Mycologia Iranica 6(2): 113 – 118, 2019
Original Article
DOI: 10.22043/mi.2020.121823
Pyrenophora lolii, a new species for the mycobiota of Iran
A. Vasighzadeh
B. Sharifnabi ✉
Department of Plant Protection, Faculty of
Agriculture, Isfahan University of Technology,
Isfahan, Iran
M. Javan-Nikkhah
Department of Plant Protection, College of
Agriculture and Natural Resources, University of
Tehran, Karaj, Iran
E. H. Stukenbrock
Christian-Albrechts University of Kiel and Max
Planck Institute of Evolutionary Biology, Kiel and
Plön, Germany
Abstract: Pyrenophora lolii causes leaf spot on
grasses including Festuca spp., Lolium spp., Dactylis
spp., Avena sativa and wheat (Triticum aestivum).
Infected oat leaves (Avena sativa) showing leaf spot
symptoms were collected from the margin of barley
fields in Golestan province of Iran during the spring
of 2016. A morphological examination of the
Pyrenophora specimen was carried out using light
microscopy. Inoculation of oat leaves with the
isolates of Pyrenophora lolii in greenhouse induced
leaf spot on leaves. In order to confirm the
morphological
identification,
sequences
of
glyceradehyde-3-phosphate dehydrogenase (gpd) gene
and Internal transcribed spacer (ITS) regions were
amplified using gpd1/2 and ITS1/4 primers,
respectively. The phylogenetic analysis based on
these sequences showed that the isolated
Pyrenophora specimen clustered together with
sequences of P. lolii. Based on result of
morphological examination and phylogenetic
analysis, it was concluded that the causal agent of leaf
spot of A. sativa (oat) was P. lolii.
INTRODUCTION
Avena sativa L. is an annual grass (Dimberg et al.
1996) and placed in the family Poaceae and its wild
ancestor is A. sterilis which is endemic to Iran, Iraq,
and Turkey. Oat has been cultivated for centuries
worldwide (Zhou et al. 1999) for its grain as food and
fodder, as well as for medicine (Coffman 1977).
Avena sativa is infected by two species of fungal
pathogens of the genus Pyrenophora; P. avenae
causing leaf blotch and black leaf spot, and P. lolii
causing leaf spot (Mehta 2001).
Species in the genus Pyrenophora (anamorph:
Drechslera), cause disease on Poaceous plants
(Zhang & Berbee 2001) and is placed in the family
Pleosporaceae (Berbee 1996). Some species of this
genus are agents of destructive diseases on Poaceae
including barley and wheat (Ariyawansa et al. 2014).
In previous studies, phylogenetic analysis of
Pyrenophora was carried out based on sequence data
of ITS, GPDH, RPB2, nrSSU and nrLSU DNA
regions (Ariyawansa et al. 2014, Zhang & Berbee
2001).
Pyrenophora lolii cause leaf spot on grass (Jones
2013) and is found in different parts of the world
including Sweden, New Zealand, England and Wales
(Jones 2013, Lam 1984). Pyrenophora lolii infect
Festuca spp., Lolium spp., Dactylis spp., Avena sativa
(Oat) and Triticum aestivum (Wilkins 1973, Gönner
et al. 1993, Tonin et al. 2015, Jones 2013) but it is
more common on Lolium spp. Yield loss of this
fungus in mixed infection with D. catenaria on
Lolium perenne can reach up to 15 %. (Jones 2013).
The goal of this study was to characterize and
identify a new-found Pyrenophora species infecting
oat in Iran, using morphological characterization and
phylogenetic analyses based on GPDH and ITS
sequences.
MATERIALS AND METHODS
Key words: Dreschlera siccans, oat, phylogeny, ITS,
GPDH
Submitted 25 Aug. 2019, accepted for publication 13 Dec.
2019
Corresponding Author E-mail: sharifna@iut.ac.ir
© 2019, Published by the Iranian Mycological Society
http://mij.areeo.ac.ir
Sampling and morphological characterization
Infected A. sativa leaves exhibiting leaf spot
symptoms were collected from margins of barley
114
fields during the spring of 2016 in the Golestan
province located in the Northeast of Iran (Sadabad
village), at an altitude of 160 m above sea level. After
sterilization with 1% sodium hypochlorite solution,
infected leaves were transferred on 2% water agar and
incubated
at
20 °C and with 12 h darkness and 12 h NUV light
(Akhavan et al. 2016). After 10 days, spores emerged
on the surface of leaves and these were used for
morphological identification. The morphological
characteristics of the specimen are summarized in
Table 1.
Pathogenicity test
To assess the pathogenicity of the isolated fungal
spores, A. sativa plants were grown in the greenhouse
under 11 h light and 13 h darkness at 20 °C. For
sporulation of P. lolii, it was cultured on Potato
Dextrose Agar, where after cubes of 6-day-old colony
margin were transferred to 2% WA (water agar) and
incubated at 12 h NUV light and 12 h darkness at
20 °C. For plant inoculation, the concentration of
spore suspension was adjusted to 5 × 105 conidia per
mL and sprayed on plant leaves at the third to fourth
leaf stage (Akhavan et al. 2016). After inoculation,
pots were covered by a plastic bag for 24 h in order to
create a 100% humidity condition. The two days
following inoculation, leaves were surveyed daily.
When symptom developed after 10 days, infected
leaves were cultured on 2% WA under 12 h NUV
light and 12 h darkness at 20 °C for re-isolation
(Akhavan et al. 2016).
DNA extraction, PCR amplification
For DNA extraction, one isolate was cultured on
PDB (Potato Dextrose Broth) and incubated at 20 °C
and with 12 h darkness and 12 h NUV light (Akhavan
et al. 2016). After 10 days, DNA was extracted using
the CTAB method (Murray & Thompson 1980). For
molecular identification, ITS region and gpd gene
were amplified and sequenced.
For amplification of the ITS1 - 5.8S – ITS2
region, forward primer ITS1 (5'-TCCGTAGGTGAA
CCTGCGG-3') and reverse primer ITS4 (5'-TCCTC
CGCTTATTGATATGC-3') were used (White et al.
1990) and for amplification of the gpd gene, forward
primer gpd1 (5'-CAACGGCTTCGGTCGCATTG-3')
and reverse primer gpd2 (5'-GCCAAGCAGTTGGTTG
TGC-3') were used (Berbee et al. 1999).
Reaction conditions for both ITS region and gpd
gene consisted of an initial denaturation step at 95 °C
for 3 min, followed by 30 cycles of 30 s at 95 °C, 30 s
at 58 °C, and 1 min at 72 °C, followed by an extra
extension at 72 °C for 7 min. PCR analysis was
performed in a reaction mixture containing 10 µL
master mix, 6 µL H2O, 2 µL DNA (3 ng/µL), 20
pmol of each of the forward and reverse primers, and
final volume was 20 µL.
The sequencing was done by Sanger sequencing.
For the ITS region, the sequenced fragment was
approximately 560 bp including the internal
Mycologia Iranica - Vol. 6, 2019
transcribed spacer 1, partial sequence, 5.8 S gene and
internal transcribed spacer 2, complete sequence; and
28 S ribosomal DNA gene, partial sequence. The
gpd1/2 primers amplified a 600 bp fragment
including 417 bp of the coding region and 180 bp of
two introns. ITS and gpd sequences were deposited in
NCBI with accession numbers of MN420825 and
MN433343, respectively.
Phylogenetic analyses
To assess the phylogenetic position of the Iranian
Pyrenophora specimens on A. sativa, both their ITS
and gpd sequences were compared with Pyrenophora
ITS and gpd sequences available in GenBank. The
ITS and gpd sequences of the Pyrenophora specimen
were blasted using blast analyses (https://blast.ncbi.
nlm.nih.gov/Blast.cgi) (2020). Alignment of sequences
for both ITS and gpd was conducted by using
MEGA7 and the align by clustal W option (Kumar et
al. 2016) and the result of alignment were used for
phylogenetic analyses utilizing a maximum likelihood
(ML) method based on the Tamura-Nei model
(Tamura & Nei 1993) and neighbor-joining (Nj)
method (Saitou & Nei 1987). The analysis was
conducted with MEGA 7 using the bootstrap method
option with 1,000 replicates (Kumar et al. 2016). The
tree was drawn by using integrated sequences of gpd
and ITS and rooted with Pleospora alfalfae.
RESULTS
Morphological characterization
The results of the morphological analysis of the
specimen are included in Table 1 and depicted in
Fig.1.
Pyrenophora lolii Dovaston, Transactions of the
British Mycological Society 31 (3-4): 251 (1948)
MycoBank MB 290353
Symptom on the infected oat leaves was an
ellipsoid brown spot form. Red-brown, long and
narrow conidiophores usually arise singly. They were
enlarged at base. A group of 7-20 conidia grow at the
top of conidiophores. The length of the conidiophores
was 120-140 µm and the base of conidiophore was
enlarged, globose, 10-20 µm wide. Cylindrical and
straight conidia are 10-20 µm thick, 48-110 µm long
and nearly round at both ends, pale to yellowishbrown, often having 3- 8 pseudosepta. The length and
width of each cell of conidium is 10 to 20 µm. The
diameter of the colony reaches to 7 cm after 5 days on
PDA. The color of the colony is dark brown with the
grey aerial mycelium developing later.
Specimens examined. IRAN, Golestan province,
Sadabad village, on Avena sativa, 15 April 2016, A.
Vasighzadeh.
The morphology of specimen on Avena sativa
from Iran agrees with the description of P. lolii on
Lolium perenne (Shoemaker 1962) and it is shown in
Table 1.
VSIGHZADEH ET AL.: Pyrenophora lolii , a new species for Iranian mycobiota
Pathogenicity test
Inoculation of the Pyrenophora specimens on
A. sativa leaves induced brown pin-like symptoms
after 4 days.
This symptom developed after 10 days as oval and
brown leaf spot with chlorotic margin (Fig. 1). By
culturing of the symptomatic leaves on 2% WA,
fungal spores emerged on the surface of the culture
medium.
Phylogenetic analyses
After the blast analysis in NCBI, some of the
sequences of P. lolii which were identical to the
Pyrenophora specimen sequence, included in the
alignment. The ITS sequence of Pyrenophora
specimen in this study was 100% similar to
previously published P. lolii sequences which were
included in the phylogenetic analysis. The gpd
sequence of P. lolii in this study was compared to gpd
sequences of four isolates of P. lolii and was also
115
100% similar to sequences of three isolates of P. lolii.
We only identified a one-nucleotide difference
between Pyrenophora specimen sequence and
Pyrenophora lolli strain HMCI which the sequence
differentiation was 0.19 %. The trees obtaining from
neighbor-joining (data not shown) and maximum
likelihood methods based on gpd and ITS sequences
showed the same tree topologies.
Based on phylogenetic analysis of integrated
sequences of ITS and gpd, we found that the
Pyrenophora specimen on A. sativa clustered with
P. lolii isolates which had been isolated from
L. multiflorum (Fig 2.). The P. lolii isolates on
L. multiflorum and Avena sativa formed a sister clade
with P. chaetomioides and P. avenae which is a
known pathogen of A. sativa (bootstrap values 100%
for both methods). Pyrenophora lolii was distant to a
monophyletic lineage on barley, wheat, and
bromegrass (P. teres, P. graminea, P. tritici-repentis
and P. bromi).
Table 1. Comparison of the most important features of the specimen on Avena sativa from Iran with Pyrenophora lolii.
Pyrenophora lolii
Pyrenophora lolii
Features
On Avena sativa (this study) µm
On Lolium perenne µm
Length of conidiophores
120-140 (250)
(15) 100-150 (270)
Width of conidiophores
10-20
(8) 12-15 (22)
Length of conidia
48-110
(54) 90-110 (154)
Width of conidia
10-20
(12) 16-18 (21)
Septa number of conidia
(3) 4-6 (8)
(3) 4-6 (8)
Size of each cell of conidia
10-20
14-18
b
a
c
c
Fig. 1. Pyrenophora lolii. a. Light micrographs of Pyrenophora lolii on Avena sativa; conidia (on 2 % TWA, at 20 ˚C, 12 h
darkness, and 12 h NUV light) —Scale bars = 25 μm. b. A 7-day-old colony of P. lolii on PDA. c. The symptom of P. lolii on oat
leaves, 10 days after inoculation in the greenhouse.
DISCUSSION
In previous studies, the connection between
Pyrenophora and Drechslera which are teleomorph
and anamorph, respectively, was proved based on ITS
and gpd data (Zhang & Berbee 2001). For instance,
ITS and gpd sequences of P. lolii and its related
anamorph, D. siccans, were identical (Zhang &
Berbee 2001). In this study, as it was excepted,
Pyrenophora spp. and its related anamorph,
Drechslera spp., clustered together in the phylogenetic
tree.
In previous studies, P. avenae, P. chaetomioides,
and P. lolii which are related to D. avenae, D.
avenacae and D. siccans anamorphs, were proposed
to be one taxon based on numerical taxonomy
(Ibrahim & Threlfall 1966) but the molecular study
based on the ITS and gpd data showed that P. lolii
formed a sister group with a clade comprising P.
chaetomioides and P. avenae and they could not be
considered as one monophyletic lineage (Zhang and
Berbee 2001). This result was similar to the
phylogenic analyses of ITS and gpd sequences of
these taxa in this study.
According to the phylogenetic analysis based on
ITS and gpd data, the Pyrenophora specimen on
Avena sativa in this study grouped with P. lolii. This
species is closely related to P. chaetomioides and
116
Mycologia Iranica - Vol. 6, 2019
Fig.2. The cladogram was constructed based on integrated ITS and gpd sequences of Pyrenophora spp. and shows the
phylogenetic position of the Pyrenophora specimens on Avena sativa from Iran among Pyrenophora spp. The tree was drawn
using a Maximum Likelihood method and rooted with Pleospora alfalfae. Bootstrap values were calculated for 1,000 replicates.
Number (*/*) on and under branches are bootstrap values for the neighbor-joining and Maximum Likelihood method,
respectively. Numbers (*-*) in front of branches are accession numbers for gpd and ITS sequences, respectively. P. =
Pyrenophora. D. = Drechslera.
which is host similarity among these species. Results
of phylogenetic analysis of both sequences of the
Pyrenophora specimen agree with the morphological
characterization and the specimen was identified as
P. lolii.
Several species of Pyrenophora/Drechslera were
found in Iran. Some of these species are destructive
pathogens on wheat and barley such as P. triticirepentis, P. graminea and P. teres (Ershad 2009).
This study is the first record of P. lolii on Avena
sativa in Iran.
ACKNOWLEDGEMENTS
The authors thank the Isfahan University of
Technology (IUT), the Iranian Ministry of Science,
Research and Technology (MSRT) and Iran National
Science Foundation (INSF) for providing financial
support and Dr. Elaheh Seifollahi for her help during
sampling.
REFERENCES
Akhavan A, Turkington TK, Askarian H, Tekauz A ,
Xi K, Tucker JR, Kutcher HR, Strelkov SE. 2016 .
Virulence of Pyrenophora teres populations in
western Canada. Canadian Journal of Plant
Pathology 38: 183-196.
Ariyawansa HA, Kang JC, Alias SA, Chukeatirote E,
Hyde KD. 2014. Pyrenophora. Mycosphere 5:
351–362.
Berbee ML, Pirseyedi M, Hubbard S. 1999.
Cochliobolus phylogenetics and the origin of
known, highly virulent pathogens, inferred from
ITS and glyceraldehyde-3-phosphate dehydrogenase
gene sequences. Mycologia 91: 964-977.
Berbee ML. 1996. Loculoascomycete origins and
evolution of filamentous ascomycete morphology
based on 18S rRNA gene sequence data.
Molecular Biology and Evolution 3: 462-470.
Dimberg LH, Molteberg EL, Solheim R, Frølich, W.
1996. Variation in oat groats due to variety,
VSIGHZADEH ET AL.: Pyrenophora lolii , a new species for Iranian mycobiota
storage, and heat treatment. I: Phenolic compounds.
Journal of Cereal Science 24: 263-272.
Coffman FA. 1977. Oat history, identification, and
classification. Technical Bulletin No. 1516.
United States Department of Agriculture,
Washington DC, USA.
Ershad D, 2009. Fungi of Iran. Agricultural Research
Education and Extension Organization Press,
Tehran, Iran.
Gönner MV, Schlösser E. 1993. Oxidative stress in
interactions between Avena sativa L. and
Drechslera spp. Physiological and Molecular Plant
Pathology 42: 221-234.
Ibrahim FM, Threleall RJ. 1966. The application of
numerical taxonomy to some graminicolous
species of Helminthosporium. Proceedings of the
Royal Society of London. Series B. Biological
Sciences 165(1000): 362-388.
Jones M. 2013. The grass crop: the physiological
basis of production. Springer, the Netherland.
Kumar S, Stecher G, Tamura K. 2016. MEGA7:
molecular evolutionary genetics analysis version
7.0 for bigger datasets. Molecular Biology and
Evolution 33: 1870-1874.
Lam A. 1984. Drechslera siccans from ryegrass fields
in England and Wales. Transactions of the British
Mycological Society 83: 305-311.
Mehta YR. 2001. Molecular and pathogenic
variability of Drechslera isolates from oats.
Fitopatologia Brasileira 26: 590-596.
Murray MG, Thompson WF. 1980. Rapid isolation of
high molecular weight plant DNA. Nucleic Acids
Research 8: 4321-4326.
117
Saitou N, Nei M. 1987. The neighbor-joining method:
a new method for reconstructing phylogenetic
trees. Molecular Biology and Evolution 4: 406425.
Spear ER, Mordecai EA. 2018. Foliar pathogens are
unlikely to stabilize coexistence of competing
species in a California grassland. Ecology 99:
2250-2259.
Tamura K, Nei, M. 1993. Estimation of the number of
nucleotide substitutions in the control region of
mitochondrial DNA in humans and chimpanzees.
Molecular Biology and Evolution 10: 512-526.
Shoemaker RA. 1962. Drechslera Ito. Canadian
Journal of Botany 40: 809-836.
Tonin RB, Reis EM, Gómez DE. 2015. In vitro
mycelia sensitivity of Drechslera siccans, isolated
from wheat, to fungicides. RIA, Revista de
Investigaciones Agropecuarias 41: 192-200.
White TJ, Bruns T, Lee S, Taylor J. 1990.
Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. PCR
protocols: a guide to methods and applications 18:
315-322.
Wilkins PW. 1973. Infection of Lolium and Festuca
spp. by Drechslera siccans and D. catenaria.
Euphytica 22: 106-113.
Zhang G, Berbee ML. 2001. Pyrenophora
phylogenetics
inferred
from
ITS
and
glyceradehyde-3-phosphate dehydrogenase gene
sequences. Mycologia 93: 1048-1063.
Zhou X, Jellen EN, Murphy JP. 1999. Progenitor
germplasm of domesticated hexaploid oat. Crop
Science 39: 1208-1214.
118
Mycologia Iranica - Vol. 6, 2019
گزارش جدیدی از گونه Pyrenophora loliiبرای میکوبیوتای ایران
آسیه وثیق زاده ،1بهرام شریف نبی ،✉1محمد جوان نیکخواه ،2اوا
اشتوکنبروک3
-1گروه گیاهپزشکی ،دانشکده کشاورزی ،دانشگاه صنعتی اصفهان ،اصفهان ،ایران
-2گروه گیاهپزشکی ،پردیس کشاورزی و منابع طبیعی ،دانشگاه تهران ،کرج ،ایران
-3موسسه بیولوژی تحولی مکس پالنک و دانشگاه کریسشن – آلبرشیتز کیل ،کیل ،آلمان
چکیده :قارچ ( Pyrenophora loliiآنامورف )Dreschlera siccansعامل لکه برگی گراسهایی همچون
Lolium ،Festuca spp.
Avena sativa ،Dactylis spp. ،spp.و Triticum aestivumمی باشد .برگهای آلوده یوالف ( )Avena sativaبا عالیم لکه برگی از
حاشیه مزرعه جو در استان گلستان در بهار سال 95جمعآوری شد .بررسی ریخت شناسی نمونه Pyrenophoraجدا شده از برگ-
های یوالف با کاربرد میکروسکوپ نوری انجام شد .مایه زنی برگهای یوالف با نمونه Pyrenophoraدر شرایط گلخانه عالیم لکه
برگی را ایجاد کرد .به منظور تایید بررسیهای ریخت شناسی ،توالی ژن گلیسرآلدهید -3فسفات دهیدروژناز ) (gpdو ناحیه ITSبه
ترتیب با کاربرد آغازگرهای gpd1/2و ITS1/4تکثیر شدند و در مطالعات فیلوژنی مورد استفاده قرار گرفتند .آنالیزهای فیلوژنی بر
اساس هر دو توالی ،نمونه Pyrenophoraجدا شده در این پژوهش را با توالی P. loliiگروهبندی کرد .براساس نتایج بررسی ریخت
شناسی و آنالیز فیلوژنی ،قارچ عامل لکه برگی در یوالف در این پژوهش P. loliiمی باشد.
کلمات کلیدی :ژن ،GPDHناحیه ژنومی ،ITSیوالف،
Dreschlera siccans
مکاتبه کننده :بهرام شریف نبی
تاریخ دریافت 1398/06/04 :تاریخ پذیرش1398/10/24 :
Email: sharifna@iut.ac.ir