, R., S113
Faske, J., S16
Faske, T., S16, S35
Fatokun, C. A., S92
Fauquet, C. M., S10, S154
Faust, J. E., S128
Fazekas, M., S51, S51
Fei, S., S184
Feliciano-Rivera, M., S35
Felise, H. B., S160
Fellers, J., S151
Feng, C., S35, S53, S72, S139,
S145
Fernandez-Ortuno, D., S35
Ferreira, A., S35, S77
Fessehaie, A., S36, S36, S80
Fetters, T., S208
Ficke, A., S36
Figueiredo, J., S36
Figueroa Betts, M., S160
Filho, A. B., S87
Fillippeli, E., S201, S201
Fischer, K., S10, S154
Fischer, K. F., S154
Fisher, J., S39
Fisher, T. W., S88
Fitzpatrick, B. M., S45
Fjellstrom, R., S202
Fleites, L. A., S111
Fletcher, J., S18, S59, S110, S155
Flor, N. C., S36
Flores, F., S7, S36
Floyd, C. M., S66
Foote, P., S123
Forbes, G., S121, S156
Forouhar, F., S77
Forster, H., S2, S2, S163, S166
Fortnum, B. A., S100
Fortunato, A. A., S96
Foster, B., S162
Fought, L., S37
Fountain, J. C., S37
Fox, G. M., S196
Fraaije, B. A., S12, S35, S49, S51
Fraaije, M. W., S12
Fragoso, R. B., S111
Francis, D. M., S20
Francis, M. I., S37
Franco, B., S87
Franco, S., S13
Franco-Lara, L., S176
Frank, M., S37
Frantz, G., S134
Frantz, J., S144
Frantz, J. M., S74
Frare, G. F., S74
Frederick, R. D., S37, S61
Frei, U., S184
French, R., S125, S149
French-Monar, R. D., S173
Frey, J. E., S120
Friesen, T., S151
Friesen, T. L., S144, S151
Fry, J., S91
Fry, W., S77
Fry, W. E., S20, S26, S52
Fu, Y., S145
Fuchs, M., S93
Fudal, I., S158
Fukuda, S. K., S82
Gabriel, D., S108, S111
Gadoury, D. M., S8, S38, S88
Galagan, J., S64, S75
Galbraith, D. W., S154
Gale, L. R., S75
Gallegly, M., S207
Galvez, L. C., S38, S38
Gamba, F., S118
Gambhir, A., S185
Ganiger, M. C., S96
Gao, B., S38
Gao, F., S38
Gao, Q., S38, S139
Garavito, M. F., S39
Garbelotto, M., S115, S167
Garber, N., S39
Garber, R. H., S153
Garces, F., S39
García, A., S39
Garcia, A., S107
Garcia, L., S39
García, M., S95
Garcia-Pedrajas, M. D., S64, S98
Garczynski, S. F., S88
Gardiner, D., S75
Garg, A., S11
Garneni, S., S133
Garner, J., S16
Garrett, K., S121
Garrett, K. A., S41, S120
Garrido, P., S30
Gartemann, K., S22
Garzon, C., S7, S30
Garzon, C. D., S7, S154
Gaska, J., S67, S67
Gaskins, V., S58
Gasparoto, M., S11
Gassmann, W., S38
Gavin, J. C., S135
Gazaway, W. S., S153
Gbur, E. E., S153
Ge, X., S39
Gearhart, K., S39
Gebhart, G. D., S190
Gent, D. H., S40, S84, S138
George, S., S122
Gergerich, R., S67
Gergerich, R. C., S201
Gerik, J. S., S135
Gevens, A. J., S40, S174, S174
Ghabrial, S., S60, S119
Ghabrial, S. A., S107
Ghadamyari, S., S87
Ghimire, S., S40, S51
Giampan, J. S., S87
Giesler, L. J., S185
Gilbertson, R. L., S12, S138
Gilchrist, E., S40, S40
Gildow, F., S100
Gildow, F. E., S126
Giles, C. G., S203
Gill, J. J., S31
Giraud, F., S32, S32, S76
Gisi, U., S162
Gitaitis, R. D., S9, S122
Glaettli, A., S37
Glass, K. M., S21
Gleason, M. L., S15, S26, S54,
S112, S112, S112, S189
Glenn, A. E., S10, S18, S147
Glover, K. D., S59
Glover, R., S154
Glucksman, S., S40
Glynn, E., S17
Glynn, J. M., S41
Glynn, N., S106
Glynn, N. C., S173, S175
Gmitter, F. G., S61
Goates, B. J., S41
Gobena, D. J., S41
Goenaga, R. J., S176
Goesmann, A., S120
Goh, J., S41
Goheen, D. J., S167
Goheen, E. M., S166
Gold, S. E., S64, S98
Golino, D., S3
Gomez, A., S75
Gomez, L., S41
Gonçalves, F. P., S41
Gongora, C., S188
Gonzáles, E. R., S35
Gonzales, M. A., S41
Gonzalez, A. D., S4
Gonzalez, C., S42
Gonzalez, C. F., S31
Goodin, M., S11
Goodwin, S. B., S25, S25, S45
Goss, E. M., S42, S157
Gossen, B. D., S59, S99, S117,
S125
Goswami, R. S., S25, S42, S185
Gottwald, T., S172, S200
Gottwald, T. R., S15, S15
Gougherty, A., S42
Gourley, J., S70
Govindarajulu, A., S42
Gowda, S., S56
Graham, J. H., S13, S15, S37,
S42, S57, S172, S173
Graham, M., S96
Graham, T. L., S20
Gramacho, K. P., S42
Granke, L., S43
Granke, L. L., S43
Grau, C. R., S186
Gray, M. E., S3, S74
Gray, S., S61, S100
Green, J., S11
Greenberg, J. T., S160
Grégoire, C., S196
Greve, C., S9
Grimes, J., S39
Grisham, M. P., S43
Groom, J., S60
Gross, D., S76
Gross, D. C., S136
Gross, J., S54
Gross, N. W., S43
Gross, P. L., S43
Grosser, J. W., S37, S86
Groth, D., S65
Groth, D. E., S201
Grove, G. G., S40, S73, S138
Groves, R., S19, S119
Grubisha, L. C., S39, S43
Grunwald, N. J., S42, S150, S156
Grybauskas, A., S137
Gu, B., S158
Gu, G., S44
Gualandi, R. J., S2, S44
Guaragna, M., S59
Guardado, A., S86
Gubler, W. D., S17, S124, S129
Gudmestad, N., S182, S203
Gudmestad, N. C., S135, S183
Guerra, D. S., S121
Gugino, B., S77
Gugino, B. K., S195
Gulati-Sakhuja, A., S44
Guo, B., S37, S57
Guo, J., S140, S141, S141, S146
Guo, L., S44
Guo, Q., S70, S75
Guo, Y., S3, S44, S105
Gupta, V., S44
Gurung, S., S3, S45, S45, S76
Gutha, L. R., S45
Gutierrez-Aguirre, I., S81
Guzman-Barney, M., S176
Gwinn, K. D., S2, S44, S131
Ha, B., S61
Ha, Y., S45
Hadwiger, L., S45
Hadziabdic, D., S45
Hagan, A. K., S16, S20, S46, S46
Haghighi, S., S46
Hajimorad, M. R., S135
Hajmansoor, S., S6, S32, S62
Halbert, S. E., S46, S77, S107
Halbrendt, N. O., S46
Hall, D. G., S6
Halterman, D., S19, S23, S46
Ham, J., S47
Hamilton, J., S153
Hamilton, J. P., S127
Hammerschmidt, R., S181
Hammond, J., S10, S71, S154
Hammond, R. W., S149, S207
Han, A., S76
Han, P., S47
Han, S., S96, S158
Hancock, J. F., S83
Hand, E. K., S17
Hanke, D. E., S70
Hanna, L., S111
Hansen, J., S3
Hansen, M., S135
Hanson, L. E., S47
Hanson, S., S47, S62, S68, S107
Hao, J., S60, S72, S74, S82
Hao, L., S47
Hao, W., S47, S51
Harbertson, J. F., S45
Harding, M. W., S48
Hari, K., S59
Harman, G., S165
Harmon, C. L., S177
Harmon, P. F., S36, S130, S138,
S152, S175, S201
Harnsomburana, J., S11
Harris, D. K., S61
Harrison, L., S48, S207
Harrison, N. A., S69, S175
Hartman, G., S190
Hartman, G. L., S48, S107, S128,
S131, S185
Hartman, J. R., S6
Hartung, J., S143
Hartung, J. S., S42, S111, S143
Harveson, R. M., S48, S48
Hasey, J. K., S48
Hatfield, J. L., S179
Hau, B., S82
Haudenshield, J., S190
Haudenshield, J. S., S48, S107,
S131, S185
Hauff, R., S128
Hausbeck, M., S43, S133, S176
Hausbeck, M. K., S34, S43, S201
Hawkins, N., S49
Hayes, R. J., S107
He, B., S135
He, G., S105
He, Y., S185
He, Z., S49, S92
Hearne, S., S113
Heaton, E., S180
Hed, B., S49, S49
Hedley, P. E., S80
Heiman, D., S64
Helliwell, E. E., S49
Henderson, D. C., S10, S154
Henn, A., S5, S49
Henne, D. C., S50, S138, S203
Henrissat, B., S64
Henry, G., S158
Henry, R., S50
Herman, M., S41
Hernandez, F. R., S4
Hernandez, M. G., S89
Hernandez-Martinez, R., S87, S101
Hernandez Nopsa, J., S50, S147,
S186, S186, S191
Hernandez-Zepeda, C., S50
Herrero, S., S121
Hershman, D., S137
Hert, A., S66
Heungens, K., S42
Hewezi, T., S132, S179, S186,
S187
Hickman, L. L., S28
Higbee, B., S119
Higgins, B., S122
Highland, H., S50
Highland, H. B., S200
Higuchi, K., S143
Hijmans, R., S121, S156
Hilf, M. E., S70
Hill, J., S120, S187
Hill, J. H., S96
Hillman, B., S98
Hily, J., S50
Himmelstein, J., S51, S207
Hinton, D. M., S10
Hirsch, R. L., S148
Hladky, L., S128
Hobbelen, P. H., S51, S51
Hodges, T., S154
Hoeschele, I., S190
Hoffmann, L., S32, S32, S76
Hogenhout, S. A., S27, S184,
S184
Hoitink, H., S124
Hoke, S., S32
Holah, N., S181
Holah, N. S., S186
Holb, I. J., S51, S51
Holbrook, C. C., S105
Holland, R., S26, S31
Hollier, C. A., S201
Honeycutt, C., S92
Honeycutt, W., S67
Hong, C., S47, S51, S97, S207
Hooftman, R., S81
Hoogenboom, G., S92
Hooks, C. R., S133
Hopkins, D., S52, S201
Horevaj, P., S52
Horn, B. W., S85, S92, S157
Horsman, L., S134
Horton, T. R., S29
Horvath, B., S6, S205
Horvath, B. J., S28, S80, S152
Hoshi, A., S52
Hosseini, P., S127
Houchins, D., S108
Howard, R. J., S48
Howard, S., S38
Howe, P. J., S186
Hoy, J. W., S39
Hoy, M. A., S22
Hresko, M., S38
Hsu, H., S59
Hsu, S., S71
Hu, C., S52, S68
Hu, J. S., S82
Hu, X., S61, S133
Huang, C., S52, S52, S53
Huang, G., S116, S141
Huang, X., S53, S74
Huber, D. P., S132
Huber, L., S76
Hudson, M., S106
Hudson, W., S78
Hughes, M. A., S177
Hughes, T., S26
Hughes, T. J., S186, S186
Hulbert, S. H., S142, S167
Hulvey, J., S41
Humann, J. L., S53
Humphry, M., S158
Hurburgh, C. R., S109
Hussey, R., S141
Hussey, R. S., S116, S186
Hutmacher, R. B., S153
Hutton, D., S75
Hwang, J., S150
Hwang, S., S99
Hynes, R. H., S99
Hyten, D., S189
Hyten, D. L., S61
Ibekwe, A., S144
Ibrahim, D., S84
Idris, A. M., S50, S53, S172
Iglesia-Garcia, A., S142
Iglesias, C., S95, S95
Iglesias-Garcia, A., S53
Ignatov, A., S207
Ilori, C. O., S92
Inderbitzen, P., S64
Inderbitzin, P., S53
Ingram, D. M., S5, S49
Irey, M., S24, S200
Irish-Brown, A., S69
Isakeit, T., S54, S106
Isakeit, T. S., S153
Ishiga, T., S54, S160
Ishiga, Y., S54, S54, S129, S160
Ishii, Y., S52
Ishimaru, C., S59
Ishimaru, C. A., S54, S120, S124
Ismail, S., S54
Ithal, N., S58
Ivey, M., S124
Ivey, M. L., S33
Ivors, K., S55
Ivors, K. L., S52
Izzo, A., S134
Jackson, A., S202
Jackson, K., S55
Jackson, K. L., S142
Jackson, T., S182, S183
Jackson, T. A., S180, S187
Jacobs, J. M., S55, S55
Jacobsen, B. J., S87, S165, S168
Jacon, G., S27
Jagadeeswaran, G., S3
Jaime-Garcia, R., S55
Jain, R., S59
Jalan, N., S55
Jan, F., S53, S146
Jang, S., S97
Janisiewicz, W. J., S56, S58
Janousek, C. N., S17
Jansky, S., S19
Jaraba-Navas, J., S75
Jardine, D., S185
Jarugula, S., S56
Jayaraj, J., S18, S205
Jayasena, A. S., S56
Jeffers, S. N., S150
Jellison, J., S21
Jenkins, D. M., S66
Jeon, A., S56
Jeon, J., S97
Jeong, M., S25
Jeong, R., S56
Jeyaraman, R., S56
Ji, C.-Y., S96
Ji, P., S55 S57, S142
Jia, M., S202
Jia, Y., S202, S204
Jiang, T., S57
Jiang, Y., S206, S207
Jimenez, P., S13, S19, S129
Jin, J., S187
Jin, X., S57
Jin, Y., S93, S93
Jo, Y., S57
Johal, G., S11
Johnson, B. J., S1
Johnson, C., S97
Johnson, D. A., S31
Johnson, E. G., S57, S173
Johnson, K., S148
Johnson, K. B., S166
Johnson, R. M., S43
Johnson, S. B., S194, S194
Jomantiene, R., S29, S206
Jones, D., S24
Jones, J., S36
Jones, J. B., S13, S22, S103,
S172, S173
Jones, S., S147
Jonkers, W., S75
Jordan, R., S59, S143, S143
Jordan, R. L., S10, S154
Jordan, S. A., S174, S175
Joseph, L., S26
Joshua, J., S84
Jossey, S., S57
Judelson, H., S140
Judge, C. A., S102
Jumpponen, A., S41
Jung, G., S92, S102, S197
Jung, H., S160
Jung, K., S25, S65
Jurick, W., S58
Jurick, II, W. M., S56
Juvale, P. S., S187
Kabbage, M., S45, S58, S58,
S160
Kabeil, S. S., S1
Kablan, L., S58
Kachroo, A., S38, S56, S58,
S117, S119, S139
Kachroo, P., S56, S58, S139
Kadooka, C. Y., S128
Kaitheri Kandoth, P., S58
Kakizawa, S., S52
Kakvan, N., S6
Kale, S. D., S158
Kalmowitz, K. E., S60
Kalpana, K., S59
Kamber, T., S59, S120
Kamenidou, S., S59
Kammerer, S., S152
Kamo, K., S59
Kamo, K. K., S67
Kandel, Y. R., S59
Kaneshiro Sueno, W., S78
Kang, S., S25, S64, S79, S97,
S97, S150
Kang, Z., S53, S133
Kantartzi, S. K., S28
Karakaya, A., S15
Karakkat, B., S59
Karasev, A., S61
Kariuki, G. M., S60
Karki, H., S47
Karthikeyan, G., S101
Kaufman, H. W., S153
Kaur, P., S60
Kav, N. N., S130
Kazan, K., S75
Kearney, S., S17
Keese, R. J., S60
Kegley, A. J., S120
Keinath, A. P., S60, S201, S201
Keller, K., S67
Keller, M., S40
Kelley, E., S60
Kemerait, R., S57
Kemerait, R. C., S37, S92, S200,
S202
Kendall, A., S60
Kendrick, M. D., S61
Kenerley, C. M., S27
Kennelly, M., S91, S91
Kenney, M. J., S61
Kerlan, C., S61
Kerns, J., S64, S64, S137, S137
Kerns, J. P., S120
Kerrigan, J. L., S204
Kersbergen, R., S194
Kersey, C., S61
Khalaf, A. A., S61
Khan, M., S25
Khan, M. F., S102
Vol. 100, No. 6 (Supplement), 2010
S213
Khan, N. I., S61
Khattab, A., S4
Khazaeli, P., S62
Khozeini, F., S83, S124
Khudokormova, Z. N., S65
Kilcrease, J., S62
Killiny, N., S62
Kim, D., S25, S97
Kim, H., S58, S62, S97, S160
Kim, J., S62
Kim, K., S41, S62
Kim, R., S116
Kim, S., S97, S97, S161
Kim, W., S25
Kim, Y., S63, S63, S96, S143,
S143, S158, S163
Kinard, G., S71, S72, S101
Kinyua, Z. M., S60
Kirk, W. W., S9
Kirkpatrick, T., S75
Kirkpatrick, T. L., S114, S153
Kiseleva, M. I., S65
Kiss, L., S19
Kistler, C., S75
Kito, H., S63
Klappach, K., S63
Klassen, W., S176, S178
Kleinhenz, M. D., S20
Klessig, D., S77
Kliebenstein, D. J., S165
Kliejunas, J. T., S94
Klindworth, D., S93
Kline, T., S160
Klittich, C. J., S63
Kloepper, J., S18, S146
Kloepper, J. W., S63, S65, S165,
S170, S170, S170, S170
Klosterman, S. J., S64, S79, S98,
S107
Kluepfel, D. A., S141
Knapp, S. J., S105
Knauf-Beiter, G., S66
Kobayashi, D., S98
Koch, P., S64, S64, S137
Koebnik, R., S8
Koehrsen, M., S64, S75
Koike, S. T., S14, S107, S140,
S155
Kojima, N., S52
Kolander, T. M., S187
Kolomiets, M., S96
Kolomiets, T. M., S64, S65
Koné, D., S142
Kong, M., S64
Kong, P., S51, S207
Kong, S., S65, S97
Koornneef, A., S160
Kopperud, K., S11
Korban, S. S., S107
Korus, K. A., S187
Kouassi, N., S65
Kousik, C., S134, S201
Kousik, C. S., S65
Koval, N. C., S34
Kovalenko, E. D., S65
Krakowsky, M., S114
Kramberger, P., S81
Krampis, K., S190
Kreiser, B., S83, S134
Krishna Kumar, V., S65
Krishnam Raju, S., S65, S170
Kriss, A. B., S65, S66
Kroll, J., S9
Kubota, R., S66
Kuhar, T. P., S205
Kuhn, P., S66
Kuldau, G. A., S44
Kumar, K., S146
Kunjeti, S. G., S66, S207
Kunkel, D., S162
Kunta, M., S66
Kuo, K., S71
Kurle, J. E., S66, S187, S188
S214
PHYTOPATHOLOGY
Labavitch, J. M., S9
Labbé, C., S198
Lacey, L., S55
Lackermann, K., S67, S67
Laflamme, G., S194
Lahaye, T., S36, S159
Lahlali, R., S99
LaHue, S. S., S28
Laird, E., S94
Lak, M., S84
Lakatos, P., S51
Lakshman, D. K., S6, S67
Lalancette, N., S195
Lambert, D. H., S194, S194
Lamondia, J. A., S195
Lamour, K., S41
Lamppa, R. S., S185
Laney, A. G., S67, S201
Lange, H. W., S20
Langston, D. B., S9, S126
Lansdell, T., S54, S59
Lapochkina, I. F., S65
Larkin, R. P., S67, S92
Larsen, H., S101
Larsen, M., S42
Larsen, R., S68, S128
Lartey, R. T., S68
Lassiter, E. S., S68
Lauter, N., S157
Lava Kumar, P., S92, S113, S117,
S117, S126
Lawrence, A., S112
Lawrence, C., S158
Lawrence, K. K., S170
Lawrence, K. S., S20, S21, S65,
S86, S118, S153
Lawrence, N., S114
Le, M. H., S68
Leach, J. E., S127, S157, S164,
S167
Lea-Cox, J., S51
Leandro, L., S30, S187, S188
Leandro, L. F., S80
Ledbetter, C., S119
Lee, I., S29, S69, S135, S208
Lee, I.-M., S206
Lee, M., S24, S25, S68, S71
Lee, R., S77
Lee, R. D., S57
Lee, R. F., S46, S107
Lee, S., S204
Lee, S. A., S68
Lee, Y., S25, S41, S62, S64, S65,
S96, S97, S97, S97
Lefebvre, F., S198
Legler, S., S19
Legreve, A., S26, S58
Lehman, M., S181
Leisner, S. M., S107, S144
LeJeune, J. T., S155
Lemmetty, A., S88
Leng, Y., S69
Leonard, W., S69
Leonberger, A. J., S69
Leon-Reyes, A., S160
Lesniak, K. E., S69
Leung, H., S11, S157
Leveau, J., S158
Levesque, C., S153
Lévesque, C. A., S150
Levy, L., S73
Lewandowski, D., S69
Lewandowski, D. J., S70, S191
Lewis Ivey, M., S155
Lewis, K. J., S132
Lewis, P., S70
Lewis, R., S116
Lewsey, M. G., S70, S147
Li, B., S70, S75
Li, C., S104
Li, F., S140, S140
Li, G., S133
Li, H., S35, S39
Li, J., S71, S72
Li, N., S38
Li, Q., S133
Li, R., S71, S72, S101, S140,
S140
Li, S., S70, S70 S75, S127
Li, W., S70, S134
Li, X., S69, S70
Li, Y., S71, S105
Li, Z., S70
Liang, Y., S130
Liang, Z., S118
Liao, C., S71
Liao, H., S72
Liberti, D., S201
Liesche, R., S53
Lijuan, Z., S30
Lim, H., S71
Lim, K., S188
Limay-Rios, V., S180
Lin, H., S30, S41, S71, S72, S72
Lin, J., S105
Lin, L., S71, S72
Lin, Y., S1
Lindeberg, M., S72
Lindow, S. E., S136
Ling, K., S72, S139, S139
LiPuma, J. J., S31
Little, C. R., S90, S106
Little, D., S102
Littlefield, L. J., S153
Liu, B., S73, S73
Liu, H., S44, S72, S74, S140
Liu, J., S105
Liu, L., S188
Liu, Q., S73
Liu, R., S73
Liu, T., S105
Liu, W., S73
Liu, X., S74
Liu, Y., S74
Liu, Z., S73, S74
Livingston, W. H., S195
Locke, J. C., S74
Long, D., S131
Long, S., S120
Loope, L., S128
Lopes, S., S74
Lopes, U. V., S42
Lopez Nicora, H., S74
López, C. E., S127
Lord, W., S196
Lore, J., S136
Lorek, J., S158
Loschinkohl, C., S1
Lou, V., S30
Lourenço, S. A., S41
Louws, F., S73
Louws, F. J., S21, S73, S106,
S109
Lozano, G. L., S39
Loza-Reyes, E., S35
Lu, H., S151
Lu, P., S202
Lu, S., S44, S151, S151
Lu, X., S70, S75, S100
Lu, X. H., S74
Lubberstedt, T., S184
Lucas, J. A., S12, S51
Lund, S. P., S136
Luo, L., S72
Luster, D. G., S75, S127, S202
Ma, B., S75, S76
Ma, J., S75
Ma, L., S64, S75
Ma, P., S70, S75
Ma, W., S206
Ma, Z., S132
Macaulay, K., S70
MacDonald, A., S70
MacDonald, J. D., S104
MacDonald, W. L., S167
MacGuidwin, A., S26
MacGuidwin, A. E., S180
MacHardy, W. E., S196
Mackasmiel, L., S84
Mackenzie, S., S75
MacLean, D., S70
Madariaga, M., S110
Madden, L., S132, S137
Madden, L. V., S6, S6, S65, S66,
S81, S113
Maddux, L. D., S41
Madeiras, A. M., S195
Maejima, K., S52
Maffia, L. A., S121
Magarey, P. A., S38
Magculia, N., S136
Magill, C. W., S106
Magnusson, V., S18
Mahmoodi, B., S13, S113
Mahoney, N. E., S95
Mahto, B. N., S76
Mahtour, A., S76
Mahuku, G., S119
Maia, G. S., S40, S174
Maier, T., S58
Maier, T. R., S186
Majerczak, D. R., S27, S184
Malapi-Nelson, M. M., S76
Malvick, D., S185
Malvick, D. K., S14, S66, S187
Mammella, M. A., S76
Manabayeva, S. A., S76
Mandal, M. K., S58
Manjunath, K. L., S46, S77, S107
Mankolo, R. N., S91
Mann, R. B., S167
Manners, J., S75
Manning, V. A., S160
Mano, E. T., S77
Manoranjitham, K., S101
Manosalva, P., S77
Manosalva, P. M., S164
Mansfield, M., S77
Mansfield, M. A., S195
Mansfield, M. E., S77
Mantoe, L., S66
Manulis-Sasson, S., S22
Marahatta, S. P., S133
Maraite, H., S32, S32
Marek, S., S131
Marek, S. M., S7
Marett, C. C., S190
Margenthaler, E., S129
Marin, D. R., S118, S119
Marino, D. A., S77
Markell, S., S185
Marlow, G., S111
Marois, J., S181
Marois, J. J., S122, S143, S178,
S186, S204
Maroof, S., S189, S190
Maroon-Lango, C. J., S1, S78
Marques, A., S114, S207
Marquez-Villavicencio, M., S78
Marra, R. E., S195
Marrero, G., S78
Marsh, A. G., S66, S207
Martin, B., S152
Martin, F., S14
Martin, F. N., S14, S76, S78,
S150, S150, S152
Martin, G. B., S92
Martin, K., S11
Martin, R., S67
Martin, R. R., S106, S110
Martinez, M. C., S178
Martinez, M. T., S89
Martinez-Espinoza, A., S78
Martínez-Romero, E., S20
Martinka, M., S67, S67
Martins, T. D., S78
Maruthachalam, K., S8, S64, S79,
S79, S107
Marx, B. D., S115
Massola, N. S., S86
Mastouri, F., S165
Matheron, M. E., S79
Matias, A. C., S99
Matthews, B. F., S127
Mattupalli, C., S79, S188
Maul, J., S51, S207
Mauzey, S. J., S79
Mavrodi, D., S142
Mavrodi, O., S142
Mavrodieva, V., S73
Mayfield, A. E., S177
Mayfield, D. A., S15, S54
Maynard, C. A., S10, S29, S89,
S91
Mayo, J. B., S120
Mazzola, M., S134
Mbofung, G. C., S80
McAvoy, E., S107
McCabe, K. J., S124
McCall, D., S28, S205
McCall, D. S., S80, S152, S208
McClean, A., S80
McCluskey, K., S80
McCoy, S., S122
McCue, K. F., S101
McCullough, P., S78
McDonald, M., S59, S60, S99,
S117, S125
McDonnell, K., S17
McDowell, J., S158
McFarland, K., S195
McGahan, D., S16
McGhee, G. C., S166
McGrath, J., S47
McGrath, M., S80, S161
McGrath, M. T., S20, S102, S196,
S196, S197
McInroy, J. A., S63, S170, S170
McKenna, F., S34
McKenzie, D., S163
McLeod, A., S20, S52
McMahon, M. B., S75
McMillan, R. T., S174
McMullen, M., S4, S137
McNally, R. R., S80
McNellis, T. W., S68
McPhee, K., S25
McRoberts, N., S81
McSpadden Gardener, B., S158
S159
McSpadden Gardener, B. B., S20,
S96
Meacham, T., S61
Meah, B., S11
Medina, C., S110
Meekes, E., S81
Mehl, H. L., S81
Mehle, N., S81
Mehra, L., S81
Meinhardt, S., S151
Meitz, J. C., S20
Mejia, J. F., S5
Mekete, T., S74
Mekuria, T., S81, S90, S90
Melanson, R., S47
Melcher, U., S10, S18, S110,
S121, S154, S154
Melito, S., S26
Mellinger, C., S134
Melnick, R., S82
Melotto, M., S24, S91
Melzer, M. J., S82
Mendoza, P., S99
Meng, B., S104
Meng, F., S55, S55
Meng, Q., S82
Meng, Y., S56
Mengistu, N., S191
Menkir, A., S113, S117
Mentreddy, S. R., S91
Mergoum, M., S104
Merighi, M., S27, S184
Mersha, Z., S82, S82, S82, S115
Mertely, J. C., S83, S174
Meyer, F. W., S197
Meyer, P. W., S188
Meyers, B. C., S66, S207
Mian, R., S192
Michailides, T. J., S48, S83, S141
Michel, A. P., S33
Miglino, R., S81
Mila, A., S14
Mila, A. L., S100
Miles, T. D., S9, S83
Milgroom, M. G., S20
Milks, D., S55
Miller, J. D., S180
Miller, M., S65
Miller, S. A., S13, S20, S33, S13,
S124, S140, S155
Miller, S. I., S160
Miller-Butler, M., S83
Millhouse, J., S191
Milling, A., S55, S115
Milus, E. A., S203
Mimee, B., S193
Minenkova, O., S143, S143
Ming, R., S182
Mingora, C., S94
Minsavage, G. V., S103
Minzenmayer, R. R., S54
Mirzaee-Qomi, M., S83
Mirzaee-Qomi, P., S84
Misaghi, I., S122
Misra, A. K., S96
Mitchell, F. L., S88
Mitchell, M. N., S84
Mitchell, T., S10
Mitchum, M., S187
Mitchum, M. G., S58, S108,
S116, S132, S186
Mitkowski, N. A., S23
Mitra, A., S38, S67
Mittal, S., S54, S129
Mitter, N., S10
Miwa, E., S143
Mmbaga, M. T., S84, S84
Mock, N., S95
Mock, R., S56, S71, S72, S101
Modarresi Chahardehi, A., S84
Moeller, J., S188
Mohammad Deimi, A., S84
Mohammadi, A., S85, S85
Mohammadi, M., S85
Mohan, K., S113
Mohd Jaaffar, A., S85
Momol, T. M., S22, S173
Monacell, J. T., S85, S92, S157
Mondal, S. N., S85, S86, S174
Monfort, W. S., S121
Monger, W., S154
Monteiro-Vitorello, C. B., S21
Montenegro, N., S86
Montesinos-Herrero, C., S86
Montoya Piedra, Y., S125
Montpetit, J., S196
Moore, G. A., S61
Moore, G. G., S157
Moore, S. R., S86
Moore, W. F., S112
Moorman, G., S51
Moorman, G. W., S153
Mora, R., S110
Moraes, S. G., S86
Moral, J., S86
Morales Ruiz, S. S., S125
Morales, J. G., S40, S87
Morales-Payan, J., S176
Morales-Pedraza, L. G., S87
Morano, L. D., S115, S116
Moreau, J., S26
Moreira, A. S., S87
Moreira, R., S42
Morgan, D., S83
Morid, B., S32, S62, S101
Morris, T., S105
Morrison, E. N., S87
Morse, J. G., S118
Moscou, M., S157
Motavalli, P., S41
Mottet, M.-J., S196
Mou, B., S107
Moulin, M., S70
Mount, L. L., S174
Mousavi, L., S87
Moya, E. A., S87
Moyer, M. M., S38, S88
Mozafari, J., S84, S87
Msikita, W., S88
Mueller, D. S., S14, S182
Mueller, J. D., S153
Muilenberg, M. M., S208
Mukhina, Z. M., S64
Mulat, H. K., S88
Mulcahy, A., S93
Munkvold, G., S28, S30, S36,
S36, S148, S185
Munkvold, G. P., S109, S180,
S184
Muñoz, M. P., S110
Munyaneza, J. E., S88
Muramoto, J., S19
Murphy, A., S147
Murphy, A. M., S70
Murphy, D., S200
Murphy, J. A., S109, S197
Murphy, J. F., S118, S130
Murray, J., S182
Musson, G. H., S37, S100
Mutschler, M. A., S147
Myers, B. A., S88
Myers, K. L., S52
Myers, M., S42
Mysore, K. S., S54, S54, S129,
S160
Nadler, S. A., S113
Naidu, R. A., S3, S45, S56, S81,
S101
Nair, S., S116
Nakajima, T., S63
Namba, S., S52
Narvaez, D. F., S143, S178, S186,
S204
Natwick, E. T., S12, S138
Naumann, T. A., S88
Nava, C., S89
Navarre, D., S38
Navi, S. S., S89, S144
Neal, J. C., S102
Neate, S. M., S43
Negeri, A., S11
Neil, G., S93
Nelson, B. D., S4, S102, S102,
S111
Nelson, L., S191
Nelson, M. E., S40, S73, S138,
S188
Nelson, R. J., S156
Nelson, R. S., S142
Nelson, S. C., S82
Nemchinov, L. G., S208
Nepal, A., S89, S89, S189
Nettleton, D., S58, S136
Neu, K., S23
Neufeld, K., S89
Neufeld, K. N., S202
Neves, A. A., S77
Newhouse, A. E., S89
Newman, M. A., S153, S202
Newsom, L. J., S57, S131
Ngugi, H. K., S11, S46, S49, S49,
S68, S100
Nguyen, H. V., S160
Niblack, T., S185
Niblack, T. L., S74
Nichols, A., S28
Nichols, A. E., S80, S208
Nickerson, J., S70
Nie, J., S69, S70
Nieto, D., S89
Nightengale, S. P., S20
Nikolaeva, O., S61
Nischwitz, C., S89
Nishiguchi, M., S90
Nissen, L., S199
Nissinen, A., S88
Nita, M., S90, S90
Njambere, E. N., S90
Noel, M., S136
Nolan, S., S17
Noll, L. W., S90
Norman, D. J., S15, S90, S103
Northern, L., S89
Northern, L. C., S91
Norton, R., S196
Nosir, W., S95
Nusbaum, C., S68
Nuss, D. L., S135
Ñústez, C. E., S87
Nutter, F., S183
Nutter, F. W., S42, S186, S188
Nutter, Jr., F. W., S181, S188
Nyochembeng, L. M., S91
O’Brien, G. K., S178, S204
O’Connell, S., S109
O’Keeffe, T. L., S95
O’Neal, M., S182
O’Neill, N. R., S208
Oak, S. W., S150
Obasa, K., S91, S91
Obulareddy, N., S91
Ochoa Corona, F. M., S7, S7, S7,
S19, S30
Ockey, S. C., S91
Oerke, E., S156
Ogata, D. Y., S82
Ogunsola, K. E., S92
Oh, C., S92
Oikonomakos, I., S7
Ojiambo, P., S89, S190
Ojiambo, P. S., S128, S144, S144,
S202, S204
Ok, C., S92, S102, S197
Okano, Y., S52
Olanya, M., S67, S92
Olarte, R. A., S92, S157
Olatinwo, R. O., S92
Olaya, G., S55, S66, S93, S93
Oliveira, F. R., S21
Oliver, J. E., S93
Oliver, R., S151
Olivera Firpo, P., S93, S93
Olson, B. D., S161
Olson, H. A., S94
Olson, M. E., S48
Ong, K., S123
Ong, K. L., S199
Ongena, M., S158
Opit, G., S30
Oriolani, E., S99
Ormeño-Orillo, E., S20
Orr, G., S166
Ortega, M. A., S189
Ortega-Beltran, A., S39
Ortiz, B. V., S86
Ortiz, C., S94
Osborne, L., S137, S185
Osborne, L. E., S59
Oshima, K., S52
Osorio, D. L., S42
Ospina-Giraldo, M. D., S94, S94
Osterbauer, N., S135
Oswald, A., S20
Otero, M. L., S99
Otrosina, W. J., S94
Otto-Hanson, L. K., S122
Oudemans, P., S27, S101
Oudemans, P. V., S94
Vol. 100, No. 6 (Supplement), 2010
S215
Ouimette, D. G., S95
Owen, J., S142
Owens, A., S112
Owens, R., S95
Owens, R. A., S135
Ownley, B. H., S2, S44, S45,
S131
Padgett, G. B., S115, S153
Pagan, C., S113
Pahalawatta, V., S95
Palenchar, J., S174
Palencia, E., S10
Palm, M., S168
Palmateer, A. J., S22, S104, S125,
S125, S174, S175
Palmer, C., S60
Palmer, J., S11
Palmero Llamas, D., S95
Palmero, D., S29, S39, S95
Palm-Hernandez, M. E., S175
Palou, L., S86
Palumbo, J. D., S95
Pan, Q., S96
Pan, R.-Q., S96
Pan, R.-W., S96
Panchal, S., S91
Pandelova, I., S160
Pandey, A. K., S96
Pandey, R., S67
Panstruga, R., S158
Pappu, H., S122
Pappu, H. R., S1, S5, S10
Pardo, J. M., S5
Paret, M. L., S96
Park, B., S97
Park, C., S198
Park, D., S146
Park, J., S25, S64, S96, S97, S97,
S97
Park, S., S65, S77, S96, S97, S97,
S159, S164
Park, Y., S96
Parker, J. K., S29, S97
Parker, P. E., S15, S15
Parker, S. R., S141
Parkunan, V., S97
Pasche, J. S., S135, S183
Pastor-Corrales, M. A., S98
Pataky, J. K., S98
Patel, N., S98
Paul, P., S132, S137
Paul, P. A., S33, S33, S33, S65,
S66, S113, S184
Paulitz, T. C., S85, S142
Paveley, N. D., S51, S51
Payne, A. F., S98
Payne, G. A., S108, S138
Paz, J. O., S92
Paz, Z., S64, S98
Peckham, G. D., S66, S98
Pedersen, J. F., S37, S37, S185
Pedley, K., S190
Pedley, K. F., S61, S96
Peduto, F., S129
Peet, M. M., S109
Peever, T., S53
Pegues, M. B., S46
Peiman Williams, M., S47
Peiris, K., S134
Pelz-Stelinski, K. S., S172
Pena, A., S37
Peña, G., S7, S29
Peña, J., S53
Peña-Reyes, E., S99
Peng, G., S99
Pennerman, K., S99
Percich, J. A., S14
Perdomo, R. D., S4
Peres, N. A., S75, S86, S174
Perez, B. A., S21, S99, S99
Perez, F. G., S52
Pérez, J., S40, S171
S216
PHYTOPATHOLOGY
Périnet, P., S196
Perry, C. D., S202
Perry, K., S99, S100
Perry, K. L., S10, S147, S154
Perumal, R., S106
Peter, K., S100
Peters, R. D., S100
Peterson, G. L., S41
Peterson, N., S53
Peterson, P. D., S100
Pfeufer, E. E., S100
Phillips, D., S35, S182
Phillips, D. V., S145
Phillips, N. A., S1
Phipps, P. M., S100, S153
Pierson, E. A., S159
Pierson, L. S., S159
Pieterse, C. M., S160
Pintye, A., S19
Pirahesh, S., S101
Pires, J., S42
Plamann, M., S80
Plata-Caudillo, J. A., S101
Ploetz, R. C., S176, S177
Pokharel, R., S101
Polashock, J., S27, S94, S101
Ponciano, G. P., S101
Poojari, S., S101
Pooran DeSouza, S., S102
Popko, J. T., S92, S102, S197
Porchas, M., S79
Poromarto, S. H., S102, S102
Porter, L., S25
Porter, L. D., S102
Post, A. R., S102
Postnikova, O. A., S208
Potnis, N., S36, S103
Poudel, B., S103, S128
Powell, C. A., S145
Powell, G., S70
Powell, W. A., S10, S29, S89,
S91
Prabavathy, V. R., S116
Prathuangwong, S., S8, S103
Pratt, P. D., S108
Pratt, R., S103
Pratt, R. C., S27, S184
Presting, G. G., S78
Pretorious, Z. A., S156
Preuett, J. A., S202
Prezelj, N., S81
Price, J. A., S103
Prinster, M., S108
Pritsch, C., S110, S118
Probst, C., S39, S103
Proffer, T. J., S69
Prom, L. K., S106
Prosser, S. W., S104
Pryor, B. M., S5
Puckett, R., S83
Puppala, N., S114
Puri, K., S4
Puri, K. D., S104, S189
Purvis, M., S57, S142
Purwar, S., S104
Putnam, M. L., S104, S206
Pye, M. F., S104
Qi, M., S104
Qin, H., S105
Qiu, J., S105, S105, S105
Qiu, W., S38
Qu, F., S105
Quello, K. L., S183, S189
Quito, D., S106
Radwan, G. L., S106
Radwan, O., S106
Rahman, A., S106
Rahman, M., S21, S106
Rahman, M. H., S130
Raid, R. N., S36, S78, S106,
S107, S175, S175, S175, S196
Raikhy, G., S107
Rajashekara, G., S13, S140
Rakhshandehroo, F., S4, S31,
S84, S87
Ramadugu, C., S107, S46, S77
Ramirez, A. L., S110
Ramírez, C., S170, S171
Ramírez, M., S171
Ramos, A. T., S21
Rao, S., S107
Raper, R. L., S16
Rappaport, K., S73
Rascon, A., S62
Rascon, A. A., S107
Rasmussen, J., S151
Rasmussen, J. B., S185
Rathburn, H. B., S88
Rauscher, G., S107, S108
Rav David, D., S33
Ravanlou, A., S108
Ravnikar, M., S81
Rayamajhi, M. B., S108
Rayapati, N. A., S90, S90
Rebello, G., S108
Rebollar-Alviter, A., S6
Recknor, J., S58
Records, A. R., S136
Reddy, M., S65, S146
Reddy, M. S., S170
Redinbaugh, M., S105
Redinbaugh, M. G., S27, S184,
S184
Reed, S., S71
Reese, B. N., S108, S138
Reeves, P. A., S164
Rehman, M., S172
Reiners, S., S20
Rémus-Borel, W., S196
Replogle, A., S108
Repshare, J. M., S115, S116
Restom Gaskill, D. A., S175
Restrepo, S., S7, S8, S19, S21,
S22, S29, S29, S39, S86, S127,
S129
Reyes Gaige, A., S118
Reynaldi, S., S40, S40
Reynolds, K., S74
Rezaee, S., S13, S83, S101
Rezende, J. A., S87
Rezzonico, F., S120
Rice, K. A., S88
Richard, J., S108
Richardson, K., S108
Richardson, P., S51, S207
Richwine, N., S42
Ridenour, J. B., S109
Rideout, S. L., S48, S207
Ridout, C. J., S157
Riegel, D. G., S136
Riley, D. G., S122
Riley, R. T., S147
Riley, T., S172
Rinehart, T., S71
Rinehart, T. A., S45
Rintoul, T. L., S153
Rioux, D., S197, S197
Rioux, S., S193
Risede, J., S58
Ristaino, J. B., S52, S68, S161
Ritenour, M. A., S12
Ritson, R., S182
Rivard, C., S73
Rivard, C. L., S109
Rivera, L. I., S176
Rivera, M. C., S21
Rizzo, D., S167
Roane, C. W., S206
Robbins, R. T., S114
Roberts, J. A., S109, S197
Roberts, M., S162
Roberts, P., S52
Roberts, P. D., S52, S175
Robertson, A., S183
Robertson, A. E., S14, S109,
S123, S123, S182, S190
Robertson, C. L., S134, S203
Robertson, J., S59
Robertson, N. L., S109
Robideau, G. P., S150, S153
Roca, M., S99
Roca, M. M., S176
Rocateli, A. C., S16
Rockhold, D. R., S101
Rodrigues, J. V., S110
Rodriguez-Burgos, P., S176
Rodriguez, S., S110, S118
Rodriguez-R, L. M., S8
Rodriguez-Salamanca, L., S176
Roenhorst, A., S81
Rogers, C. A., S208
Rogers, E. E., S68, S110
Rogers, S., S110
Rogers, S. L., S12, S35
Roig, J., S41
Rojas, P. F., S110
Rojas-Bertini, C., S110
Rollins, J., S151
Rollins, J. A., S201
Roman, F., S34
Romer, P., S36
Rommens, C. M., S101
Rong, X., S159
Roose, M., S107
Roper, C. M., S85
Rosales Villavicencio, I., S99,
S110
Rosenberger, D. A., S197
Ross, D., S51
Ross, R. E., S18, S205
Ross, S., S208
Rossi, L., S166
Rossi, V., S19, S194
Rosskopf, E., S176
Rosskopf, E. N., S19
Rothrock, C., S31, S75, S111,
S129
Rothrock, C. S., S121, S153,
S203
Rothwell, N. L., S9
Rott, P. C., S111
Roubtsova, T. V., S104
Rouxel, T., S158
Rowhani, A., S3, S3, S5
Roy, A., S111
Roy-Chowdhury, M., S202
Royer, M., S111
Ruaro, L., S111
Rudolph, K., S111
Rugh, A. L., S197
Ruhl, G., S69
Ruhl, G. E., S168
Ruhl, G. S., S32
Rupe, J., S26, S31, S111, S129
Rupe, J. C., S70, S203
Rush, C., S50, S68
Rush, C. M., S2, S103, S120,
S138, S149, S203
Russell, P., S15
Russell, S. A., S136
Russell, T., S131
Russo, J., S46, S179
Ryu, C., S54, S160
Saad, A. T., S111
Saalau Rojas, E., S26, S112,
S112, S112, S189
Sabanadzovic, S., S2, S103, S112,
S112, S112, S112, S112, S123
Saberi-Riseh, R., S84
Saborío, F., S63
Saffarian Abbas Zadeh, M., S113
Saha, S., S72
Sainju, U. M., S68
Sakthikumar, S., S156
Salaudeen, M. T., S113
Salazar, J. J., S4
Salgado, J. D., S113
Salih, M. M., S4
Sampangi, R., S113
Sanchez, K., S113
Sanchez, K. R., S27
Sanders, Jr., F., S113
Sanderson, K. R., S100
Sanderson, P. G., S163
Sandoval, C., S110
Sangchote, S., S121
SanMiguel, P. J., S17
Sano, T., S95
Sanogo, S., S114, S114
Santa-Cruz, J., S114
Santhanam, P., S64
Santos, M., S39, S95
Santos, V. C., S77
Sastoque, L., S42
Sattler, S. E., S37, S37, S185
Savary, S., S136, S156
Saville, B. J., S87
Sayler, R. J., S114
Schaad, N., S114, S116, S207
Schaafsma, A. W., S180
Schell, M. A., S45, S66
Schena, L., S76
Scherf, J. M., S115
Scherm, H., S55, S81
Schilder, A. M. C., S9, S9, S83
Schlehuber, S., S37
Schlub, K. A., S115
Schlub, R. L., S82, S82, S115
Schmale, D. G., S208
Schmidt, D., S115
Schmidt, F. J., S43
Schmidt, L. S., S115
Schneider, K. L., S78
Schneider, R. W., S22, S115,
S134, S203
Schneider, W., S117
Schneider, W. L., S126, S149,
S209
Scholez, J. E., S43
Scholthof, H. B., S76
Schreiber, H. L., S115, S116
Schroeder, B. K., S31, S53
Schroeder, K. L., S142
Schubert, T., S116, S172
Schuenzel, E., S116
Schultheiss, H., S54, S129
Schultz, P. B., S205
Schumacher, R., S114
Schwab, E. B., S16
Schwandt, J., S166
Schwartz, D., S75
Schweri, K. K., S116
Sciumbato, G. L., S153
Scocco, E. A., S116
Scorza, R., S209
Scott, R., S136
Scully, B., S37, S57
Sechler, A., S114, S116, S207
Seebold, K. W., S153
Seem, R. C., S8, S38, S88
Sekar, J., S116
Selote, D., S117
Semer, C. R., S40
Sengodagounder, V., S88
Sepúlveda, P., S99, S110
Serdani, M., S104
Serrato, L. M., S176
Sessa, G., S22
Sétamou, M., S66
Sether, D. M., S82
Sexton, P. J., S194
Shabana, Y., S176
Shah, K. H., S117
Shah, S., S130
Shamsbakhsh, M., S31
Shan, W., S158
Shao, J., S29, S95
Sharma, K., S117, S117, S117
Sharma, S. K., S96
Shatters, R. G., S6
Shaw, B. D., S62, S133
Shaw, J. N., S86
Shaw, M. W., S49
Shennan, C., S19
Sherman, D., S117
Sherman, D. J., S209
Shi, X., S118
Shim, W., S62, S76, S94, S109,
S133
Shimabuku, R., S82
Shiraishi, A., S118
Shishkoff, N., S136
Shock, C., S113
Shoemaker, R., S189
Shokes, F. M., S153
Shrestha, B., S47
Shu, X., S38
Shuai, B., S118
Shyu, C., S11
Sierotzki, H., S49, S162
Sikora, E., S30
Sikora, E. J., S118
Silva Junior, G. J., S118, S119
Silva, P., S110, S118
Silva, V., S187
Silva-Rojas, H., S6
Silva-Rojas, H. V., S119
Silveira Baggio, J., S119
Simard, M., S197
Simmons, A., S103
Simon, A., S105
Simon, J., S198
Simon, L., S121
Simonson, A., S59
Simpson, C. E., S204
Singer, S., S50
Singh, A., S119
Singh, D., S93
Singh, R., S85
Sistani, K., S30
Sisterson, M., S119
Sivasithamparam, K., S35, S39, S60
Skaltsas, D., S119
Skaria, M., S66
Skatenok, O. O., S64
Skelsey, P., S120
Skelton, L. L., S155
Skipper, C. E., S115, S116
Sliva, D., S131
Smart, C. D., S20, S20
Smeda, J., S108
Smith, A. G., S70
Smith, A. J., S120
Smith, B. J., S83, S134
Smith, C. A., S196
Smith, D., S137
Smith, D. L., S98, S120
Smith, D. S., S69
Smith, J., S16
Smith, J. A., S82, S174, S176,
S177
Smith, J. E., S120
Smith, L., S44
Smith, M. J., S120
Smith, R. J., S3
Smith, T. R., S175
Smither, M. R., S1
Smits, T. H., S54, S59, S120
Snelling, J., S157
Sniezko, R. A., S120, S167
Snook, M., S10
Socha, C., S19
Sokhandan Bashir, N., S121
Sokhandan, N., S87
Solano, F., S62
Soler, J., S40
Soltani Nejad, S., S113
Song, W., S97
Sopee, J., S121
Soto-Arias, J., S148
Souza, A. G., S121
Sowa, D. A., S48
Spadafora, V. J., S166
Spaine, P. C., S94
Spaine, P. O., S82
Spanu, P., S56
Sparks, A., S121
Speers, C., S69
Spiers, J., S71
Spolti, P., S121
Spooner, D., S19
Spósito, M. B., S118, S119
Spurlock, T. N., S121, S203
Sreedharan, A., S122
Srinivasachary, A., S136
Srinivasan, R., S122
Srivastava, P., S122
St. John, R., S91
St. Martin, S., S189
St. Martin, S. K., S190, S192
Stacey, G., S68
Stack, J., S181
Stadnik, M. J., S122
Stafford, C. A., S122
Stammler, G., S37
Stanghellini, M., S122
Stansly, P. A., S77
Starr, J., S123
Starr, J. L., S199
Steadman, J., S122
Stebbins, T. C., S169
Steddom, K., S123
Steffenson, B., S157
Steger, A., S26, S31
Stein, J. M., S15
Stenger, D. C., S68
Stensvand, A., S8
Stephenson, R. C., S112, S123
Stevens, M., S147
Stevenson, K. L., S9, S105, S105,
S126
Stewart, P., S60
Stewart, S. M., S123, S123, S190
Stockwell, V. O., S59, S120
Stolz, M., S23
Stone, A. L., S42, S209
Stone, C. L., S37
Stone, E. A., S85, S92, S157
Stowell, L., S152
Stoxen, S., S156
Strausbaugh, C. A., S123
Stubbs, G., S60
Stuchi, E. S., S41
Su, H., S123
Suárez, A., S171
Subbarao, K., S53
Subbarao, K. V., S8, S24, S53,
S64, S79, S79, S107
Subedi, N., S124
Suciu, D., S117
Sudarshana, M. R., S3
Sugawara, K., S52
Suh, S., S130
Sui, D. D., S107
Suizu, Y., S90
Sullenger, A. R., S9
Summers, C., S129
Sumner, D. R., S153
Sun, J., S73, S73
Sun, X., S24
Sundaram, S., S104
Sundin, G., S23
Sundin, G. W., S69, S80, S166
Sung, S. S., S94
Sunkar, R., S3
Sutherland, A. M., S124
Sweany, R., S28
Sweany, R. R., S124
Sweets, L., S137, S185
Sykes, V. R., S80, S152
Syverson, R. L., S124
Szabo, L. J., S27, S156
Taber, S., S174
Taberner, V., S86
Tabor, G., S164
Talezari, A., S124
Tallapragada, P., S208
Talledo Albujar, M. J., S125
Tally, A. H., S165
Tande, C., S59
Tarnowski, T. L., S125, S125
Tatineni, S., S125
Taylor, E., S172
Taylor, R. J., S183
Te Beest, D., S125
Tech, K., S95
Tedford, E., S163
Teichmann, B., S198
Teixeira, D. C., S74
Teliz, D., S89
Tello Marquina, J., S29, S39
Tello, J., S95, S95
Temsah, M., S111
Tenuta, A., S185
Tertuliano, M., S199
Tewolde, H., S30
Thaher, N. H., S125
Thannhauser, T., S100
Thaxton, P. M., S153
Thies, J. A., S65
Thomas, A., S9, S126
Thomashow, L. S., S85, S141
Thomma, B. P., S64
Thompson, D. C., S126
Thonart, P., S158
Thorne, J., S68
Thowthampitak, J., S103
Thru Ppoyil, S., S190
Thru Ppoyil, S. B., S126
Tian, B., S126
Time, I., S126
Timmer, L., S174
Timmer, L. P., S85
Tisserat, N., S153
Tisserat, N. A., S127
Tolin, S. A., S173, S205
Tomaso-Peterson, M., S126
Tong, L., S77
Tooley, P. W., S208
Torres Puyo, C., S118, S110
Toth, I. K., S80
Townley, M. A., S77
Trapero-Casas, A., S86
Travers, S. E., S4
Travis, J. W., S46, S49, S49,
S100
Tredway, L. P., S75
Trelles Di Lucca, A., S125
Tremblay, A., S127
Tremblay, G., S193
Trigiano, R., S71
Trinidad Chipana, E., S125
Tripathy, S., S190
Triplett, L. R., S127
Trivedi, P., S127
Trogolo, J. A., S61
Tronsmo, A., S8
Trujillo, C. A., S127
Tsai, C., S184
Tschaplinski, T., S160
Tucker, D., S189
Tucker, D. M., S190
Tudzynski, B., S18
Tuffen, M. G., S127
Turechek, W., S127
Turner, R. S., S78
Turoop, L., S128
Tweddell, R., S65
Tweddell, R. J., S193
Twieg, E., S73
Twizeyimana, M., S128, S190
Tychon, B., S32, S32, S76
Tylenko, D. P., S100
Tyler, B., S190
Tyler, B. M., S158
Tyler, D., S30
Tylka, G., S30, S185
Vol. 100, No. 6 (Supplement), 2010
S217
Tylka, G. L., S190
Tzanetakis, I., S67
Tzanetakis, I. E., S103, S112,
S128, S201
Tzeng, K., S71
Uchida, J., S118, S128
Uddin, W., S106
Udvardi, M., S40
Ueng, P., S59
Ullah, H., S128
Ullman, D. E., S122
Uppala, S., S128
Uppalapati, R., S160
Uppalapati, S., S129
Uppalapati, S. R., S54, S54
Urashima, A. S., S78
Urbez-Torres, J. R., S129
Uribe, P., S14
Urrea, K., S129
Urrea, K. E., S203
Urrea, R., S129
Uyemoto, J. K., S3
Vadivel, K., S129
Vahling, C., S30
Vaiciunas, J., S94
Vaira, A., S71
Valdivia, C., S41
Valenzuela-Solano, C., S87, S101
Vallad, G., S129, S140
Vallad, G. E., S177, S177
Valverde, R. A., S112, S115
Van Bruggen, A., S177
Van den Bosch, F., S51, S51
Van der Does, D., S160
van Kan, J. A., S151
Van Rij, N., S181
van Santen, E., S18
van Schadewijk, T., S81
Van Wees, S. C., S160
Vanasse, A., S193
Vargas, A. M., S7, S22, S29
Vargas, H. A., S40
Vasquez, S., S95
Vaughn, V. L., S76
Vazquez-Mundo, M., S29
Vega, B., S130
Velasquez, N. Y., S130
Vemulapati, B., S130
Venkata, B., S11
Vera Cruz, C. M., S11, S157
Vercauteren, A., S42
Verhalen, L. L., S153
Verma, S. S., S130
Vermeire, M., S58
Veronese, P., S64
Vicente, M., S86
Vico, I., S56, S58
Vidaver, A. K., S38, S187
Vidic, U., S81
Vigaya Satya, R., S117
Vijay Krishna Kumar, K., S170
Villamor, D. V., S130
Villani, S., S27
Villanueva, L. M., S130
Vincelli, P., S6
Vincent, M., S80
Vining, K. J., S77
Vitoreli, A., S34
Vitoreli, A. B., S177
Vitoreli, A. M., S110
Vittal, R., S131
Vivancos, J., S196
Vodkin, L., S106
Vowell, T., S96
Vu, A. L., S131
Wach, M., S163
Wade, L., S166, S166
Wadl, P. A., S45
Wager-Page’, S. A., S131
S218
PHYTOPATHOLOGY
Wakefield, L. M., S38
Walcott, R., S200
Walgenbach, P. J., S131
Walker, D. R., S122
Walker, G. P., S122
Walker, K., S63
Walker, K. A., S131
Walker, N., S131
Walkinshaw, C., S203
Walkinshaw, C. H., S131
Wall, G. C., S178
Wallace, R. W., S173
Waller, L., S190
Wallhead, M. W., S113, S132
Wallis, C., S132
Waltz, C. F., S78
Wan, A., S132
Wang, B., S133
Wang, C., S62, S133
Wang, D., S10, S104, S133, S154
Wang, G., S31
Wang, H., S132, S190
Wang, J., S23, S71, S108, S132
Wang, K., S133
Wang, L., S24
Wang, M., S53, S105, S132
Wang, N., S7, S44, S55, S122,
S127, S141
Wang, Q., S49, S73, S74, S146
Wang, S., S133
Wang, X., S45, S132, S133, S135
Wangdi, T., S54, S160
Ward, L., S70
Ward, N. A., S134, S203
Ward, T., S157
Watson, A., S162
Watson, A. K., S197
Watt, B., S194
Webster, C., S134, S134
Wedge, D. E., S134
Weems, J. D., S191
Weerakoon, M., S134
Wegulo, S., S134, S147, S186,
S186, S191
Wegulo, S. N., S50
Wei, W., S135, S206, S207,
S208
Weil, C., S11
Weiland, J., S153
Welch, K. D., S135
Welker, R., S73
Welker, R. M., S109
Weller, D. M., S85, S141
Welliver, R., S42, S135
Wen, A., S135
Wen, J., S146
Wen, R., S135
Weng, Z., S154
Werres, S., S42
Westerdahl, B. B., S135
Westerman, R. P., S17
Westphal, A., S136
Westwood, J., S70
Westwood, J. H., S147
Whalen, M. C., S101
Wharton, P. S., S14
Wheeler, G., S108
Wheeler, T., S200
Wheeler, T. A., S136, S138,
S153, S173
Whitaker, B., S58
White, F., S159
White, J., S75
White, T. L., S145, S145, S178
Whitehead, J., S28
Whitfield, A. E., S184
Whitham, S., S187
Whitham, S. A., S96
Wick, R., S32
Wick, R. L., S195, S196
Widmer, T. L., S34, S136, S202,
S206
Wiemann, P., S18
Wiest, A., S80
Wiglesworth, M., S166
Wilcox, W. F., S38, S88, S136
Williams, B., S58, S58, S160
Williams, D., S60
Williams, J., S38
Williams, J. L., S136
Williams, W. P., S137
Willie, K., S105
Willocquet, L., S136, S156
Willyerd, K. T., S137
Wilson, C., S137, S137
Wilson, R., S137
Windels, C. E., S16
Windham, A., S71
Windham, G. L., S137
Windham, M., S71
Wingo, R. M., S124
Winterhagen, P., S38
Wintermantel, W. M., S103,
S128, S138
Winters, S. A., S153
Wise, K., S50
Wise, R., S56, S157
With, K. A., S120
Woeste, K. E., S17
Wolf, J., S208
Wolfe, D. W., S26
Woloshuk, C. P., S62, S108,
S109, S138
Woltjen, C. D., S191
Wong, F. P., S152, S168
Wood, A., S182
Wood, E., S14
Woodruff, W., S94
Woods, J. L., S138
Woodward, J. E., S136, S138,
S200, S204
Woodward, S., S95
Workneh, F., S50, S138, S203
Worley, E., S40
Worthington, C. J., S157
Wrather, A., S70
Wright, A. F., S138
Wright, D., S181
Wright, D. L., S122, S143, S178,
S186, S204
Wright, E. R., S21, S99
Wright, R., S183
Wu, B., S24
Wu, B. M., S139
Wu, J., S139
Wu, T., S173
Wu, W., S135, S206, S207, S208
Wulff, N. A., S74
Wyenandt, A., S198
Wyenandt, C. A., S139, S196
Xia, J., S139
Xia, J. Q., S72, S139
Xia, Y., S139
Xiang, Q., S140
Xiao, C., S63, S63, S143, S143,
S163
Xie, C., S140
Xie, Y., S47
Xing, J., S204
Xiong, Z., S154
Xu, D., S132, S140, S140, S146
Xu, D.-G., S96
Xu, J., S52, S151
Xu, L., S140
Xu, S., S93, S151
Xu, X., S13, S140
Xue, B., S116, S141
Yadagiri, K., S204
Yaghmour, M. A., S141
Yajima, W., S130
Yakabe, L. E., S141
Yamaoka, N., S90
Yan, Q., S141
Yang, B., S159
Yang, C., S96, S144
Yang, D., S198
Yang, J., S40, S141
Yang, K., S158
Yang, M., S141
Yang, W., S141, S142
Yang, X., S89, S132, S142, S144
Yang, Y., S49, S73, S74, S146
Yao, C., S63, S142
Ye, X., S105
Yin, C., S142
Yin, J., S55, S57, S142
Yin, Y., S63, S143, S143, S163
Yokota, K., S143
Young, C., S7
Young, H., S37
Young, H. M., S143, S178, S204
Young, R. F., S31
Young, S., S64
Younkins, A., S209
Yu, J., S191
Yu, K., S139
Yu, X., S136
Yuan, L., S204
Yuan, Q., S143, S143
Zablotowicz, R. M., S1
Zaccaron, M. L., S144
Zaid, A. M., S144
Zale, J., S131
Zamanizadeh, H., S4, S6, S32,
S62, S84, S101, S124
Zearfoss, A. D., S144, S144,
S204
Zelaya-Molina, L. X., S191
Zellermann, E., S22
Zellner, W. L., S144
Zenbayashi, K. S., S63
Zeng, Q., S64, S68, S75, S144
Zhai, L., S145
Zhang, A. B., S89
Zhang, C., S96, S164, S187
Zhang, G., S145, S182, S191
Zhang, J., S139, S146
Zhang, L., S138
Zhang, M., S145
Zhang, N., S90
Zhang, P., S73
Zhang, S., S145, S145, S145,
S146, S178
Zhang, X., S68
Zhang, Z., S38, S151, S192
Zhao, W., S23
Zhao, Y., S29, S69, S80, S104,
S133, S135, S206, S206, S207,
S208
Zheng, D., S107
Zheng, Y., S3, S53, S146, S146
Zhong, S., S4, S69, S104, S189
Zhou, C., S146
Zhou, G., S132, S140, S146
Zhou, J., S128
Zhou, L., S41, S190
Zhou, M., S23
Zhou, S., S75
Zhou, X., S52, S139, S146,
S146
Ziebell, H., S70, S147
Ziems, A. D., S187
Zimba, P. V., S43
Zimmerman, B. H., S39
Ziska, L., S179
Ziska, L. H., S208
Zitomer, N. C., S147
Zitter, T. A., S147
Zou, W.-C., S96
Zuluaga, A., S147
Zuñiga-Davila, D., S20
Zwingman, M., S147
Zwonitzer, J., S114