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Submitted 9 March 2014, Accepted 5 April 2014, Published online 20 April 2014
Corresponding Author: Kevin D Hyde – e-mail – kdhyde3@gmail.com
Ji-Chuan Kang – e-mail – jichuank@yahoo.co.uk 351
Pyrenophora
Ariyawansa HA1,2,3, Kang JC1, Alias SA4, Chukeatirote E2,3and Hyde KD2,3,5,6
1The Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of
China, Guizhou University, Guiyang 550025, Guizhou Province, China.
2School of Science, Mae Fah Luang University, Chiang Rai. 57100, Thailand.
3Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand.
4Institute of Biological Sciences, University of Malaya, 50603, Kuala Lumpur.
5Centre for Mountain Ecosystem Studies (CMES), Kunming Institute of Botany, 8Chinese Academy of Science, Kunming
650201, Yunnan, China.
6World Agroforestry Centre, East Asia Office, Kunming 650201, Yunnan, China.
Ariyawansa HA, Kang JC, Alias SA, Chukeatirote E, Hyde KD 2014 – Pyrenophora. Mycosphere
5(2), 351–362, Doi 10.5943/mycosphere/5/2/9
Abstract
This is the first in a series of papers in which we revisit genera of fungi to provide baseline
data for future study. In this article we examine the genus Pyrenophora and provide details of
morphology, phylogeny and the current status of species. Pyrenophora is a genus of saprobic and
plant pathogenic fungi with a worldwide distribution, commonly associated with leaves, wood,
cereals and other grasses. A phylogeny for Pyrenophora (sexual state of Drechslera) and allied
genera is presented based on analysis of ITS, GPDH, RPB2, nrSSU and nrLSU DNA sequence
datasets. Pyrenophora is a monophyletic genus in Pleosporaceae. Pyrenophora sexual states
cluster with their expected Drechslera asexual states. As a genus can now only have one name we
synonymise Drechslera under Pyrenophora.
Key words – GPDH – RPB2 – Epitypification – Nomenclature
Introduction
Pyrenophora (sexual state = Drechslera) causes disease on many graminicolous hosts
(Zhang & Berbee 2001) where they are commonly observed in their asexual state (Zhang & Berbee
2001). Several recent studies using multigene analysis and some coupled with morphology have
provided the groundwork for classification in Pyrenophora (Berbee 1996, Zhang & Berbee 2001,
Zhang et al. 2012, Hyde et al. 2013). We have been working on the genera of Pleosporales in order
to provide a natural classification via morphological and phylogenetic characterization (Zhang et al.
2012, Ariyawansa et al. 2013a, b, c, Hyde et al. 2013, Ariyawansa et al. 2014). In this paper we
bring together data on the genus Pyrenophora.
History
The type species of Pyrenophora, P. phaeocomes (Rebent.) Fr., was described as Sphaeria
phaeocomes by Rebentisch (1804) and placed in Xylariaceae. Later, Fries (1849) reclassified the
genus as Pyrenophora and placed in Pleosporales. Wehmeyer (1961) placed Pyrenophora in the
family Pleosporaceae. Barr (1987) redefined Pleosporaceae to comprise Clathrospora,
Kirschsteiniothelia, Lewia and Pleospora and grouped Cochliobolus, Pyrenophora and
Mycosphere 5 (2): 351–362 (2014) ISSN 2077 7019
www.mycosphere.org Article Mycosphere
Copyright © 2014 Online Edition
Doi 10.5943/mycosphere/5/2/9
352
Setosphaeria in the family Pyrenophoraceae. Berbee (1996) disagreed, suggesting these genera
belong to Pleosporaceae and this has been followed by later researchers (Zhang et al. 2012, Hyde
et al. 2013). Pyrenophora is characterized by immersed to semi immersed ascomata and neck
covered with brown to reddish-brown setae, lack of pseudoparaphyses, clavate to saccate asci,
usually with a large apical ring, and muriform terete (cylindrical, frequently circular in section but
narrowing to one end) ascospores. Morphologically, the terete ascospores of Pyrenophora can be
readily distinguished from Clathrospora and Platyspora. The lack of pseudoparaphyses and smaller
ascospores of Pyrenophora can easily be differentiated from those of Pleospora (Sivanesan 1984).
Pyrenophora has usually clustered in Pleosporaceae with Bipolaris and Setosphaeria (Zhang &
Berbee 2001). Pyrenophora species can easy be distinguished from species in Bipolaris as
ascospores are filiform and Setosphaeria as ascospores are phragmosporous and hyaline
(Wehmeyer 1953, Zhang & Berbee 2001).
Sexual and asexual states
Pyrenophora has been linked to asexual morphs in Drechslera. Drechslera species were
initially categorized in Helminthosporium on the basis of their dark colour, transversely septate
conidia and a graminicolous habitat (Drechsler 1923, Shoemaker 1959, 1961). Consequently,
graminicolous Helminthosporium species were segregated into three genera, Bipolaris, Drechslera,
and Exserohilum, defined based on their association with their sexual states Cochliobolus,
Pyrenophora and Setosphaeria respectively (Zhang & Berbee 2001).
Importance and role
Pyrenophora species are phytopathogens or as saprobes are involved in nutrient cycling.
Many species cause disease on their graminicolous hosts and are usually present in their asexual
state (Drechslera) (Zhang & Berbee 2001). Some species of Pyrenophora are serious plant
pathogens (Zhang & Berbee 2001). Pyrenophora teres (= Drechslera teres) is a necrotrophic
pathogen of economically important crops, such as barley (Kingsland 1991, Gupta & Loughman
2001). Pyrenophora graminea (= Drechslera graminea) causes barley stripe resulting in significant
yield losses (Tekauz 1983). Pyrenophora graminea lives within barley kernels as mycelium, and
when seeds germinate, hyphae enter the seedling through the coleorrhiza, causing a systemic
infection (Pecchia et al. 1998, Leisova 2005). Pyrenophora tritici-repentis causes tan spot of wheat
(Lamari & Bernier 1989, Balance et al. 1996) which occurs in all the major wheat growing areas of
the world and causes 3 to 50% yield losses (Lamari & Bernier 1989) and its prevalence has
increased recently.
Number of species
Currently 198 species of Pyrenophora and 135 species of Drechslera are listed in Index
Fungorum (2014).
Molecular data
Rapid identification of diseases cause by Pyrenophora has been determined via different
DNA markers. Identification of molecular genetic markers in Pyrenophora teres f. teres associated
with low virulence on ‘Harbin’ barley was assessed by random amplified polymorphic DNA
(RAPD) (Weiland et al. 1999) and five RAPD markers were obtained that were associated in
coupling with low virulence. The data suggested that the RAPD technique can be used to tag
genetic determinants for virulence in P. teres f. teres (Weiland et al. 1999). Specific polymerase
chain reaction (PCR) primers were developed from amplified fragment length polymorphism
(AFLP) fragments of Pyrenophora teres, the causal agent of net blotch on barley leaves (Leisova et
al. 2005). The primers were designed to amplify DNA from P. teres f. teres (net form) and allow its
differentiation from P. teres f. maculata (spot form), which is morphologically very similar to P.
teres f. teres in culture (Leisova et al. 2005). The PCR assay was certified with 60 samples of
Pyrenophora species. The amplification with four designed PCR primer pairs provided P. teres
353
form-specific products. No cross-reaction was observed with DNA of several other species, such as
P. tritici-repentis and P. graminea (Leisova et al. 2005). Pyrenophora graminea is the causal agent
of barley leaf stripe disease (Lubna et al. 2012, Mokrani et al. 2012). Two leaf stripe isolates PgSy3
(exhibiting high virulence on the barley cultivar 'Arabi Abiad') and PgSy1 (exhibiting low virulence
on Arabi Abiad), were mated and 63 progeny were isolated and phenotyped for the reaction on
Arabi Abiad (Lubna et al. 2012). From 96 AFLP markers, three AFLP markers, E37M50-400,
E35M59-100 and E38M47-800 were linked to the virulence locus VHv1 in isolate PgSy3. Lubna et
al. 2012 suggested that the three markers are closely linked to VHv1 and are unique to isolates
carrying the virulence locus. Pecchia et al. (1998) developed an efficient PCR protocol for
amplification of the IGS region in P. graminea and to characterize this region by restriction
fragment analysis. During the study based on the length of the IGS-PCR product, ca. 3.8 or 4.4 kb,
two groups of isolates were identified from six cultures i.e I3/88 (Italy; CBS 100862), I7/88 (Italy;
CBS100861), 60/93 (Austria; CBS 100866), I10/95 (Tunisia; CBS 100863), I28/95 (Tunisia; CBS
100864), I33/95 (Tunisia; CBS 100865). The RFLP patterns of isolates obtained with the 6-base
cutting enzymes ApaI, BglII, DraI, EcoRV, HindIII and SacI were similar within each group and
different between the two groups (Pecchia et al. 1998). Restriction patterns of IGS-PCR products
digested with the 4-base cutting enzyme AluI were polymorphic among isolates in spite of their
IGS-PCR product length (Pecchia et al. 1998).
DNA sequence-based phylogenetics has dramatically influenced both the taxonomy and
systematic of Pyrenophora (Zhang & Berbee 2001, Zhang et al. 2012). In phylogenetic analysis
based on 18s rRNA Pyrenophora clustered within Pleosporaceae (Zhang & Berbee 2001) and thus,
excluded from Pyrenophoraceae (Zhang & Berbee 2001). Later, phylogenetic analysis of the ITS
and gdp data showed that Pyrenophora is monophyletic (Zhang & Berbee 2001). In the same study
Zhang & Berbee 2001 has shown that the asexual states of the Pyrenophora, Drechslera clustered
with their predicted sexual relatives.
Aim of study
The genera of ascomycetes are relatively confused as most 20th century classifications were
based on morphological characters and thus personal opinions. Molecular data has changed this
approach and we can now use morphological and molecular characters to develop more natural
classifications. This is the first in a series of papers in which we detail provide data on a genus,
including molecular data and the morphology of type material with illustrations.
Materials and Methods
Specimen examination
The basic methodology used in this study was the same as Ariyawansa et al. (2013c, d).
Type specimens were loaned from the Museum of Evolution, Uppsala University, Sweden (UPS).
Ascomata were rehydrated in 5% KOH prior for examination and sectioning. Hand sections of the
fruiting structures were mounted in water for microscopic studies and photomicrography. The
fungus was examined in a Nikon ECLIPSE 80i compound microscope and photographed by a
Cannon 450D digital camera fitted to the microscope. Measurements were made with the Tarosoft
(R) Image Frame Work program and images used for figures were processed with Adobe
Photoshop CS3 Extended version 10.0 software (Adobe Systems Inc., The United States).
Phylogenetic analyses
Multiple sequence alignments were generated with MAFFT v. 6.864b
(http://mafft.cbrc.jp/alignment/server/index.html). The alignments were checked visually and
improved manually where necessary. Two different datasets were used to estimate two
phylogenies; a Pleosporineae family tree and a Pyrenophora phylogeny. The first tree focuses on
phylogenetic placement of Pyrenophora in Pleosporaceae and Pleosporineae, the second one was
354
Table 1 Taxa used in the phylogenetic analysis and their corresponding GenBank numbers
Taxon
voucher/cultur
e
ITS
LSU
SSU
RPB2
GPDH
Alternaria alternata
CBS 916.96
DQ678082
KC584507
KC584375
Alternariaster helianthi
CBS 119672
KC584368
KC584626
KC584493
Alternariaster helianthi
CBS 327.69
KC584369
KC584627
KC584494
Bipolaris maydis
ACCC 38152
KC445317
KC445317
Clathrospora diplospora
IMI 68086
U43481_
U43464
Clathrospora elynae
CBS 196.54
GU323214
GU296142
KC584496
Clathrospora elynae
CBS 161.51
KC584370
KC584628
KC584495
Clathrospora heterospora
CBS 175.52
KC584320
KC584577
KC584445
Cochliobolus
heterostrophus
CBS134.39
AY544645
AY544727
DQ247790
Cochliobolus sativus
DAOM226212
DQ678045
DQ677995
DQ677939
Comoclathris compressa
CBS 156.53
KC584372
KC584630
KC584497
Comoclathris compressa
CBS 157.53
KC584373
KC584631
KC584498
Comoclathris magna
CBS 174.52
DQ678068
KC584578
DQ677964
Coniothyrium palmarum
CBS:400.71
EU754153
EU754054
DQ677956
Coniothyrium palmarum
CBS 758.73
EU754154
EU754055
Cucurbitaria berberidis
CBS 394.84
GQ387605
GQ387544
Cucurbitaria berberidis
CBS 363.93
GQ387606
GQ387545
Dendryphiella salina
CBS 142.60
KC793339
KC584583
KC793340
Didymella exigua
CBS 183.55
EU754155
EU754056
Dothidotthia aspera
CPC 12933
EU673276
EU673228
Dothidotthia
symphoricarpi
CPC 12929
EU673273
EU673224
Drechslera andersenii
CBS 258.80
AY004804
AY004835
Drechslera andersenii
CBS 967.87
AY004805
Drechslera andersenii
DAOM 229292
JN943646
JN940084
JN940958
Drechslera avenae
CBS 189.29
AY004795
AY004827
Drechslera avenae
CBS 279.31
AY004796
AY004828
Drechslera biseptata
DAOM 208987
AY004786
AY004817
Drechslera biseptata
CBS 308.69
JN712464
JN712530
AY004819
Drechslera biseptata
CBS 599.7
AY004787
AY004818
Drechslera biseptata
CBS 108940
AY004788
Drechslera campanulata
BRIP15927
AF163058
Drechslera catenaria
DAOM 63665A
AY004802
AY004833
Drechslera catenaria
CBS 191.29
AY004803
AY004834
Drechslera dactylidis
DAOM 92161
AY004781
AY004812
Drechslera dematioidea
CBS 108963
AY004789
JN712532
AY004820
Drechslera dematioidea
DAOM 229295
JN943648
JN940094
Drechslera dematioidea
CBS 108962
JN712465
JN712531
Drechslera dematioidea
CBS 108962
AY004790
JN712531
AY004821
Drechslera dictyoides
DAOM 63666
AY004806
JN940080
AY004836
Drechslera erythrospila
CBS 108941
AY004782
AY004813
Drechslera erythrospila
DAOM 55122
AY004783
AY004814
Drechslera fugax
CBS 509.77
AY004791
AY004822
Drechslera nobleae
CBS 259.80
AY004792
AY004823
Drechslera nobleae
DAOM 229296
JN943647
JN940095
Drechslera nobleae
CBS 966.87
AY004793
AY004824
Drechslera nobleae
CBS 316.69
AY004794
AY004825
Drechslera phlei
CBS 315.69
AY004807
AY004837
Drechslera phlei
DAOM 225627
JN943656
JN940077
JN940964
JN993627
Drechslera poae
DAOM 145373
AY004801
JN940082
JN940961
JN988321
AY004832
Drechslera poae
DAOM 169240
JN943651
Drechslera siccans
DAOM 115701
AY004797
JN940078
JN940963
JN993626
Drechslera siccans
DAOM 115702
AY004799
Drechslera sp.
DAOM126766
AY004800
AY004831
Drechslera sp.
DAOM126772
AY004784
AY004815
Drechslera sp
CBS313.69
AY004785
AY004816
Drechslera triseptata
NZ6120
AF163059
355
Taxon
voucher/cultur
e
ITS
LSU
SSU
RPB2
GPDH
Halojulella avicenniae
BCC 20173
GU371822
GU371831
GU371786
Halojulella avicenniae
BCC 18422
GU371823
GU371830
GU371787
Leptosphaeria maculans
DAOM 229267
DQ470946
DQ470993
DQ470894
Leptosphaerulina
australis
CBS 317.83
GU301830
GU296160
GU371790
Neophaeosphaeria
filamentosa
CBS 102202
GQ387577
GQ387516
GU371773
Ophiosphaerella
herpotricha
CBS 620.86
DQ678062
DQ678010
DQ677958
Paraphoma radicina
CBS 111.79
EU754191
EU754092
KF252180
Phaeosphaeria eustoma
CBS 573.86
DQ678063
DQ678011
DQ677959
Phoma betae
CBS 109410
EU754178
EU754079
GU357804
Phoma exigua
CBS 431.74
EU754183
EU754084
GU371780
Pleospora calvescens
CBS 246.79
EU754131
EU754032
KC584500
Pleospora chenopodii
CBS 206.80
JF740266
JF740095
KC584501
Pleospora halimiones
CBS 432.77
JF740267
JF740096
KC584503
Pleospora herbarum
CBS 191.86
DQ491516
DQ247804
DQ247812
DQ247794
AY316969
Pleospora incompta
CBS 467.76
GU238087
GU238220
KC584504
Pleospora typhicola
CBS 132.69
JF740325
JF740105
KC584505
Pyrenochaeta corni
CBS 248.79
GQ387608
GQ387547
Pyrenophora bromi
DAOM 127414
AY004809
JN940074
JN940954
AY004839
Pyrenophora
chaetomioides
DAOM 208989
AF081445
JN940091
AF081371
Pyrenophora dictyoides
DAOM 75616
JN943654
JN940079
JN940962
JN988322
Pyrenophora japonica
DAOM 169286
AF071347
AF081369
Pyrenophora lolii
CBS 318.69
AY004798
AY004829
Pyrenophora phaeocomes
DAOM 222769
JN943649
DQ499596
DQ497614
Pyrenophora semeniperda
DAOM 213153
AF081446
JN940089
JN993630
AY004826
Pyrenophora tetrarrhenae
DAOM 171966
JN943663
JN940090
JN993620
Pyrenophora tritici-
repentis
DAOM 226213
JN943670
AY544672
AF081370
Pyrenophora tritici-
repentis
DAOM 208990
AF071348
JN940071
AY004838
Pyrenophora tritici-
repentis
DAOM 107224
AY004808
DQ384097
Pyrenopora graminea
11
Y10748
Pyrenopora teres
PM2
Y08746
AY004830
Setosphaeria monoceras
CBS 154.26
AY016368
AY016352
Setosphaeria turcica
ATCC 64835
KF278475
KF278475
Trematosphaeria pertusa
CBS 122371
GU301876
GU348999
GU371801
generated to show the placement of Pyrenophora and its asexual state Drechslera. All sequences
obtained from GenBank were previously used in Zhang & Berbee (2001), Schoch et al. (2009) and
Woudenberg et al. (2013) and are listed in Table 1.
Maximum-parsimony analysis was performed by using PAUP v. 4.0b10 (Swofford 2002) to
obtain the most parsimonious tree. Trees were inferred using the heuristic search option with 1000
random sequence additions. Maxtrees were setup to 5000 and branches of zero length were
collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics for parsimony
(Tree Length [TL], Consistency Index [CI], Retention Index [RI], Relative Consistency Index [RC]
and Homoplasy Index [HI] were calculated for trees generated under different optimality criteria.
Kishino-Hasegawa tests (KHT) (Kishino & Hasegawa 1989) were performed in order to determine
whether trees were significantly different. Maximum parsimony bootstrap values (MP) equal or
greater than 50 % are given below or above each node in blue (Fig. 1 and 2).
Maximum likelihood analyses including 1000 bootstrap replicates were run using RAxML
v. 7.2.6 (Stamatakis 2006, Stamatakis et al. 2008, Stamatakis & Alachiotis 2010). The online tool
Findmodel (http:// www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html) was used to find
out the best nucleotide substitution model for each partition. For both SSU (Pleosporineae family
356
Fig. 1 – RAxML tree based on based on the LSU, SSU and RPB2 sequences of 51 strains
representing the Pleosporineae. Bootstrap support values >50% are shown above or below the
branch. The tree is rooted to Trematosphaeria pertusa. The original isolate numbers are noted after
the species names. Bold indicates ex-type strains.
122371) in the Pleosporineae phylogeny. The resulting trees were printed with TreeView v. 1.6.6
(Page 1996). The resulting replicates were plotted on to the best scoring trees obtained. Maximum
Likelihood bootstrap values (ML) equal or greater than 50 % are given below or above each node
in red (Fig.1 and Fig.2).
Results and discussion
Phylogeny
The final Pleosporineae alignment included 51 strains, representing eight families, and
consisted of 2795 characters, of which 2008 characters were constant, 143 variable characters were
parsimony-uninformative and 644 characters were parsimony-informative. Kishino-Hasegawa
(KH) test showed length= 3145 steps, CI= 0.391, RI= 0.649, RC= 0.254 and HI= 0.609. All trees
were similar in topology and not significantly different (data not shown). In the SSU alignment a
large insertion at position 480 in the isolate Ophiosphaerella herpotrichia (CBS 620.86) was
excluded from the phylogenetic analyses. A best scoring RAxML tree is shown in Fig. 1 and 2 with
the value of -6929.59087and -8276.09706 respectively. Phylogenetic trees obtained from maximum
likelihood and maximum parsimony analyses yielded trees with similar overall topology at species
relationship in agreement with previous work based on maximum likelihood (Zhang & Berbee
2001, Schoch et al. 2009, Hyde et al. 2013, Woudenberg et al. 2013). The support values for the
different phylogenetic methods vary, with the RAxML bootstrap being higher than the maximum
parsimony bootstrap support values in most cases.
357
Fig. 2 – RAxML tree based on a combined dataset of ITS, LSU and GPDH. Bootstrap support
values >50% are shown above or below the branch. The tree is rooted with Pleospora herbarum.
The original isolate numbers are noted after the species names. Bold indicates ex-type strains.
For defining the taxonomy of Pyrenophora and its asexual state Drechslera, 49 strains were
included in the alignment. The maximum parsimony dataset consists of 2194 characters; of which
1781 characters were constant, 95 variable characters were parsimony-uninformative and 318
characters were parsimony-informative. Kishino-Hasegawa (KH) test showed length= 5108 steps,
CI=0.399, RI=0.508, RC= 0.203 and HI=0.601. All trees were similar in topology and not
significantly different (data not shown). Phylogenetic trees obtained from maximum likelihood and
maximum parsimony analyses yielded trees with similar overall topology at species relationship in
agreement with previous work based on maximum likelihood (Zhang & Berbee 2001, Schoch et al.
2009, Hyde et al. 2013).
Taxonomy
Pyrenophora Fr., Summa veg. Scand., Section Post. (Stockholm): 397 (1849). MycoBank: MB
4596 = Drechslera S. Ito, Proc. Imp. Acad. Japan 6: 355 (1930)
358
Sexual state – Ascomata immersed, becoming erumpent to near superficial, solitary or
scattered, globose to subglobose, broadly or narrowly conical, smooth-walled, ostiolate. Ostiole
papillate, covered with brown to reddish-brown setae, which are darkened at the base. Peridium
comprising 2–4 layers of brown, thick-walled cells of textura angularis. Pseudoparaphyses not
observed. Asci 8-spored, bitunicate, fissitunicate, clavate to sub-cylindrical, with a short, broad
pedicel, with a distinct ocular chamber surrounded by a large apical ring. Ascospores 2–3-seriate,
muriform, constricted at the septum, smooth-walled, surrounded by a mucilaginous sheath. Asexual
state: hyphomycetous, Conidiophores macronematous, mononematous, sometimes caespitose,
straight or flexuous, often geniculate, unbranched or in a few species loosely branched, brown,
smooth in most species. Conidiogenous cells polytretic, integrated, terminal, frequently becoming
intercalary, sympodial, cylindrical, cicatrized. Conidia solitary, in certain species also sometimes
catenate or forming secondary conidiophores which bear conidia, acropleurogenous, simple,
straight or curved, clavate, cylindrical rounded at the ends, ellipsoidal, fusiform or obclavate, straw-
coloured or pale to dark brown or olivaceous brown, sometimes with cells unequally coloured, the
end cells then being paler than intermediate ones, mostly smooth, rarely verruculose, pseudoseptate
(description of asexual state from Ellis 1971).
Type species – Pyrenophora phaeocomes (Rebent.) Fr., Summa veg. Scand., Section Post.
(Stockholm): 397 (1849) MycoBank: MB 222199
= Sphaeria phaeocomes Rebent., Prodr. fl. neomarch. (Berolini): 338 (1804)
Notes – The genus Pyrenophora clusters in the suborder Pleosporineae of the family
Pleosporaceae with a relatively high bootstrap support (Fig 1, 60%). Phylogenetic analysis (Fig. 2)
shows that sexual Pyrenophora states cluster with asexual Drechslera states, i.e. Pyrenophora
dictyoides (DAOM 75616) clusters with Drechslera dictyoides (DAOM 63666). The putative strain
of Pyrenophora phaeocomes (DAOM 222769), which is the type species of the genus clusters with
other Pyrenophora species and forms a sister clade with Drechslera biseptata. As a genus can now
only have one name Drechslera is synonymized under Pyrenophora.
To establish the phylogenetic placement of Pyrenophora species at the higher level,
combined analysis of LSU (LROR/LR5), SSU (NS1/NS4) and RPB2 (fRPB2-SF/fRPB2-7cR)
sequence datasets are recommended. For resolving species we recommend combined of ITS
(ITS1/ITS4), LSU (LROR/LR5) and GPDH (gpd1/gpd2) datasets. Phylogenetic inferences from
sequence data of parts of the 18S nrDNA (SSU), 28S nrDNA (LSU), the internal transcribed spacer
regions 1 and 2 and intervening 5.8S nrDNA (ITS) and glyceraldehyde-3-phosphate
dehydrogenase(GAPDH) genes has shown that GAPDH and ITS regions provide more resolution
for species of Pyrenophora as compared to LSU.
Pyrenophora phaeocomes (Rebent.) Fr., Summa veg. Scand., Section Post. (Stockholm): 397
(1849) MycoBank: MB 222199
= Sphaeria phaeocomes Rebent., Prodr. fl. neomarch. (Berolini): 338 (1804
Sexual state – Ascomata 380–450 × 370–430 μm (
x
= 395 × 380 µm, n = 10), solitary or
scattered, initially immersed, becoming erumpent to near superficial, globose to subglobose,
broadly or narrowly conical, coriaceous, smooth-walled, ostiolate. Ostiole usually broadly
papillate, central ostiolar canal filled with periphyses and covered with setae. Setae brown to
reddish-brown, darkened at the base, septate and tapered towards the apex. Peridium 40–70 μm (
x
= 45 µm, n = 20) wide, comprising two types of cells, outer cells of 1–2 layers of heavily
pigmented cells of textura angularis, inner layer composed of small, light brown to hyaline cells of
textura angularis. Pseudoparaphyses not observed. Asci 300–400 × 130–160 μm (
x
= 345 × 140
µm, n = 20), 8-spored, bitunicate, fissitunicate, clavate to sub-cylindrical, with a short, broad
pedicel, thickened and rounded at apex with a distinct ocular chamber surrounded by a large,
distinct, apical ring. Ascospores 78–96 × 27–34 μm (
x
= 88 × 30 µm, n = 40), biseriate to
overlapping triseriate, ellipsoidal with broadly rounded ends, hyaline to light brown when
immature, becoming brown to chestnut brown when mature, muriform with 5–6 transverse septa
359
and single longitudinal septa in one or all cells, constricted at the septa, smooth-walled, relatively
thick-walled, with a 5–9 μm thick mucilaginous sheath. Asexual state – not observed, but see notes.
Material examined – SWEDEN, on leaves of Anthoxanthum (Poaceae), 7 August 1951, J.
Ax. Nannfeldt (UPS 170980, neotype).
Distribution – Putative collections of Pyrenophora phaeocomes have reported from
Belgium, Czech Republic, Denmark, Norway, Portugal, Sweden (GBIF, 2014), but these
identifications have not been confirmed by molecular data.
Type specimen – UPS (neotype), Putative collections of Pyrenophora phaeocomes are
available in BG, BPI, C and O
Sequence data – There is no extype sequence data.
Molecular data is available in GenBank for a putative strain of Pyrenophora phaeocomes
(DAOM 222769). However it is not clear if this species was correctly identified. DAOM 222769
was initially used by the Assembling the Fungal Tree of Life (AFTOL) project in 2007.
AFTOL ID: AFTOL: 283
ITS: JN943649.1 (ITS1/ITS4)
LSU: JN940093.1 (LROR/LR5)
SSU: JN940960.1 (NS1/NS4)
EF1a: DQ497607.1 (983/2218R)
RPB2: DQ497614.1 (fRPB2-SF/fRPB2-7cR)
Notes – Pyrenophora phaeocomes is the type species of Pyrenophora. Sivanesan (1987)
stated that P. phaeocomes has a Drechslera asexual state, but the species was not identified. In our
study we did not observe the asexual state of Pyrenophora phaeocomes on the neotype.
Industrial relevance
Some Pyrenophora species have been used as biocontrol agents. Bromus tectorum is a
dominant winter annual weed in cold deserts of the western United States. (Meyer et al. 2007).
Bromus tectorum and other annual brome grasses have invaded many ecosystems of the western
United States, and because of an annual-grass influenced alteration of the natural fire cycle on arid
western range lands near monocultures are created and conditions in which the native vegetation
cannot compete have been established (Meyer et al. 2007).
Biosecurity
Some species of Pyrenophora are considered as economically important plant pathogens. i.e
Pyrenophora avenae causes seedling blight of oats in different climatic zones (Motovilin, 2000).
Because of the destruction of leaf tissue, photosynthesis is reduced in diseased plants, resulting in
light or shriveled grains. Direct attack of kernels by the fungus also results in light or shriveled
kernels. Severe disease attacks have caused yield losses as high as 30-40 percent (Motovilin &
Strigekozin, 2000). Drechslera cactivora (stem rot and fruit rot on Cactus species), Drechslera
curvispora, Drechslera gigantean, Drechslera longirostrata (seed rot), Drechslera maydis
(Southern corn blight), Drechslera musae-sapientium (leaf spot), Drechslera nodulosa (seed rot),
Drechslera patereae, Drechslera pedicellata (root rot), Drechslera sorghicola (grain mould),
Drechslera stenospila (leaf spot), Pyrenophora cerastii, Pyrenophora chrysospora and
Pyrenophora tetramera (net blotch) are listed in New Zealand Ministry for Primary Industries as
unwanted organisms(http://www.biosecurity.govt.nz/ Accession Date – 2 April 2014).
Biochemistry
A new phytotoxic sesquiterpenoid penta-2,4-dienoic acid, named pyrenophoric acid, was
isolated from solid wheat seed culture of Pyrenophora semeniperda, which is a fungal pathogen
proposed as a mycoherbicide for bio-control of cheat grass (Bromus tectorum) and other annual
bromes (Masi et al. 2014). This genus should be assessed for its chemical diversity and novel
compounds.
360
Fig. 3 – Pyrenophora phaeocomes (neotype) A, B. Ascomata on host specimen. C. Close up of
ascoma. D. Side view of ascoma with neck covered with setae. E, F. Sections of ascomata. G.
Section of peridium. H. Ostiole, with central periphyses. J. Light brown seta. K-M. Asci with 8
ascospores, distinct ocular chamber and apical ring. N-Q. Mature and immature muriform
ascospores. Scale bars: E-F=200 μm, G=30 μm, H=80 μm, J=50 μm, K-M=50 μm, N-Q=15 μm.
Acknowledgments
We are grateful to the Mushroom Research Foundation, Chiang Rai, Thailand, for
supporting this research. MFLU Grant no. 56101020032 is thanked for supporting studies on
Dothideomycetes. Hiran A. Ariyawansa and Ji Chuan Kang are grateful to the International
collaboration plan of Science and Technology at Guizhou Province (Contract no.[2012] 7006) and
the construction of innovation talent team of Science and Technology at Guizhou Province
(Contract no. [2012] 4007). Hiran Ariyawansa is grateful to A.D Ariyawansa, D.M.K Ariyawansa,
Sajeewa S.N. Maharachchikumbura, D.S. Manamgoda and D. Udayanga for their valuable
suggestions.
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