Mycological Society of America
Phomopsis amaranthicola, a New Species from Amaranthus sp.
Author(s): E. N. Rosskopf, R. Charudattan, Y. M. Shabana, G. L. Benny
Source: Mycologia, Vol. 92, No. 1 (Jan. - Feb., 2000), pp. 114-122
Published by: Mycological Society of America
Stable URL: http://www.jstor.org/stable/3761455
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Mycologia, 92(1), 2000, pp. 114-122.
? 2000 by The Mycological Society of America, Lawrence, KS 66044-8897
Phomopsis amaranthicola, a new species from Amaranthus sp.
E. N. Rosskopfl
U.S.D.A.-A.R.S., Horticultural Research Unit, 2199 S.
Rock Road Ft. Pierce,Florida 34945
R. Charudattan
Department of Plant Pathology, 1453 Fifield Hall,
P.O. Box 110680, University of Florida, Gainesville,
Florida 32611
Y. M. Shabana
Plant PathologyDepartment,Faculty of Agriculture,
Mansoura University,El-Mansoura, Egypt 35516
G. L. Benny
Department of Plant Pathology, 1453 Fifield Hall,
P.O. Box 110680, University of Florida, Gainesville,
Florida 32611
Abstract: In 1992, a pycnidial fungus isolated from
diseased amaranth plants was found to be the causal
agent of the observed stem and leaf blight. Inoculations performed in the greenhouse revealed that this
organism caused a disease characterized by a general
blight. The isolate was tentatively identified as a
member of the genus Phomopsis and its morphological characteristics were compared to those reported
in the literature for Phomopsis species collected from
amaranth. The Florida isolate, ATCC 74226, was
found to produce extremely large alpha conidia. Beta
conidia were produced, as well as a third type of conidium of intermediate shape. Phylogenetic analysis,
based on the sequences within the internal transcribed spacer regions of the ribosomal DNA, revealed that isolate ATCC 74226 differed substantially
from the other Phomopsis species tested, including
those that are known to produce the third type of
conidium. Based on the morphological characteristics and DNA sequence information the isolate is described here as a new species of Phomopsis.
Key Words: Coelomycete, internal transcribed
spacer region, ITS, pigweed
biological control. Surveys were conducted to seek
potential agents for development as mycoherbicides.
A pycnidial fungus isolated from diseased pigweed
(Amaranthus sp.) was found to be the causal agent
of a stem and leaf blight. Inoculations revealed that
the disease caused by this organism began as leaf lesions, which expanded, coalesced, and moved to the
leaf petiole, causing premature leaf abscission. Symptoms were observed within 5 d of inoculation. Symptoms then appeared on stems, with lesions girdling
the stem, causing stem constriction and toppling of
plants. The severity of the symptoms observed in the
field and then reproduced in subsequent inoculations warranted further investigation of the fungus as
a potential biological control agent for Amaranthus
spp.
Approximately 65 species of Phomopsis are plant
pathogens, of which 30 are found in the United
States. The identification of species in this genus,
based primarily on morphological characteristics, is
hindered by plasticity of characters. Difficulty with
identification is also compounded due to teleomorphic associations with Diaporthe spp. being
known for only about 20% of the named Phomopsis
species. Evidence that host association is inadequate
for speciation in this genus was provided by Brayford
(1990), who showed that Phomopsis occurring on
twigs and bark of Ulmus L. in the United Kingdom
and other parts of Europe belonged to different morphological and genetic groups. Both groups were isolated from a variety of tree species, showing that
more than one species could be found on a single
host and more than one host could support a single
species.
Species of Phomopsischaracteristically produce two
types of conidia, referred to as a conidia and ( conidia. Three species reported to have a third type of
conidium, the C conidium, are P hordei Punith., P
oryzae Punith., and P phyllanthi Punith. These species were isolated from Hordeum vulgare L., Oryza sativa L., and Phyllanthus L. spp., respectively (Punithalingam 1975). A single species of Phomopsis
(Sacc.) Bubak has been reported to infect amaranth.
This species, P amaranthi Ubriszy & V6r6s was reported from Hungary from Amaranthus retroflexusL.
(Ubriszy and Voros 1966).
Rehner and Uecker (1994) tested the relationship
INTRODUCTION
Many plant species in the genus Amaranthus are troublesome weeds and are considered good targets for
Accepted for publication September 2, 1999.
' Email: erosskopf@msn.com
114
115
ROSSKOPF ET AL: P. AMARANTHICOLASP. NOV.
TABLEI. Descriptions of cultures used for comparison of the internal transcribed spacer regions.a
Isolate
Location
Host genus
Conidial types
75b
Amaranthus
USA-Iowa
a (AF079770)J
BG
FP1
FP3
PHOc
Amaranthus
Amaranthus
Amaranthus
USA-Florida
USA-Florida
USA-Florida
Glycine
Amaranthus
Amaranthus
USA-Arkansas
USA-Florida
ot (AF079771)
ot (AF079772)
a (AF079773)
N/A (AF079775)
N/A (AF079774)
a, 3, and C (AF079776)
oa, 3, and C (AF079777)
Unknown
aoand P
a and 3
Unknown
at and (3
at and P
a and P
a
a
Unknown
a and p
a and P
a and P
a and P
ot and P
a and P
Mad
Bi
Poe
624f
537f
528f
522f
512f
476g
468g
649f
642f
597f
484g
456g
452g
gm2h
GAP8h
GLB06h
Oryza
Glycine
Kalmia
USA-Florida
USA-New Jersey
Puerto Rico
USA-New Jersey
USA-New Jersey
USA-New Jersey
USA-New Jersey
Canada
Magnifera
Sassafras
Juniperus
Vaccinium
Vaccinium
Convolvulus
USA-Maryland
Dominican Republic
USA-Maryland
USA-Mississippi
USA-Texas
USA-Georgia
USA-Georgia
USA-Georgia
Glycine
Solanum
Capsicum
Stokesia
Capsicum
Prunus
Prunus
Prunus
All sequences for isolates with labels composed of a three digit number were obtained from GenBank and were deposited
by S. Rehner and F. Uecker. Sequences for isolates gm2, GAP08, and GLB06 were also obtained from the GenBank. These
isolates were deposited by W. Uddin (1996).
b Isolate
provided by C. Block, North Central Regional Plant Introduction Station, Ames, Iowa.
a
c Phoma
medicaginis, ATCC 52798.
Isolate of Microsphaeropsisamaranthi provided by G. Weidemann, University of Arkansas.
e
Phomopsis oryzae, IMI 158929.
fAs reported in Uecker 1988.
g As obtained from the personal notebooks of F. Uecker, courtesy of R. Pardo-Schultheiss, USDA, Beltsville, NM.
h Isolate information from W. Uddin 1997.
i Described herein as Phomopsis amaranthicola.
JGenBank accession number of new deposits
d
of Phomopsis spp. with host association
through
se-
quencing of the internal transcribed spacer (ITS) regions of the ribosomal DNA. Their work resulted in
three groups based on the ITS phylogeny. Group A
consisted of two subclades. High similarity of one
subclade, referred to as Al, if viewed as a single species, could be indicative of a broad host range, including plants belonging to the genera Paulownia
Siebold & Zucc., Epigaea L., Kalmia L., Cornus L.,
Tsuga Carriere, Lindera Thunb., Vaccinium L., and
Picea Dietr., to name a few. Members of the second
subclade, A2, were all associated with cultivated Vaccinium spp. Group B, with the exception of a single
isolate, originated from southern temperate to tropical regions and produced elongate paraphyses in the
conidiomata. Isolates in Group C were obtained from
a wide range of herbaceous cultivated plants. Results
of this study suggest that there is a great deal of variation within the genus that could be attributed to
geographical distribution.
MATERIALSAND METHODS
Isolates.-Single-spore isolates were recovered from agar
slants and soil tubes by plating onto Difco potato dextrose
agar (PDA). A single isolate, ATCC 74226 (isolate B, TABLE
I), having consistent growth in all subcultures, was used for
determination of morphological characters. Plate cultures
were inoculated using a 5-mm3 mycelial plug taken from 10d-old PDA cultures. These were then incubated at 25 ? 2
C with a 12-h light cycle. Conidia were harvested from
plates using 10 mL of sterile deionized water per plate and
gentle scraping with a rubber policeman. A hemacytometer
was used to count conidia. Data obtained included proportions of each type of conidium and measurements of conid-
116
MYCOLOGIA
ia. Conidial germination was evaluated by placing a thin
coating of PDA onto a sterile glass slide. A droplet of a
conidial suspension, obtained from plates in the same manner as above, was placed onto the PDA and a sterilized cover
slip was placed on top. The slides were allowed to incubate
for 20-24 h on a bench top and the proportions of germinating conidia were pooled with the spore germination
data obtained directly from the suspensions.
The dimensions of pycnidia were measured at the first
sign of sporulation. Measurements were also made of conidia and pycnidia taken from inoculated A. hybridus L.
tissue that was allowed to senesce and dry slightly to induce
pycnidial formation, as had been observed in the field.
Stem pieces with pycnidia were placed in moist chambers
to induce sporulation. In an attempt to induce a perithecial
state, infected tissue samples were allowed to dry and were
stored in sand or soil, at both 4 C and at 30 C, in the absence of light. The available isolates were also paired on
PDA and V-8 agar plates (Miller 1955).
The physical dimensions of conidia were measured using
cultures of the ATCC 74226 isolate grown on PDA and from
diseased plant tissue. Samples were obtained from 1- to 2wk-old colonies from plates that were incubated at 25 + 2
C. Four samples of conidial droplets from each of five replicate cultures were used. Droplets were mounted on glass
slides in water and conidia were measured in two randomly
chosen fields until 100 measurements of each type of conidium were taken.
Isolate ATCC 74226 was grown on artificial growth media,
including Difco potato dextrose agar (PDA), V-8 agar, Difco
(Detroit, Michigan), Difco corn meal agar (CMA), Difco
oatmeal agar (OMA), oatmeal agar (OMAII), and amaranth-infusion agar, with (AIAS) and without sucrose (AIA),
for comparison of growth characteristics. Amaranth infusion agars were prepared by boiling 50 g of amaranth leaves
and stems in 500 mL of deionized water for 30 min. The
strained extract (200 mL) was added to 800 mL of deionized water containing 14 g of agar (Fisher Scientific, Pittsburgh, Pennsylvania). Sucrose was then added to a portion
of the medium to achieve a concentration of 2%. Oatmeal
agar was prepared using 60 g/L of oat flakes and 14 g/L
agar. Growth rate of the isolate was recorded daily.
The following isolates, obtained from symptomatic tissues
of accessions of Amaranthus spp. were compared for growth
of mycelium and spore production: 75A, 72A, and 72B provided by Dr. Charles Block of the North Central Regional
Plant Introduction Station, Ames, Iowa; BG from Belle
Glade, Florida; FP1 and FP3 from Fort Pierce, Florida; AI
from Immokalee, Florida; and BRZ from Jaboticabal, Sao
Paulo, Brazil. Isolates 75A, 72A, and 72B were tentatively
identified by Dr. Block as members of the genus Phomopsis.
Each isolate was transferred onto six petri plates of each
medium. Plates were inoculated using a 5-mm diam mycelial plug from cultures grown on PDA.
DNA analysis.-The following isolates were grown in liquid
potato-dextrose broth (PDB) shake cultures: B (ATCC
74226) from Gainesville, Florida; FP1 and FP3 from Fort
Pierce, Florida; 75A from Ames, Iowa; MA Microsphaeropsis
amaranthi from Arkansas; Pho, Phoma medicaginis Malbr. &
Roum. (ATCC 52798); and PO, Phomopsis oryzae (IMI
158929).
Flasks (250 mL) containing 50 mL of PDB were inoculated with three, 5-mm diam mycelial plugs from 10-d-old
PDA cultures of each of the isolates derived from singlespore cultures stored at 9 C. After 7-10 d of growth in shake
culture, the contents of the flasks were filtered through sterile cheesecloth, squeezed dry, and rinsed three times with
sterile deionized water. Mycelium was then placed into 13mL plastic tubes, stored for 24 h in a -80 C freezer, and
lyophilized for 24-48 h. The dry mycelium was then mixed
with liquid nitrogen, ground to a fine powder, and DNA
was extracted using the method of Koenig (1997).
The crude pellet was dissolved in TE buffer containing
2M NaCl and reprecipitated in two volumes of ethanol. The
DNA was then pelleted and the tubes were allowed to dry.
The pellet was resuspended in 100 iLLof TE buffer (10 mM
Tris, 1 mM EDTA). Samples were then placed at 4 C until
the DNA was dissolved.
Approximately 100 ng of template DNA per 100 KL reaction mixture were used in symmetric PCR. The internal
transcribed spacer regions (ITS) of the nuclear ribosomal
repeat were analyzed using primers ITS4 and ITS5 (White
et al 1990, Bruns et al 1991). Primers were synthesized at
the University of Florida Interdisciplinary Center for Biotechnology Research Oligonucleotide Synthesis Laboratory
(Gainesville, Florida). Polymerase chain reactions were performed using final concentrations of the components in the
reaction mixture as follows: 20 mM Tris-HCl (pH 8.4); 50
mM KC1; 1.5 mM MgCl,; 1 pJMof each primer; 200 piM
each of dATP, dCTP, dGTP, and dTTP; and 2.5 U of Taq
polymerase (Gibco-BRL, Gaithersburg, Maryland) per 100
pLLof reaction mixture. A GenAmp 6000 (Perkin-Elmer Applied Biosystems, Foster City, California) was used for the
amplification. The cycling conditions used included an initial denaturation step of 2 min at 94 C, with 32 cycles of 94
C for 45 s, 55 C for 30 s, and 72 C for 45 s each. The last
cycle included a 10-min incubation at 72 C and then storage
at 4 C. Unincorporated nucleotides and primers were separated from double stranded PCR products using Wizard
PCR Preps (Promega Inc., Madison, Wisconsin) according
to the manufacturer's instructions. Sequences, provided by
the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Laboratory, were
aligned manually. Sequences were deposited with GenBank
(AF079770-AF079777). Initially, sequences from both ITS1
and ITS2 were aligned as a single data set (TreeBASE
SN254). Alignment of these sequences resulted in a data
matrix of 498 sites in 24 isolates. Alignment of both ITS1
and ITS2 required the insertion of gaps to maximize sequence similarity. Because of ambiguities in alignment,
short sequence segments were excluded from the analysis.
Phylogenetic analyses were performed using PAUP version
3.1.1 (Swofford 1993). The positions in the alignment were
used as uniformly weighted characters; single gaps were
treated as missing characters; and regions that could not be
aligned or regions with large, continuous gaps were excluded from the analysis. The analysis was based on 254 informative characters.
Microsphaeropsis amaranthi was used as
the outgroup. The analysis included 24 sequences, 13 cho-
ROSSKOPF ET AL: P. AMARANTHICOLASP. NOV.
117
sen from those deposited in GenBank by Rehner and Uecker (1994), and three deposited by Uddin (1996), which
were chosen based on a wide range of host associations (TABLEI). The analysis was performed using 1000 replicate
heuristic searches with random sequence addition. Support
for groupings was determined by bootstrapping of 500 replicate data sets, unless noted, using random input of sequences and by determining the decay indices using
TreeRot (Sorenson 1996).
RESULTS
Phomopsis amaranthicola Rosskopf, Charudattan,
FIGS.1-5
Shabana, et Benny, sp. nov.
Coloniae in agaro "potato dextrose" albidae,floccosae,
denique fumosus. Mycelioseptalis,ramosa.Pycnidiaabundanti, producens concentricus 0.5-cm separatus,solitaria.
Pycnidia globosa, ostiolata et unilocularia, 371-287 Ixm, exudato sporali dilutus armeniacus. Paries pycnidialis brunneis vel piceus, cellulis multistratosis, eustromaticus. Conidiophores hyalinae, simplicia, recta vel ramosa. Cellulae conidiogenae hyalinae, procreans a conidia multilocus, phiali-
dicae, exorientes stratis ex intimis cellularum cavitatis
pycnidii. Alpha conidia hyalina, fusiforme-elliptica, 1-7 gut-
tulata,unicellularia,6.6-24.0 X 2.2-6.6 Ixm,average14.1 X
5.7 ,xm (mode 13.2 X 6.6 uLm).Beta conidia hyalina,filiformia, hamatusvel recta, 24.2-28.6 X 1.1-2.2 ,pm,average
27.8 X 1.6 pLm(mode 28.6 X 1.1 pIm).C conidia sparsa,
hyalina,variabilisguttulata,18.0-22.0 ,Im, average19.9 X
2.4 (mode 18.2 X 2.6). Pycnidiain caules emortuisimmersa, erumpescentia,singularis,unilocularia.
Colonies produced on potato dextrose agar white,
floccose, turning gray to brown. Mycelium septate
and branched. Pycnidia abundant, produced in concentric rings 0.5-cm separating, solitary. Pycnidia globose, ostiolate, and unilocular, 371-287 ,um, with conidia released in a peach-colored matrix. Pycnidial
wall brown to black and multicelled. Conidiophores
cells
hyaline, occasionally branched. Conidiogenous
hyaline, having multiple loci of alpha conidium pro-
duction, phialidic, lining the inner-wall of the cavity.
Alpha conidia hyaline, fusiform-elliptic, containing
1-7 guttules, aseptate, 6.6-24.0 X 2.2-6.6 ,xm, x =
14.1 x 5.7 pLm(mode 13.2 X 6.6 ,xm). Beta conidia
hyaline, filiform, hamate or straight, 24.2-28.6 x 1.12.2 ixm, x = 27.8 x 1.6 ,pm (mode 28.6 X 1.1). C
conidia sparse, hyaline, guttulate, 18.0-22.0 pxm,x =
19.9 X 2.4 (mode 18.2 X 2.6). Pycnidia on dead
stems immersed and erumpent, single, and unilocular.
HOLOTYPE.USA. FLORIDA:Alachua County,Gainesville, on Amaranthus hybridus, 7 Oct 1998, E. N. Rosskopf
FLASF56785 (MycologicalHerbarium,Universityof Florida, Gainesville,Florida).ATCC74226.
Symptoms produced
on Amaranthus hybridus that
were spray-inoculated with conidial suspensions of
Florida isolate B consisted of round to elliptical le-
FIG. 1. A. Leaf lesions on Amaranthus hybridus L. resulting from inoculation with Phomopsis amaranthicola. B.
Stem lesions on Amaranthus hybridusL. resulting from inoculation with Phomopsis amaranthicola.
sions with either tan or light brown centers surrounded by red-brown rings (FIG. 1A). Coalescing, large
lesions developing on leaf petioles and stems caused
a stem and leaf blight (FIG. 1B), which led to premature defoliation, girdling of stems, and plant mortality. Leaf symptoms were apparent 5-7 d after incoulation. Stem lesions developed within 2 wk of inoculation. Occurrence of plant mortality was variable
(data not shown).
Growth on PDA and all other media was initiated
with white mycelium growing in a circular pattern.
Pycnidia were produced within 14 d after culture initiation, beginning closest to the point of inoculation
and formed in concentric rings approximately 5-mm
apart (FIG.2). Pycnidia produced on all types of media, as well as those produced on plant material, were
ostiolate (FIGS. 3, 4). There were no significant differences between pycnidia produced on agar and natural substrate.
Alpha, P, and C conidia (FIGS. 5A-C) were produced within the same pycnidium when all spore
types were present. In pycnidia containing all three
types of conidia, an average of 87% of all conidia
produced were a conidia. Beta conidia contributed
10% and C conidia an average of 3% to the total
conidial counts. Conidia were produced on hyaline,
phialidic conidiophores, which were occasionally
branched (FIGS. 5D, E). Alpha conidia were occasionally produced from multiple loci of conidiogenesis
within the conidiophore.
Suspensions containing mixtures of the conidial
types were plated on water agar in order to obtain
single conidial isolates and examine the germination
of conidia. Germination of conidia on PDA-covered
glass slides produced the following results. No C conidia were observed to germinate, although they did
118
MYCOLOGIA
I4UP,
_?
FIGS.2-4. Growth and sporulation of Phomopsis amaranthicola. 2. White mycelium with dark pycnidia produced in concentric rings around the point of inoculation on potato dextrose agar (X 1). 3. Pycnidia on a stem section of an inoculated
A. hybridusplant. Stem sections were placed on wetted Whatman 3 filter paper and placed in a moist chamber until sporulation (XlO). 4. Sporulating
pycnidium
in a squash mount (X600).
FIG. 5. Conidia and condiophores of Phomopsis amaranthicola. A. a conidium observed by light microscopy (X6000).
Note guttules within the conidium. B. P conidium (X 6000). C. C conidia (X 6000). D. Conidiophores with a conidia (X 2400).
E. Conidiophore with a conidium (X6000).
119
SP. NOV.
ROSSKOPFET AL: P. AMARANTHICOLA
TABLEII. Comparison of morphological characteristics of Phomopsis amaranthi and P amaranthicola
Phomopsis species
Statistic
P amaranthi
P amaranthicola
Range
Range
Averagea
Modeb
a
Pycnidia (>Lm)
300-600
168-762
371
297
x
X
X
X
200-300
148-584
287
198
Alpha conidia (RIm)
6.7-9.0
6.6-24.0
14.1
13.2
X
X
X
X
2.7-3.6
2.2-6.6
5.7
6.6
C conidia (txm)
Beta conidia (pLm)
18-27
24.2-28.6
27.8
28.6
X
X
X
X
0.5-1.5
1.1-2.2
1.6
1.1
None
18.0-22.0
19.9
18.2
present
X 2.0-3.1
X 2.4
X 2.6
Averages for P amaranthicola obtained from material obtained from cultures and infected plant material.
b Mode for P amaranthicola
obtained from material obtained from cultures and infected plant material.
exhibit incipient morphological changes associated
with germination, including irregular swellings. After
more than 20 h, less than 30% of 3 conidia showed
morphological changes and production of threadlike mycelium. This mycelial growth, like that reported for P helianthe (Muntanola-Cvetkovic et al 1985),
disintegrated and did not produce colonies. Alpha
conidia were found to germinate approximately 87%
of the time using this method.
Measurements of conidia and conidiomata were
obtained for the Florida amaranth isolate B (ATCC
74226) from PDA (TABLE II) with the comparative
measures reported for Phomopsis amaranthi from amaranth in Hungary. The average size of a conidia of
the Florida isolate are larger than the range reported
for P amaranthi. In addition to the size differences
in both cx conidia and 3 conidia of both isolates,
there is the marked absence of the third type of conidium from the description of the Hungarian isolate.
Pairing of isolates on PDA and V-8 juice agar did
not result in the production of a sexual stage, nor
did there appear to be any zonation. Instead, all of
the paired isolates grew in an intermingled fashion.
Infected stem pieces stored in soil and sand and exposed to temperatures of 4 C and 30 C did not produce perithecia. Removal of these tissues and subsequent plating resulted in regrowth of P amaranthicola from all pieces.
The Florida isolate (ATCC 74226) grew more slowly than any of the other test isolates on all of the
media tested (data not shown). The isolate of P oryzae and the Florida isolates were the only isolates that
produced ax, 3, and C conidia. Isolate BRZ produced
both a0and 3 conidia. Florida isolates produced only
a conidia on OMA. The presence of all three types
of conidia in pycnidia produced on plant tissue was
confirmed. The remaining isolates, BG, FP1, FP3,
72A, 72B, and 75A, produced only cx conidia on all
of the media tested. Only the isolate ATCC 74226
produced pycnidia at regular intervals in concentric
rings, rather than distributed throughout the plate
(FIG.2). All isolates produced mycelium in PDB, and
produced pycnidia only at the liquid-air interface
along the sides of the glass flasks. Growth rates of
Florida isolate B (ATCC 74226) on the media tested
are as follows: 1.9 cm/d on corn meal agar, no pycnidia produced after 30 d; 2.2 cm/d on AIA, pycnidia
produced in 7 d; 2.4 cm/d on PDA, pycnidia produced in 14 d; 2.6 cm/d on V8 agar, pycnidia produced in 6 d; 2.7 cm/d on AIAS, no pycnidia produced; 3.3 cm/d on OMA, pycnidia produced in 6
d; 3.7 cm/d on OMAII, pycnidia produced in 5 d.
DNA amplification and sequence analysis.-Comparisons were performed to determine the relationship
of the Florida isolate with other members of the genus Phomopsis, as well as selected isolates of related
genera. Length variations within the sequences of the
ITS regions of the chosen isolates were observed.
The isolates tested had ITS1 sizes ranging from 140175 bp. Sequences of the ITS2 region ranged in size
from 144-157 bp in this region. The sequences of
the isolates in this study had base composition which
ranged from 45-56% GC in ITS1 and 49-59% in
ITS2. Initial heuristic searches on 24 sequences
found 53 equally parsimonious trees of length 251,
with a consistency index (CI) of 0.946 and a retention index (RI) of 0.974 (FIG. 6). Analysis was repeated by separating the two regions into character
subsets. Heuristic searches based on the sequences of
ITS1 were performed with a character matrix of 298
sites in 24 isolates. This search found 52 equally parsimonious trees with a length of 154 steps, a CI of
0.799, and RI of 0.930 (FIG. 7). Heuristic searches
based on the sequences of the ITS2 region were performed using a data matrix of 245 sites in 24 isolates.
This search resulted in nine equally parsimonious
trees of 102 steps, CI of 0.709, and a RI of 0.894.
DISCUSSION
There have been more than 400 taxa described within the genus Phomopsis, with no recent revision of
the members of the genus. The general morphological characters of the Florida isolate B (ATCC 74226)
indicate that it belongs in the genus Phomopsis. It
shares the characteristic conidial types produced by
MYCOLOGIA
120
75
BG
100
FPI
dl
FP3
PHO
MA
B
68
d23
1
7522
dl
dl
512
476
468
100
66
~1 ~~~
,-d6
I
gm2
'I
GAP08
I
dl--~~00
~~d24
~~~GLBO6
-----------------------649
642
597
81
484
dl-
456
452
FIG.6. Phylogenetic relationships among strains of Phomopsis spp. from various hosts and isolates obtained from species
of Amaranthus based on internal transcribed spacer region (ITS) sequences. Strict consensus of 53 equally parsimonious
trees (tree length = 251, consistency index = 0.946, homoplasy index = 0.054, retention index = 0.974, rescaled retention
index = 0.921). Bootstrap replication frequencies greater than 50% are indicated above branches. Values below branches,
preceded by a lowercase d represent decay indices values. Isolate information is listed in TABLEI.
other species of Phomopsisand produces these conidia in pycnidial conidiomata. The conidiophores were
branched or straight and these are shared characteristics.
Determination of speciation within the genus
Phomopsis has been highly dependent upon the host
from which the isolate was described. More recent
studies with several species have shown that the host
ranges of many of the isolates that have been named
as species are more broad than originally hypothesized, and it also has been found that a single host
plant is capable of supporting more than one species.
This somewhat complicates the process of speciation
in this genus. The lack of a true lectotype species for
the genus Phomopsis (Sutton 1980) further complicates taxon identification in the group.
Several characters of the Florida isolate B (ATCC
74226), named here as P amaranthicola, are unique
in comparison to many members of the genus. The
presence of a third type of conidium, the C conidium, is reported for only a few species (Punithalingam
1975). Multiple loci of conidiogenesis are reported
for no other species in the genus.
Alpha conidia are apparently the infectious propagules of this isolate and were found to germinate
efficiently and to be produced abundantly on several
types of media. Alpha conidia and beta conidia are
commonly produced for many species of Phomopsis
and the alpha conidia have been found to be the
most predominant type. The presence of the C conidium has been reported for only three other species (Punithalingam 1975). The species P amaranthi
SP. NOV.
ROSSKOPFET AL: P. AMARANTHICOLA
75
BG
I--75
-_
-FP1
L--FP3
|dl
PHO
MA
B
-------
100
|I -
51
d2
dl6
PO
624
537
60
-
512
68
476
-468
dl '
100 r
81 [d5 Id3 L
----60
dl
528
522
dl522
59
dl
77
d2
gm2
GAP08
-GLBO
649
---------____
642
484
456
452
597
FIG. 7. Phylogenetic relationships among strains of
Phomopsisspp. from various hosts and isolates obtained
from species of Amaranthusbased on rDNAinternal transcribed spacer region 1 (ITS1) sequences. Strictconsensus
of 52 equallyparsimonioustrees (tree length = 154, consistencyindex = 0.799, homoplasyindex = 0.201,retention
index = 0.930, rescaledretentionindex = 0.742). Bootstrap
replication frequencies greater than 50% are indicated
above branches.Valuesbelow branches,preceded by a lowercase d represent decay index values. Isolate information
is listed in TABLEI.
was not reported to have C conidia, nor was it reported that there were multiple loci of conidiogenesis for a conidium production. Type specimens of
this species could not be obtained.
Analysis of the sequence data from a variety of isolates from Amaranthus spp. and other species of
Phomopsis showed considerable variation in the ITS1
region. Nearly all nodes resolved in the strict consensus cladograms were present and received moderate
to strong support by the bootstrap analyses. In all
cases, the groupings resulted in the Florida isolate B
(ATCC 74226), P amaranthicola, falling as the sister
group to the major clade containing the Phomopsis
spp. sequenced by Rehner and Uecker (1994). The
latter were grouped in this study similarly to Rehner
and Uecker's groupings in their original work. The
Florida isolate B (ATCC 74226), although grouping
as the sister group to the other Phomopsis "species,"
relative to the Phoma-like isolates, was consistently
outside of both of the major clades (FIGS.6, 7).
121
The cladogram based on the ITS2 sequence data
was less resolved than all other trees (data not
shown). Cladograms that were based on sequence
groups that excluded the Phomopsis "species" sequenced by Rehner and Uecker (1994), resulted in
the grouping of the Florida isolate B as the well-resolved sister taxon within a clade containing P oryzae
and the Phomopsis spp. isolates from Georgia peach.
The Florida isolate B is clearly distinct from the two
major clades containing the PHO isolate and the PO
isolate (FIGS.6, 7).
A number of points complicate making conclusions about the generic placement of the named isolate. There was a high degree of variability in the
sequences of the included isolates. This made alignment of the sequences difficult and somewhat ambiguous in terms of identifying the optimal alignment. Alignments are often difficult when the paired
sequences differ by more than 30% (Hillis and Dixon
1991). In addition, due to the complicated taxonomic situation regarding the genus Phomopsis, it is impossible to determine if the terminal clades represent
species or groupings of isolates that have been referred to as species. Although the Florida isolate B
consistently occurs as the sister group to the remaining isolates of Phomopsis spp., it is difficult to determine if it belongs in this genus. The results of the
sequence analysis, coupled with the unique morphological characteristics, indicate that it is not any of
the existing species that have been sequenced, nor is
it similar morphologically to other species of Phomopsis known to occur on amaranth. Therefore, it is
necessary and justified to name it as a new species.
ACKNOWLEDGMENTS
This research,basedon the firstauthor'sPh.D.Dissertation,
was supportedin part by funds providedby SpecialGrants
in Tropical Agriculture, Caribbean Basin Administrative
Group (CBAG),USDAContractNo. 94-34135-0649and the
Universityof Florida.ContributionNo. R-06778FloridaAgriculturalExperimentStationJournal Series.
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