Australasian Plant Pathol. (2017) 46:91–101
DOI 10.1007/s13313-016-0463-y
ORIGINAL PAPER
Alternaria infectoria and Stemphylium herbarum, two new
pathogens of pyrethrum (Tanacetum cinerariifolium) in Australia
Azin Moslemi 1 & Peter K. Ades 2 & Tim Groom 3 &
Marc E. Nicolas 1 & Paul W. J. Taylor 1
Received: 3 November 2016 / Accepted: 27 December 2016 / Published online: 13 January 2017
# Australasian Plant Pathology Society Inc. 2017
Abstract Perithecia at the base of dead flower stems of pyrethrum (Tanacetum cinerariifolium) plants in yield-decline affected fields of northern Tasmania were identified as Alternaria
infectoria and Stemphylium herbarum. Identification was based
on morphological description of cultures established from single
ascospores; and conidiospores; ascospore shape and septation;
and multigene phylogenetic analyses using the internal transcribed spacer (ITS) region, translation elongation factor 1-α
(EF1), polymerase II second largest subunit (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes.
Stemphylium herbarum produced necrotic leaf lesions on both
sides of the spray inoculated pyrethrum leaves which coalesced
to encompass the entire leaf in in vivo and in vitro experiments.
Alternaria infectoria produced small necrotic leaf lesions on both
sides of the leaves two weeks after inoculation which did not
expand and hence, was considered as a minor pathogen of pyrethrum. This is the first report of A. infectoria and S. herbarum as
pathogens of pyrethrum in Australia. The role of A. infectoria
and S. herbarum in pyrethrum yield-decline in northern
Tasmania needs to be evaluated.
Electronic supplementary material The online version of this article
(doi:10.1007/s13313-016-0463-y) contains supplementary material,
which is available to authorized users.
* Paul W. J. Taylor
paulwjt@unimelb.edu.au
1
Faculty of Veterinary and Agricultural Sciences, University of
Melbourne, Melbourne, VIC 3010, Australia
2
School of Ecosystem Forest Sciences, University of Melbourne,
Melbourne, VIC 3010, Australia
3
Botanical Resources Australia Pty Ltd, PO Box 3251,
Ulverstone 7315, TAS, Australia
Keywords Alternaria infectoria . Multigene analyses .
Perithecia . Stemphylium herbarum . Tanacetum
cinerariifolium
Introduction
Pyrethrum, Tanacetum cinerariifolium is commercially used for
production of natural pyrethrins (Pethybridge et al. 2008a).
Pyrethrins are extracted from the achenes located within pyrethrum flower heads (Ambrizic et al. 2007). Approximately
3000 ha of pyrethrum, accounting for 70% of the world production of pyrethrins is grown in Australia each year (Hay et al.
2015). Pyrethrum seeds are sown in winter and the first harvest
occurs in spring of the following year (15 to 17 months after
planting). Pyrethrins yield is greatly affected by the foliar and
soil-borne pathogens that cause reduced flower production.
Ideally, pyrethrum plants can be harvested annually for three to
four subsequent years, however, in fields severely affected by
pathogens, growth is reduced after the first or the second harvesting season and the crops are then terminated (Hay et al. 2002).
During surveys conducted between April 2015 and
September 2016 in northern Tasmania, necrotic leaf lesions, necrotic crown tissues and perithecia at the base of dead flower
stems from the previous season’s harvest were consistently observed on pyrethrum plants growing in yield-decline affected
sites. Many foliar pathogens of pyrethrum such as
Stagonosporopsis tanaceti causing ray blight disease of pyrethrum (Vaghefi et al. 2012), Didymella tanaceti the causal agent
of pyrethrum tan spot (Pearce et al. 2015); Colletotrichum
tanaceti causing anthracnose disease of pyrethrum (Barimani
et al. 2013); Sclerotinia sclerotiorum and S. minor causing
sclerotinia flower blight (Scott et al. 2014); winter blight caused
by Alternaria tenuissima and pink spot caused by Stemphylium
botryosum (Pethybridge et al. 2004); and Botrytis flower blight
92
caused by Botrytis cinerea (Pethybridge et al. 2008a) have been
reported on the affected plants.
Alternaria tenuissima was first reported as a pathogen of
pyrethrum by Srinath and Sarwar (1965) with necrotic lesions
on the apical part of the pyrethrum leaves. Later, winter blight
disease of pyrethrum caused by A. tenuissuma and pink spot
disease caused by Stemphylium botryosum were reported as
foliar pathogens with A. tenuissima producing necrotic leaf
spots and S. botryosum brown water-soaked lesions with pink
and brown margins on both sides of the leaves (Pethybridge
et al. 2004; Pethybridge et al. 2008a). The teleomorph stage of
S. botryosum was detected by Pethybridge et al. (2004) on
dead plant material and the bases of old flower stems in pyrethrum fields, however, no further studies were undertaken to
determine the contribution of this pathogen to pyrethrum yield
reduction.
In a survey conducted in 2012 on foliar pathogens of pyrethrum causing necrotic leaf lesions, the isolation frequency of
Alternaria spp. declined significantly from 92% of fields in winter to 40% in summer during three sampling times. Isolation
frequency of S. botryosum was considerably lower (23% of fields
in winter) (Hay et al. 2015). However, Hay et al. (2015) reported
a flux in the population of Alternaria spp. and S. botryosum in
different seasons of the year. Although these pathogens were
mentioned as not being yield-limiting, the frequency of isolation
from necrotic leaf lesions of pyrethrum was high (Hay et al.
2015). Pethybridge et al. (2003) frequently isolated
A. tenuissima and S. botryosum from necrotic leaf spots of pyrethrum during October and November 2001, emphasizing that the
population of these pathogens was higher in winter and spring
compared to that in summer.
Alternaria are ubiquitous fungi with different lifestyles
from saprophytic to phytopathogenic causing plant disease
and post-harvest rots in wide variety of agricultural crops and
ornamental plants. Alternaria species also cause human diseases
resulting in problems in immunocompromised patients and are
responsible for childhood asthma (Lawrence et al. 2013;
Woudenberg et al. 2013). Taxonomy of Alternaria species has
been changing since Nees first described it in 1816 based on
production of multi-celled and melanised club-shaped spores in
short or long chains (Woudenberg et al. 2013). Later, Wallroth in
1833 and Preuss in 1851 added two closely related genera of
Stemphylium and Ulocladium to the Alternaria species complex
based on production of phaeodictyospores = muriform spores
(production of both longitudinal and transverse septa) and made
the identification more complicated (Lawrence et al. 2013).
However, Simmons in 2007 classified 275 species in the
Alternaria complex according to morphological differences
(Woudenberg et al. 2013). Currently, the Alternaria species complex possesses nine genera: Alternaria, Stemphylium,
Chalastospora, Crivellia, Embellisia, Nimbya, Ulocladium,
Undifilum and Sinomyces. The genus Alternaria includes eight
sections with the isolates forming a sexual stage belonging to
Moslemi A. et al.
A. infectoria (syn. Lewia) species group clading distinctly from
other Alternaria sections due to morphological differences in
conidiospores and chemical differences in toxin production
(Inderbitzin et al. 2009; Lawrence et al. 2013; Woudenberg
et al. 2013). Alternaria and Stemphylium are among the most
common asexual morphs of the order Pleosporales (Zhang
et al. 2012).
Stemphylium species are mostly saprophytic growing on
dead plant materials, however, S. botryosum, S. solani, S.
vesicarium and S. loti have been known as pathogens of agriculturally important crops and stone fruits (Wang et al. 2009).
Simmons (1967) established criteria for morphological identification of various Stemphylium spp. and introduced
Pleospora herbarum as teleomorph of S. botryosum.
However, Simmons in 1985 subsequently reclassified
Stemphylium/Pleospora holomorphs and reported
Pleaospora tarda as the sexual stage of S. botryosum and
P. herbarum the sexual stage of S. herbarum (Inderbitzin
et al. 2009).
The aims of this study were to i) identify the species associated with the perithecia isolated from the base of the pyrethrum dead flower stems using multigene phylogenetic analyses and morphological identification ii) determine the pathogenicity of these species on pyrethrum.
Methods and materials
Sample collection
In a field survey in September 2016 from fields located
in Table Cape and Burnie, northern Tasmania, seven
plants showing perithecia at the base of dead flower
stems were collected and transferred to the laboratory
for isolate identification. Pieces (5–10 cm in length) of
necrotic flower stem bases containing perithecia were
cut from each plant, placed on moisturised filter papers
with sterile water in Petri dishes and incubated at room
temperature (23–25 °C) for 2 days. Fertile perithecia
were then lifted from the surface of the flower stems
using a sterilised needle; vortexed in sterilised water
and cultured on WA for single sporing. Perithecia were
also mounted on microscopic slides with clear lactic
acid and gently tapped to release asci for morphological
identification.
Five additional Alternaria spp. strains isolated from necrotic leaf lesions of pyrethrum in northern Tasmania were also
collected. All other strains used in the study were reference
strains derived from the national centre of biotechnology information (NCBI) database at http://www.ncbi.nlm.nih.gov/.
A culture of each isolate was sent to the Queensland Plant
Pathology Herbarium (BRIP), Brisbane, Australia to obtain
culture collection reference numbers.
26–37 (SD 2.83)
μm × 13–16
(SD 1.03) μm
143–188 (SD 14.5)
μm × 27.5–33
(SD 2.1) μm
19.5–35.5 (SD 4.12)
μm × 9–22
(SD 3) μm
17–57.5 (SD 21) μm
20–27.5 (SD 2.05)
μm × 7.5–16
(SD 2.62) μm
23.5–59.5 (SD 10.6)
μm × 7–12
(SD 1.5) μm
11–21 (SD 7.11) μm
122.5–137 (SD 10.4)
μm × 16–16.5
(SD 0.40) μm
Conidiospores Length
× Width (SD)
Morphological descriptions were made of monosporic isolates
grown on synthetic poor nutrient agar (SNA) with pieces of
sterilised filter papers (double autoclaved for 20 min at
121 °C) on the surface of the agar as carbon source to induce
sporulation (Crous et al. 2009). Cultures were incubated at
21–22 °C under cool white fluorescent light in a 8/16 h regime
for 7 days (Woudenberg et al. 2013). Morphological descriptions of ascospores for the species that did not form perithecia
on SNA, were made directly from the perithecia that were
isolated from flower stems. Slides were prepared with clear
lactic acid. Microscopic images were captured using a Leica
DM2900 compound microscope with both bright field and
differential interference contrast illuminations. Colony growth
rate was measured in 2-day intervals for 7 days and pigmentation was rated according to Rayner et al. (1970) color chart.
The size of 30 conidiospores and ascospores; and 10 samples
of all other structures including asci and conidiophores were
measured (Table 1).
Conidiophores length
Taxonomy
Ascospores Length
× Width (SD)
93
Asci
Alternaria infectoria and Stemphylium herbarum of pyrethrum
Colony pigmentation
Pale grey to silvery with white
and fluffy aerial mycelia;
reverse white thick margins
with dark grey centre
Olive grey colonies with pale grey
aerial mycelia, reverse black
with thin white margins
35
30
A. infectoria
(BRIP 65180)
S. herbarum
(BRIP 65181)
Purified amplicons were sequenced and sequence intensity
was assessed using the Chromas Lite MFS computer package.
Isolate sequences were tentatively identified after conducting
Growth rate after
7 d (mm)
Multigene phylogenetic analysis
Table 1
Fungal mycelia were scraped directly from 7-day-old single
spored cultures on oatmeal agar (OA), DNA extracted using
the DNeasy Plant Mini Kit (Qiagen Pty. Ltd., Australia) following the manufacturer’s instruction, and quantified by comparing
the band density using Lambda DNA/HindIII Marker (Thermo
Ficsher Scientific, Australia) after electrophoresis in 1.5% agarose gel. The ITS region (700 bp) was amplified using primer
pairs V9G (de Hoog and Gerrits van den Ende 1998) and ITS4
(White et al. 1990). Approximately 420 bp of the EF1 was amplified by PCR primers EF-728f (Carbone and Kohn 1999) and
EF2 (O’Donnell et al. 1998); and 890 bp of the partial RPB2
genes were amplified by PCR primers RPB2-5F2 (Sung et al.
2007) and fRPB2-7cR (Liu et al. 1999). The GAPDH region was
amplified using primer pairs gpd1 and gpd2 designed by Berbee
et al. (1999). PCR thermal cycler for ITS was performed as
described by White et al. (1990), for RPB2 touchdown PCR,
for EF1 gradient PCR and for GAPDH were performed according to PCR conditions described in Woudenberg et al. (2013).
DNA amplification was carried out using an Eppendorf thermal
cycler (Pty. Ltd. Australia). PCR products were then purified
using QIAquick PCR purification kit (Qiagen Pty. Ltd.
Australia) and sequenced.
Key morphological features of A. infectoria and S. herbarum, SD = standard deviation
DNA extraction and PCR amplification
Species
Molecular identification
94
BLAST searches of the database. Sequences were aligned
manually using the sequence alignment editing program
MEGA6 (Tamura et al. 2013). Consensus sequences were
obtained from both forward and reverse sequences using
DeNovo assembly in Geneious v 7.06 (Kearse et al. 2012).
Consensus sequences were then pairwise aligned and annotated with the closely related reference sequences derived from
the database within Geneious (Table 2). Maximum likelihood
(ML) phylogenetic trees were constructed for each gene individually and in combination using MEGA6 software and
PhyML (Guindon and Gascuel 2003) within Geneious.
Due to the lack of RPB2 gene sequences for most
Stemphylium species, two separate multigene phylogenetic
trees including ML combined tree of ITS-EF1GAPDH-RPB2 for Alternaria and ITS-EF1-GAPDH for
Stemphylium were generated. However, Stemphylium
species were included in the combined phylogenetic tree constructed for Alternaria, where RPB2 gene sequences were
available. In the tree for Alternaria, species of closely related
genera to A. infectoria group including species of sections
Chalastospora, Ulocladium and Stemphylium were included.
Species of section Alternata were also included to determine
whether recovered strains isolated from the dead flower stem
bases were associated with the sexual stages of A. tenuissima
or A. alternata. Moreover, an individual EF1 phylogenetic
tree was constructed with all the aforementioned isolates
plus the five isolates recovered from necrotic leaf lesions in 2016 to determine if they were anamorphs of
A. infectoria. However, no further sequencing was carried out for these isolates.
In the combined tree for Stemphylium spp., species of
different groups (Inderbitzin et al. 2009) were included to
differentiate various groups in the Stemphylium species
complex. The best substitution model was selected using
MEGA6 with ML statistical analysis and complete deletion of the gaps. In PhyML analysis, the GTR substitution
model was selected. To assess the relative stability of
branches, bootstrap analysis with 1000 pseudoreplicates
for ML analyses in MEGA6 and 500 pseudoreplicates in
PhyML analyses was performed. Gaps were treated as
missing data. Consensus sequences were deposited in
the database and trees in TreeBASE at www.treebase.
org. GenBank accession numbers for the reference
markers are shown in Table 2.
Pathogenicity tests
Pathogenicity of A. infectoria and S. herbarum isolated from
the dead pyrethrum flower stems was assessed in two trials.
For each trial, the pyrethrum seedlings of cultivar Pyrate were
germinated from steam sterilised seeds and raised in seedling
mix in Tasmania (BRA Pty. Ltd.). Seedlings were transferred
to 10 cm diam pots with potting mix, fertilised with 5 g of
Moslemi A. et al.
Osmocote Plus (Scotts Australia Pty. Ltd.) per pot and grown
in the glasshouse for two months.
Inoculum was prepared from 5-day-old single spored cultures on SNA by adding 10 mL of sterile water to each plate,
gently scraping the colony surface with a glass spreader and
then filtering the spore suspension using cheese cloth. The
concentration of each spore suspension was quantified to
106 spore/mL using a haemocytometer. Two drops of 0.1%
Tween-20 solution was added to each spore suspension
(Pethybridge et al. 2008b).
In vivo experiment
To assess pathogenicity of each isolate to produce leaf lesions,
three replicates of healthy Pyrate pyrethrum plants were each
spray inoculated with 20 mL of 106 spore/mL spore suspension of each isolate of A. infectoria and S. herbarum. A hand
sprayer was used to spray the leaves and inoculation was
continued until just before runoff. Three control plants were
sprayed with sterilised water. Each plant was then covered
with a plastic bag to increase humidity after inoculation and
induce germination of the spores. The plastic bags were removed after 24 h and the plants were then maintained in the
glasshouse until necrotic lesions appeared on the leaves.
Necrotic lesions were cultured on potato dextrose agar
(PDA) for pathogen identification. Tissue surface sterilisation
and culture were carried out by dipping the leaf sections into
80% ethanol for 30 s, 1% active ingredient (ai) sodium hypochlorite for 1 min and then rinsed two times in sterilised water
for 1 min. They were then blotted on a sterilised paper towel
and small 5 mm2 sections of tissue were cultured onto WA and
incubated according to the condition described above.
Mycelium was subsequently subcultured onto PDA and incubated for a further 5 days at 23 °C.
In vitro experiment
An in vitro bioassay was undertaken with detached leaves of
healthy pyrethrum plants inoculated by placing 106 spore/mL
of spore suspension of each species onto the surface
sterilised leaves. Controls were treated with sterilised
water. Detached leaves were incubated in plastic containers, four leaves per container, with moistened filter
papers at 21 °C for eight days and symptoms were
assessed after eight days. A Leica DM205 FA stereomicroscope was used to assess the symptoms.
Results
Perithecia isolated from dead flower stems of all the seven
plants in 2016 were associated with both A. infectoria and
S. herbarum (Fig. 1).
Alternaria infectoria and Stemphylium herbarum of pyrethrum
Table 2
95
Accession numbers of Alternaria spp. and Stemphylium spp. used in this research
Isolates
Culture accession
No.
Location
Isolate source
Accession No.
ITS
EF1
GAPDH
RPB2
AF229485
AF347031
AF347033
KC584182
FJ839608
KC584187
JN383482
FJ266475
AF392987
KC584197
DQ323697
KC584730
KC584634
KC584636
KC584638
KC584700
KC584644
KC584699
KC584649
KC584657
KC584658
KC584662
AY278815
AY278808
AY278810
KC584099
KC584148
KC584105
AY562398
AY562401
AY278795
KC584116
AY278793
KC584470
KC584375
KC584377
KC584379
KC584442
KC584385
KC584441
KC584390
KC584398
KC584399
KC584404
A. alternariae a
A. alternata a
A. arborscens a
A. armoraciae a
A. breviramosa a
A. capsici-annui a
A. cerata a
A. conjucta a
A. ethzedia a
A. gaisen a
A. infectoria a
A. infectoria
A. obclavata a
A. oudemansii a
CBS 126989
CBS 916.96
CBS 102607
CBS 118702
CBS 121331
CBS 504.74
CBS 121340
CBS 196.86
CBS 197.86
CBS 632.93
CBS 210.86
UMAi01- BRIP 65180 Australia
CBS 124120
USA
CBS 114.07
-
Tanacetum cinerariifolium KY009902 KY009904 KY038017 KY009906
Air
KC584225 KC584701 KC584149 KC584443
FJ266488 KC584746 KC584175 KC584486
A. oregonensis a
CBS 542.94
CBS 918.96
CBS 198.67
BRIP 12521
BMP0384
CBS 192.86
CBS 714.68
CBS 482.90
CBS 191.86
UMSh02- BRIP 65181
EGS 46–182
NO 770
Strain 05
EGS 16–068
EGS 31–016
EGS 29–188
CBS 109843
EGS 41–135
USA
UK
USA
USA
Canada
Israel
India
Australia
New Zealand
USA
China
USA
USA
USA
Auckland
USA
Triticum aestivum
Dianthus sp.
soil
Medicago sativae
Medicago sativa
Gracilaria sp.
Medicago sativa
Tanacetum cinerariifolium
Aguilegia sp.
Lotus sp.
Lycopersicon esculentum
Latyrnus maritimus
Trifolium pratense
Sedum spectabile
Lupinus sp.
FJ266478
AF347032
AF229487
KJ415541
AF229478
AF442775
KC584238
AY329217
KC584239
KY009903
AY329203
AF442789
KR911818
AY329228
AY329231
AY329221
AY329232
AY329214
KC584674
KC584693
KC584737
KJ415451
JQ672389
AY324676
KC584729
AY324709
KC584731
KY009905
AY324742
AY324775
KR911808
AY324710
AY324769
AY324746
AY324713
AY324759
FJ266491
AY278809
KC584737
KJ415405
AY278824
AY443874
AF443881
AY377021
AF443884
KY038018
AF443886
AF443888
KR911813
AY317032
AY317035
AY317025
AY317036
AY317018
KC584416
KC584435
KC584477
JQ646512
AF107804
KC584471
KY009907
-
EGS 12–142
EGS 36–118
CBS 109844
CNU094009
EGS 17–137
USA
UK
USA
China
New Caledonia
Trifolium repens
Triglochin maritima
Lycopersicon esculentum
Allium sp.
Xanthosoma sagitiifolium
AY329218
AY329175
AY329229
JF331519
AY329206
AY324744
AY324753
AY324711
JF331628
AY324758
AY317022
AF443901
AY317033
JF331462
AF443903
-
A. tenuissima a
Alternaria sp. a
Curvularia australis c
Exerohilum pedicellatum b
S. alfalfae c
S. botryosum c
S. gracilariae c
S. herbarum c
S. herbarum
S. lancipes c
S. loti c
S. lycopersici c
S. majusculum c
S. paludiscripi c
S. sarciniforme c
S. sedicola d
S. solani c
S. trifolii c
S. triglochinicola c
S. tomatonis d
S. vesicarium c
S. xanthosomatis c
a
USA
India
USA
New Zealand
Australia
Australia
Switzerland
Switzerland
Japan
UK
Daucus carota
Arachis hypogaea
Lycopesicon esculentum
Armoracia rusticana
Triticum sp.
Capsicum annuum
Elymus scabrus
Pastinaca sativa
Brassica napus
Pyruc pyrifolia cv. Nijiseiki
Triticum aestivum
(Woudenberg et al. 2013); b (Lawrence et al. 2013); c (Camara et al. 2002); d (Simmons 2001)
*BRIP, Queensland Plant Pathology herbarium; Brisbane, Australia; CBS, Centraalbureau voor Schimmelcultures, the Netherlands; BMP, BM Pryor,
School of Plant Sciences, University of Arizona, Tuscon, Arizona 85,721; EGS, EG Simmons, Mycological Srevices, Crawfordsville, Indiana 47,933;
NO, Nichole O’Nille, Culture Collection, Beltsville, MD; CNU, Chungnam National University, Yuseong, KungDong, Daejeon, Chungnam, Korea;
UM, University of Melbourne; Australia
Taxonomy
Detailed taxonomic description of A. infectoria and S. herbarum
are shown in Table 1.
Alternaria infectoria: Conidia obclavate in long or short
chains predominantly without longitudinal septa and with
short branched conidiophores growing in between the conidia.
Ascospores muriform, 6–7 transverse septa with 3–4
96
Moslemi A. et al.
longitudinal septa in the central segments, end cells without
septa. Cylindrical asci with 8 ascospores, straight or somewhat
curved (Fig. 2). No perithecia formed on SNA with pieces of
filter papers and WA with sterilised flower stems.
Stemphylium herbarum: Oblong-ovoid shaped conidia, light to dark brown with meristemic growth occasionally, multicelled with transverse and longitudinal
septa. Conidiophores short and long, branched.
Ascospores yellow, 7–9 transverse and many longitudinal septa in central and lateral segments. Cylindrical
asci with 8 ascospores, curved or straight (Fig. 3).
Perithecia produced on SNA and WA with pieces of
sterilised flower stems (Fig. 1b).
Phylogeny
Fig. 1 a an old necrotic pyrethrum flower stem cut from a field grown
plant with perithecia formed at the base; b perithecia of S. herbarum
produced in vivo on a sterilised pyrethrum flower stem in WA, 10 days
after inoculation
Fig. 2 Alternaria infectoria; a
colony morphology and
pigmentation on OA; b-e conidia;
f asci containing eight ascospores;
g multi-celled ascospores. Scale
bars b-e and g 20 μm; f 50 μm
Multilocus combined phylogenetic analyses of ITS-EF1RPB2-GAPDH; and ITS and RPB2 individual trees (individual trees were not shown) confirmed that
Alternaria infectoria and Stemphylium herbarum of pyrethrum
97
Fig. 3 Stemphylium herbarum; a colony morphology and pigmentation on OA; b-g conidia; h short and long conidiophores i perithecia on SNA; j-l asci
containing eight ascospores;. Scale bars b-g and h 20 μm; i 100 μm; j-l 50 μm
A. infectoria (BRIP 65180) claded with the holotype
strain A. infectoria (CBS 210.86) within the
A. infectoria species group with bootstrap value of
100% (Fig. 4). However, in individual trees of EF1
and GAPDH, the isolate claded with A. conjuncta.
Stemphylium herbarum (BRIP 65181) claded with the
type strain of S. herbarum (CBS 191.86) in the ITSEF1-RPB2-GAPDH phylogenetic tree (Fig. 4) with
100% bootstrap value and in ITS-EF1-GAPDH phylogenetic tree (Fig. 5) with 88% bootstrap support
confirming that this was a different species to
S. botryosum that had been previously reported on pyrethrum. Individual trees showed similar results to the
combined phylogenetic trees (individual trees were not
shown). Isolate BRIP 65181 clustered closely with related species to Stemphylium spp. group C (S. herbarum
group; Inderbitzin et al. 2009) and next to the type
strain of S. herbarum (CBS 191.86).
All the five isolates cultured from leaf lesions were identified as A. alternata and clustered in the Alternata species
complex in the individual EF1 tree with 98% bootstrap support (tree was not shown). ML bootstrap supports obtained
from MEGA6 multigene analysis were similar to those obtained in PhyML analysis.
In the combined maximum likelihood tree of ITS-EF1RPB2-GAPDH constructed for the species of Alternaria, a
total of 2035 (ITS: 510, EF1: 248, RPB2: 829, GAPDH:
448) positions in the final data set were obtained with 1201
constant sites (ITS: 386, EF1: 133, RPB2: 372, GAPDH: 310)
and 613 (ITS: 97, EF1: 83, RPB2: 353, GAPDH: 80) parsimony informative sites and 221 uninformative variable
characters.
In the combined maximum likelihood tree of ITS-EF1GAPDH constructed for the species of Stemphylium, a total
of 1342 (ITS: 394, EF1: 437, GAPDH: 511) positions in the
final data set were obtained with 851 constant sites (ITS: 342,
98
Moslemi A. et al.
Fig. 4 Maximum likelihood PhyML combined phylogenetic tree of ITSEF1-RPB2-GAPDH for Alternaria and Stemphylium spp. Highest log
likelihood −8961.6128. The analysis involved 22 nucleotide sequences.
The tree was rooted to Exerohilum pedicellatum BMP0384. Bootstrap
values less than 80% were deleted. * refers to strains recovered from
the base of the pyrethrum flower stems. Scale bar indicates expected
changes per site
EF1: 150, GAPDH: 359); 376 (ITS: 133, EF1: 116, GAPDH:
127) parsimony informative sites and 115 uninformative variable characters.
Discussion
Pathogenicity tests
Alternaria infectoria and S. herbarum were isolated from necrotic leaf lesions produced in both in vivo and in vitro experiments.
Alternaria infectoria was able to produce small black spots on
the surface of the leaves 4 days after inoculation with 106 spore/
mL spore suspension in detached leaf bioassay and 2 weeks after
inoculation in in vivo glasshouse experiments. Lesions were
mostly small with regular margins and did not expand further
beyond 14 days after inoculation in both experiments (Fig. 6A).
Stemphylium herbarum also caused reddish-brown necrotic leaf lesions on both sides of the leaves after 3 days in in vitro
and 5 days after inoculation in in vivo bioassays. Lesions
expanded to encompass the entire leaf surface (Fig. 6B). No
controls were found infected in in vivo and in vitro experiments for both species.
Alternaria infectoria and Stemphylium herbarum have been
identified as new pathogens of pyrethrum in Australia. Both
pathogens were isolated from the perithecia collected from
dead pyrethrum flower stems in plants affected by yielddecline syndrome in northern Tasmania.
Alternaria infectoria caused black necrotic lesions on both
sides of the leaves in the glasshouse and detached leaf pathogenicity bioassays. However, leaf symptoms on plants in the
glasshouse were not severe and developed slowly after
2 weeks. Low isolation frequency of this pathogen from leaf
lesions of field plants and morphological similarities of conidia shape and size to other Alternaria species isolated from
pyrethrum might have been the main reasons that A. infectoria
was not identified in previous field surveys.
Pyrethrum winter blight disease has been reported to be
caused by A. tenuissima (Pethybridge et al. 2003;
Pethybridge et al. 2008a). Pethybridge et al. (2004) reported
that A. tenuissima was a minor pathogen but had the potential
Alternaria infectoria and Stemphylium herbarum of pyrethrum
99
Fig. 5 Maximum likelihood
PhyML combined phylogenetic
tree of ITS-EF1-GAPDH for
Stemphylium spp. Highest log
likelihood −5364.0913. The
analysis involved 19 nucleotide
sequences. The tree was rooted to
Curvularia australis BRIP
12521. Bootstrap values less than
75% were deleted. * refers to the
S. herbarum strain recovered
from the base of the pyrethrum
flower stems. Group C created
according to Inderbitzin et al.
(2009). Scale bar indicates expected changes per site
to reduce the leaf area and hence affect photosynthesis and
flower production.
Perithecia of plant pathogens on dead plant material are
important sources of inoculum with ascospores dispersed by
wind or water splash (Thomma 2003). However, the sexual
morphs for most species of Alternaria have not been identified
(Thomma 2003). Isolates of A. alternata from leaf lesions
Fig. 6 A Symptoms caused by
Alternaria infectoria on
pyrethrum in in vivo and in vitro
trials. a-c necrotic and black spots
produced 4 days after inoculation
in vitro on both sides of the
leaves. d necrotic lesions
produced two weeks after
inoculation in vivo. B Symptoms
caused by Stemphylium herbarum
on pyrethrum in in vivo and
in vitro trials. a necrotic spots
produced 3 days after inoculation
in vitro bioassay b-c necrotic and
reddish brown irregular lesions
produced 5 days after inoculation
in in vivo experiment on both
sides of the leaves
were in the Alternata species complex in which no sexual
stage has been recorded. More extensive surveys of dead pyrethrum flower stems and trash need to be undertaken to assess the incidence of A. infectoria. In the study by Hay et al.
(2015), different species of Alternaria were reported to be
associated with pyrethrum leaf spots but the species were
not identified. More investigations need to be carried out on
100
Alternaria spp. as foliar pathogens of pyrethrum in Australia
and the role they have in yield-decline of pyrethrum. Isolation
of A. alternata was also reported by Pethybridge et al. (2004),
however, this was shown to be a saprophyte colonizing the
dead pyrethrum plant material and was unable to infect pyrethrum leaves in glasshouse bioassays. Nevertheless,
A. alternata has been reported to be pathogenic to chrysanthemum (Pethybridge et al. 2008a).
The A. infectoria species group comprises many taxa
which are morphologically differentiated from taxa in other Alternaria sections by the production of shorter and
branched conidia (Andersen et al. 2009). Alternaria
infectoria is taxonomically distinct from other closely related genera and clustered with other members of the sexual Alternaria lineage (Andersen et al. 2009; Woudenberg
et al. 2013). In individual trees of EF1 and GAPDH the
isolate from the base of the old flower stems claded with
A. conjuncta, however, A. infectoria was morphologically
different to A. conjuncta as it produced longer
conidiospores and short conidiophores which grow between conidia (Andersen et al. 2009). Nevertheless, morphological characters of taxa within the A. infectoria species group are similar; so molecular phylogenetic studies
are required to differentiate many members of the group.
Pathogenicity of S. herbarum on pyrethrum was also confirmed with symptoms very similar to those of S. botryosum
(Pethybridge et al. 2004). Stemphylium herbarum is morphologically different to S. botryosum as it produces longer,
narrower conidia. However, no information exists about the
teleomorph of S. botryosum (syn. P. tarda). Pathogenic
Stemphylium spp. survive on the debris of the host plants where
perithecia are produced. Perithecia discharge ascospores under
suitable environmental conditions. Penetration occurs through
the epidermis or preferably stomata of the host plant and then
hyphae enter the cells resulting in production of necrotic, brown
lesions on the surface of the leaves (Kohl et al. 2009; Ahmed
2014). BLAST searches of the gene sequences of S. herbarum
(BRIP 65181) also showed high similarity to S. vesicarium,
however, the sexual stage of S. vesicarium is morphologically
different to the sexual stage of S. herbarum where the width of
asci is smaller and ascospore arrangement within asci is different. Other taxa within group C such as S. alfalfae showed
genetic similarity to S. herbarum (Inderbitzin et al. 2009) but
were morphologically different to S. herbarum as they produce
long conidia and different ascospores with unpredictable longitudinal septa. Species grouped in S. herbarum clade (group C)
were closely related and multilocus phylogenetic analyses have
not been able to distinguish intra-species relationships, although
they resolved new lineages. Hence, Camara et al. (2002) divided Stemphylium species into different groups according to differences in their nucleotide sequences, bootstrap value obtained
by Bayesian analyses and phenotypic differences based on
asexual spores.
Moslemi A. et al.
Diseased pyrethrum plants frequently showed leaf lesions
on both sides of the leaves and perithecia at the bases of the
dead flower stems at different times of the year. This raises the
question as to whether S. botryosum that was reported previously as a pathogen of pyrethrum does actually exist in pyrethrum fields of northern Tasmania. Hence, more field surveys
and sample collections are needed to identify all Stemphylium
spp. and Alternaria spp. existing in the fields and associated
with pyrethrum leaf diseases; and to determine their effect on
pyrethrum yield reduction.
The symptoms caused by Alternaria spp. and Stemphylium
spp. have been reported to be very similar (Pethybridge et al.
2008a). Care must be taken in isolation and identification of
the causal agent as many foliar pathogens in pyrethrum fields
cause similar symptoms and can be easily confused.
Acknowledgements Thanks to Botanical Resources AustraliaAgricultural Services Pty. Ltd. for providing pyrethrum seedlings and
supplementary funding for this project; and to Ruvini Lelwala for isolates
of Alternaria sp. Azin Moslemi was supported by a University of
Melbourne International Research Scholarship (MIRS).
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