Plant Pathol. J. 38(2) : 78-89 (2022)
https://doi.org/10.5423/PPJ.OA.10.2021.0154
pISSN 1598-2254 eISSN 2093-9280
The Plant Pathology Journal
©The Korean Society of Plant Pathology
Research Article Open Access Fast Track
Phylogenetic Analysis of Phaeosphaeria Species Using Mating Type Genes and
Distribution of Mating Types in Iran
Fariba Ghaderi 1*, Azadeh Habibi2, and Bahram Sharifnabi3
1
Department of Plant Protection, College of Agriculture, Yasouj University, Yasouj 7591874831, Iran
2
Department of Biodiversity, Institute of Science and High Technology and Environmental Sciences, Graduate University
of Advanced Technology, Kerman 7631885356, Iran
3
Department of Plant Protection, College of Agriculture, Isfahan University of Technology, Isfahan 8415683111, Iran
(Received on October 18, 2021; Revised on January 19, 2022; Accepted on January 20, 2022)
Phaeosphaeria species are pathogenic on wheat, barley
and a wide range of wild grasses. To analyze mating
type loci of the Phaeosphaeria species and investigate
mating type distribution in Iran, we sequenced mating type loci of 273 Phaeosphaeria isolates including
67 isolates obtained from symptomatic leaves and ears
of wheat, barley, and wild grasses from two wheatgrowing region in Iran as well as 206 isolates from
our collection from other regions in Iran which were
isolated in our previous studies. Mating type genes
phylogeny was successfully used to determine the species identity and relationships among isolates within the
Phaeosphaeria spp. complex. In this study, we reported
seven new host records for Phaeosphaeria species and
the Phaeosphaeria avenaria f. sp. tritici 3 group was first
reported from Iran in this study. Mating type distribution among Phaeosphaeria species was determined.
Both mating types were present in all sampling regions
from Iran. We observed skewed distribution of mating types in one region (Kohgiluyeh va Boyer-Ahmad)
and equal distribution in the other region (Bushehr).
*Corresponding author.
Phone) +98-7431006000, FAX) +98-9173418661
E-mail) fghaderi2003@yahoo.com
ORCID
Fariba Ghaderi
https://orcid.org/0000-0002-0072-0521
Handling Editor : Jungkwan Lee
This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0) which permits unrestricted
noncommercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Articles can be freely viewed online at www.ppjonline.org.
However, when considering our entire dataset of 273
Iranian Phaeosphaeria isolates, the ratio of mating
types was not deviated significantly from 1:1 suggesting
possibilities for isolates of opposite mating type to interact and reproduce sexually, although the sexual cycle
may infrequently occur in some regions especially when
the climatic conditions are unfavorable for teleomorph
development.
Keywords : Iran, Mat1-1 gene, Mat1-2 gene, PCR-RFLP,
phylogenetic analyses, wild grasses
Phaeosphaeria species are important pathogens of cereals with global distribution. The origin of Phaeosphaeria
species is in the Fertile Crescent coinciding with their
hosts (McDonald et al., 2012). Phaeosphaeria nodorum
(Muller) Hedjar (anamorph Parastagonospora nodorum
(Berk.) Quaedvl., Verkley & Crous) is the causal agent of
Septoria nodorum leaf and glume blotch (SNB) on wheat,
a widespread and yield-reducing disease in many of wheatgrowing regions of the world (Shipton et al., 1971; Sommerhalder et al., 2006). P. nodorum is also pathogenic
on barley and a wide range of wild grasses (Solomon et
al., 2006). Phaeosphaeria avenaria f. sp. avenaria (Paa),
(Weber) Eriksson (anamorph Stagonospora avenae f. sp.
avenae Frank) is a major leaf pathogen of oat and other cereals. Phaeosphaeria avenaria f. sp. tritici (Pat) described
by Shaw (1957), is morphologically similar to Paa but not
pathogenic on oat while pathogenic on wheat and other cereals (McDonald et al., 2012). Ueng and Chen (1994) and
Ueng et al. (1998) studies on genetic differences between
biotypes, split Pat into three groups, Pat1, Pat2, and Pat3.
Later, McDonald et al. (2012) included over 300 Phaeos-
Phaeosphaeria Species Mating Type Genes
phaeria isolates collected from wild grasses on different
continents in a three-gene phylogeny of internal transcribed
spacer (ITS), β-tubulin and β-xylosidase and mating type
loci to determine the relationships among Phaeosphaeria
spp. complex and introduced three new groups; Pat4, Pat5,
and Pat6.
Different genes have been used to analyze Phaeosphaeria species complex including mating type loci, β-tubulin,
β-glucosidase, RNA polymerase II; histidine synthase
(Bennett et al., 2003; Malkus et al., 2005, 2006; Reszka et
al., 2005; Ueng et al., 2003; Wang et al., 2007). In a recent
study, whole-genome sequencing data have been used to
explore the phylogenetic relationships among Phaeosphaeria species (Croll et al., 2021).
Phaeosphaeria species are heterothallic fungi. Sexual
reproduction in these species requires the presence of two
isolates carrying opposite forms of mating type idiomorphs,
called MAT1-1 and MAT1-2, at the same geographic location (Solomon et al., 2004; Sommerhalder et al., 2006).
The extent of sexual reproduction and the contribution of
airborne ascospores as the source of primary inoculum is
important in epidemiology and management of pathogens.
Recombination resulting from sexual mating have the potential to give rise to fitter genotypes that are more virulent
and fungicide resistant. In asexual reproduction, the main
source of the inoculum is pycnidiospores that have a limited increase in genetic diversity comparing to ascospores
(Sommerhalder et al., 2006). The presence of both mating
types and the mating type ratios have been studied to obtain
information about the sexual reproduction by heterothallic
fungi (Cowger and Silva-Rojas, 2006; Notteghem and Silué, 1992).
There are arguments about the degree of sexual reproduction in populations of Phaeosphaeria species. Studies
on P. nodorum isolates from North Africa, North America,
Australia, Europe, and Near East showed that MAT1-1
and MAT1-2 were not evenly distributed (Halama, 2002).
Skewed mating type ratios among P. nodorum isolates are
reported among populations from Central Asia (Vergnes
et al., 2006). On the other hand, random mating within
populations of P. nodorum from Texas, Oregon, and Switzerland have been proved (Keller et al., 1997a, 1997b; McDonald et al., 1994; Sommerhalder et al., 2006). McDonald
et al. (2012) studied isolates of Pat from five continents and
observed that all Pat1 isolates carried only the MAT1-1 allele, Pat3 and Pat5 isolates had both MAT1-1 and MAT1-2
alleles, and Pat4 and Pat6 isolates were all MAT1-2.
The objectives of this study were (1) to collect Phaeosphaeria isolates from wheat, barley and wild grasses in
Bushehr, Kohgiluyeh va Boyer-Ahmad and Khuzestan
79
Provinces in Iran and identify the species using morphological and molecular data, (2) to analyze mating type loci
of the collected isolates in addition to isolates collected
from wheat, barley, and wild grasses in Iran from our previous studies and use them in a phylogeny to determine the
relationships among 273 isolates within the Phaeosphaeria
spp. complex; and (3) to investigate mating type distribution among Phaeosphaeria species in Iran.
Materials and Methods
Sampling, fungal isolation, and morphological characterization. Symptomatic leaves and ears of wheat, barley,
and wild grasses were collected from Bushehr, Kohgiluyeh
va Boyer-Ahmad and Khuzestan Provinces in Iran, and
taken to laboratory. The diseased leaves and ears showing
typical symptoms of SNB were cut into segments of 5-7
mm, sterilized for 2 min in 1% sodium hypochlorite, rinsed
in sterile water, placed in glass slides with tape, and kept in
high humidity until the pycnidia produced cirri containing
pycnidiospores. Purification was carried out using singlespore method on 2% water agar medium in plastic Petri
dishes with a flame-sterilized needle. After 2-3 days of incubation, germinated spore was transferred to yeast sucrose
agar (YSA, 10 g/l yeast extract, 10 g/l sucrose, 1.2% agar).
Pure cultures of each isolate were stored on lyophilized
filter-paper strips at –80°C (Adhikari et al., 2008). Only
one single-spore strain of Phaeosphaeria sp. was isolated
from each infected plant and morphological characteristics
i.e., colony color, conidia and conidiomata morphology,
pigmentation and colony growth rate were used for species
identifications (Quaedvlieg et al., 2013).
DNA extraction. For molecular identifications, mycelium
plugs from isolates grown on YSA (10 g/l yeast extract,
10 g/l sucrose, 16 g/l agar) for 5 days were transferred to
flasks containing 50 ml yeast sucrose broth medium (YSB,
10 g/l yeast extract, 10 g/l sucrose) and incubated on an
orbital shaker for 7 days at 120 rpm at 18°C. Harvested
mycelia were freeze-dried and stored at ‒20°C until further
use. Lyophilized mycelium was ground into powder and
total DNA was extracted using CTAB method according
to Murray and Thompson (1980). Genomic DNA was visualized on a 1.2% agarose gel (1.2% agarose, 0.5× TAE)
using UV light (GelDoc, Bio-Rad Laboratories, Hercules,
CA, USA). Obtained sequences were deposited in GenBank (Tables 1 and 2).
Mating type identification and fertility. Mating type
idiomorphs, MAT1-1 and MAT1-2, were amplified and se-
80
Ghaderi et al.
Table 1. Phaeosphaeria isolates used in phylogenetic analysis of MAT1-1 gene
Species
Isolate
Region
Host
GenBank accession no.
Phaeosphaeria nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. avenaria f. sp. tritici 1 (Pat1)
P. avenaria f. sp. tritici 1 (Pat1)
P. avenaria f. sp. tritici 1 (Pat1)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici 3 (Pat3)
P. avenaria f. sp. tritici 3 (Pat3)
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici5 (Pat5)
P. avenaria f. sp. tritici3 (Pat3)
P. avenaria f. sp. tritici1 (Pat1)
P. avenaria f. sp. tritici1 (Pat1)
P. avenaria f. sp. tritici3 (Pat3)
B10
C5
E31
E2
E20
AVR 1
AVR12
L1
I32
H2
M7
A1
A12
A37
AVR6
AVR7
AVR8
AVR9
I36
I37
Pn1 to Pn35
Pn36 to Pn57
Pn58 to Pn89
Pn90 to Pn99
Pn100 to Pn112
Pn-grass-1
Pn-grass-2
P1
P3 to P5
Pt1 to Pt4
P2
Pt5
P6
P7
Pt6
P10
P11
Pt7 to Pt8
Pt9 to Pt15
Pt16 to Pt18
Pt19 to Pt20
Western Cape
Arkansas
Russia
Kyrgistan
Tadjikistan
North Dakota
North Dakota
Oregon
Denmark
North Dakota
Sweden
Saskatchewan
Alberta
Manitoba
North Dakota
North Dakota
North Dakota
North Dakota
Denmark
Denmark
Iran (KB)
Iran (Fars)
Iran (Khuzestan )
Iran (Golestan)
Iran (Bushehr)
Iran ( KB)
Iran ( KB)
Iran (Golestan)
Iran (Golestan)
Iran (KB)
Iran (Khuzestan )
Iran (Khuzestan )
Iran (Golestan)
Iran (Golestan)
Iran (Khuzestan )
Iran (Fars)
Iran (Fars)
Iran (Bushehr)
Iran (Bushehr)
Iran (Bushehr)
Iran (KB)
Wheat
Wheat Seed
Durum wheat
Wheat
Wheat
Inter. Wheat Grass
Barley
Triticale
Wheat
Crested Wheatgrass
Wheat
Wheat Seed
Wheat Seed
Wheat Seed
Smoothe Brome
Smoothe Brome
Smoothe Brome
Smoothe Brome
Wheat
Triticale
Wheat
Wheat
Wheat
Wheat
Wheat
Dactylis glomerata
D. glomerata
Phalaris arundinacea
P. arundinacea
P. arundinacea
P. arundinacea
P. arundinacea
Aegilops tauschii
Bromus hordeaceus
Aegilops tauschii
Bromus hordeaceus
P. arundinacea
Wheat
Wheat
Barley
Barley
JQ758272
JQ758289
JQ758301
JQ758295
JQ758296
JQ758317
JQ758323
JQ758361
JQ758345
JQ758327
JQ758364
JQ758228
JQ758252
JQ758253
JQ758319
JQ758321
JQ758320
JQ758322
JQ758367
JQ758368
OK000630-OK000664a
OK000665-OK000687a
OK000688-K000718a
OK000719-OK000728a
OK000729-OK000741a
OK000742a
OK000743a
OK000744a
OK000745-OK000747a
OK000748-OK000751a
OK000752a
OK000753a
OK000754a
OK000755a
OK000756a
OK000757a
OK000758a
OK000759-OK000760a
OK000761-OK000767a
OK000768-OK000770a
OK000771-OK000772a
a
Sequences generated in this study.
quenced for 67 Phaeosphaeria isolates that were obtained
in this study and 201 isolates from our collection, which
were isolated in Ghaderi et al. (2017, 2020) (Tables 1 and 2).
The amplifications were carried out using a multiplex
polymerase chain reaction (PCR) with primers (Table 3)
designed by Bennett et al. (2003). PCR amplifications were
performed in 25 μl reactions containing 2 μm primers, 0.4
mM dNTPs (Fermentas Inc., Waltham, MA, USA), 8 pg
Phaeosphaeria Species Mating Type Genes
81
Table 2. Phaeosphaeria isolates used in phylogenetic analysis of MAT1-2 gene
Species
Isolate
Region
Host
GenBank accession no.
Phaeosphaeria nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. avenaria f. sp. tritici 6 (Pat6)
P. avenaria f. sp. tritici 6 (Pat6)
P. avenaria f. sp. tritici 6 (Pat6)
P. avenaria f. sp. tritici 6 (Pat6)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici 4 (Pat4)
P. avenaria f. sp. tritici 4 (Pat4)
P. avenaria f. sp. tritici 4 (Pat4)
P. avenaria f. sp. tritici 4 (Pat4)
P. avenaria f. sp. tritici 3 (Pat3)
P. avenaria f. sp. tritici 3 (Pat3)
P. avenaria f.sp. avenaria (Paa)
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. nodorum
P. avenaria f. sp. avenaria (Paa)
P. avenaria f. sp. avenaria (Paa)
P. avenaria f. sp. avenaria (Paa)
P. avenaria f. sp. avenaria (Paa)
P. avenaria f. sp. avenaria (Paa)
P. avenaria f. sp. avenaria (Paa)
P. avenaria f. sp. avenaria (Paa)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici 5 (Pat5)
P. avenaria f. sp. tritici3 (Pat3)
P. avenaria f. sp. tritici3 (Pat3)
B14
E30
F2
I27
H26
AVR13
R11
R12
R16
R17
AVR3
AVR4
R1
R2
R3
R4
I34
I35
s258
Pn113 to Pn127
Pn128 to Pn156
Pn157 to Pn183
Pn184 to Pn197
Pn198 to Pn214
Pn-grass4 to Pn-grass5
Pn-grass6 to Pn-grass7
P15 to P18
P21
Paa1
Paa2 to Paa3
P22 to P25
P12 to P13
Paa4
Pt21
P8 to P9
Pt22-Pt23
Pt24-Pt27
Western Cape
Russia
Switzerland
Denmark
North Dakota
North Dakota
Iran
Iran
Iran
Iran
North Dakota
North Dakota
Iran
Iran
Iran
Iran
Denmark
Denmark
Netherlands
Iran (KB)
Iran (Fars)
Iran (Khuzestan )
Iran ( Golestan)
Iran (Bushehr)
Iran (Bushehr)
Iran (Bushehr)
Iran (Golestan)
Iran (Fars)
Iran( KB)
Iran( KB)
Iran (Golestan)
Iran (Khuzestan )
Iran (Khuzestan )
Iran( KB)
Iran (Fars)
Iran (Bushehr)
Iran( KB)
Wheat
Durum wheat
Wheat
Triticale
Crested wheatgrass
Barley
Dactylis glomerata
D. glomerata
D. glomerata
D. glomerata
Smoothe brome
Altai wild rye
Dactylis glomerata
D. glomerata
D. glomerata
D. glomerata
Wheat
Wheat
Oat
Wheat
Wheat
Wheat
Wheat
Wheat
Aegilops tauschii
Avena sativa
P. arundinacea
Avena sativa
P. arundinacea
Convolvulus arvensis
A. sativa
P. arundinacea
Aegilops tauschii
A. tauschii
Bromus hordeaceus
Wheat
Barley
JQ758369
JQ758401
JQ758407
JQ758442
JQ758425
JQ758493
JQ758509
JQ758510
JQ758511
JQ758512
JQ758489
JQ758490
JQ758499
JQ758500
JQ758501
JQ758502
JQ758485
JQ758486
JQ758487
OK000773-OK000787a
OK000788-OK000816a
OK000817-OK000843a
OK000844-OK000857a
OK000858-OK000874a
OK000875-OK000876a
OK000877-OK000878a
OK000879-OK000882a
OK000883a
OK000884a
OK000885-OK000886a
OK000887-OK000890a
OK000891-OK000892a
OK000893a
OK000894a
OK000895-OK000896a
OK000897-OK000898a
OK000899-OK000902a
a
Sequences generated in this study.
DNA, 0.05 U Taq DNA polymerase (MBI Fermentas), 3
mM MgCl2 and corresponding reaction Dream Taq buffer
(MBI Fermentas) (Sommerhalder et al., 2006). The PCR
condition was set up in 2 min initial denaturation at 96°C,
35 cycles of 30 s at 96°C, annealing at 55°C for 30 s, extension at 72°C for 1 min and a final 5 min extension at
72°C (Sommerhalder et al., 2006). The PCR products were
visualized on 1.5% agarose gel (1.5% agarose, 0.5× TBE).
Sequencing was performed by Macrogen (Seoul, Korea).
The ratio of MAT1-1 to MAT1-2 alleles was evaluated.
Deviations from a 1:1 ratio of the two mating types within
fields was tested using chi-square statistics.
To confirm the development of sexual phase, genetic
crosses were carried out between opposite mating types for
67 Phaeosphaeria isolates collected in this study following the procedure of Halama and Lacoste (1992). Opposite
mating types were grown on 2% water agar medium encompassing sterilized wheat straws and incubated at 10°C
82
Ghaderi et al.
Table 3. List of primers used in this study
Locus
Length of product
Sequence (5'-3')
Reference
MAT1-1
MAT1-2
MAT2-1
MAT2-2
Bxylo9Fcod
Bxylo970Rco
360
CTTCACGACCGGCCAGATAGT
CAGAGGCTTGTCGGGTTCAT
ACCCCGCCCCATGAACAAGTG
CTAGACCGGCCCGATCAAGACCAAAGAAG
CAAAGAACCCATTGTCACACAC
GCTGTTCTTCAGCCAACTT
Bennett et al. (2003)
Bennett et al. (2003)
Bennett et al. (2003)
Bennett et al. (2003)
McDonald et al. (2012)
McDonald et al. (2012)
510
962
with a 12-h photoperiod, near ultraviolet light (300-400
nm) and intensities of 400 and 600 pW/cm2 for 50 days.
Isolates were paired with themselves as control.
Genetic data analyses. Mat1-1 and Mat1-2 sequences
were generated for 273 isolates. The obtained sequences
of 273 isolates of this study and some sequences from McDonald et al. (2012) which were obtained from GenBank,
were used in phylogenetic analyses to determine the taxonomic status of Phaeosphaeria species and identifications
(Tables 1 and 2).
The phylogenetic analyses of Mat1-1 and Mat1-2 alleles
were performed separately. Sequences were edited manually and aligned by Geneious version 7 (Biomatters Ltd.,
Auckland, New Zealand). Phylogenetic analyses were
performed using heuristic searches in PAUP v. 4.0a133
(Swofford, 2002) for parsimony, neighbour-joining and
maximum likelihood analyses. For maximum likelihood
analyses, models of sequence evolution were evaluated
for both datasets by JModeltest v.2.1.4 (Posada, 2008) using the Akaike information criterion and model parameter
estimates were implemented in PAUP v. 4.0a133. The
resulted trees were midpoint rooted. The resulted trees
were observed and edited in FigTree v1.4.0. Mating Type
polymorphism within each species assessed using DnaSP
v5 (Librado and Rozas, 2009).
PCR-restriction fragment length polymorphism technique. In order to confirm identification of P. avenaria f.
sp. tritici 1 (Pat1) isolates, we used PCR‒restriction fragment length polymorphism (PCR-RFLP) assay (McDonald
et al., 2012) to differentiate between P. nodorum and Pat1.
Isolates of Phaeosphaeria sp. were surveyed using this
method.
An isolate obtained from earlier studies of McDonald et
al. (2012) was used as positive control. Partial sequence of
β-xylosidase gene (962 bp) was amplified using specific
primers (Table 3) and was used as template DNA. Speciesspecific restriction enzyme recognition sites were distinguishing by NEB Cutter v2.0. Digestion of β-xylosidase
gene PCR amplicons was carried out with 2 units of the
restriction enzyme ScaI (MBI Fermentas) at 37°C for 90
min. A 15-min treatment at 65°C was applied for inactivation. Digested PCR products were visualized on 2% agarose gels with ethidium bromide staining.
Results
Fungal isolation and identification. In total, 67 Phaeosphaeria isolates were obtained from symptomatic leaves
and ears of wheat, barley and wild grasses from Bushehr,
Khuzestan and Kohgiluyeh va Boyer-Ahmad Provinces.
Based on morphological characterization and molecular
analysis, isolates were identified as P. nodorum, Paa, Pat1,
Pat3, and Pat5. Paa, Pat1, Pat3, and Pat5 were identified
based on molecular phylogeny of MAT genes.
In Bushehr province, 48 isolates were collected from
wheat and wild grasses (Tables 1 and 2). We obtained 30
isolates of P. nodorum from wheat ears, two from Aegilops
tauschii ears and two from Avena sativa leaves. A. tauschii
and A. sativa are new hosts for P. nodorum to the world.
Four isolates from wheat ears were identified as Pat3. Pat3
was first isolated from Iran in this study. Ten pat1 isolates
were obtained from wheat and barley. Barley is a new host
for pat1 to the world.
In Kohgiluyeh va Boyer-Ahmad Province, 16 isolates
were collected from leaves and ears of wild grasses (Tables
1 and 2). Three Paa isolates, one from P. arundinacea and
two from Convolvulus arvensis leaves were obtained. Convolvulus arvensis is a new host for Paa to the world. Five
isolates of Pat5 were identified from P. arundinacea and
A. tauschii. Two P. nodorum isolates were identified from
Dactylis glomerata ears, which is a new host to the world.
Six Pat3 isolates were obtained from barley ears. Barley
is a new host for Pat3 to the world. Pat3 is isolated for the
first time from Iran.
In Khuzestan Province, three isolate were collected. One
Paa was obtained from ear of A. tauschii, which is a new
host for Paa to the world and two Pat5 were isolated from A.
tauschii and P. arundinacea.
Phaeosphaeria Species Mating Type Genes
Phylogenetic analyses. Amplification of MAT1-1 and
MAT1-2 gene fragments of 510 bp and 360 bp from all isolates (Tables 1 and 2) was conducted successfully. The obtained sequences were deposited in GenBank and accession
83
numbers were obtained (Tables 1 and 2). The aligned data
sets of MAT1-1 and MAT1-2 gene consisted of 269 and 396
characters of which 52 and 55 characters were parsimony
informative, respectively. The three phylogenetic analy-
Fig. 1. The parsimony tree constructed using MAT1-1 gene from 151 Phaeosphaeria isolates. Branch length shows the substitution rate.
Bootstrap values are labeled on the branches. The tree is midpoint rooted.
84
Ghaderi et al.
Fig. 2. The parsimony tree constructed using MAT1-2 gene from 143 Phaeosphaeria isolates. Branch length shows the substitution rate.
Bootstrap values are labeled on the branches. The tree is midpoint rooted.
Phaeosphaeria Species Mating Type Genes
85
Table 4. Summary of mating type polymorphism within each species
Mat1-1 polymorphism
N haplotype
Haplotype diversity (Hd)
Intron
Total no. of mutations
Synonymous
Non-synonymous
Phaeosphaeria nodorum
(n = 115)
6
0/7599
1
6
4
2
Pat3
(n = 4)
2
1
1
1
0
1
Mat1-2 polymorphism
Pat5
(n = 14)
2
0.5275
1
1
0
1
Pat1
(n = 10)
1
0
0
0
0
0
P. nodorum
(n = 106)
4
0/6748
1
5
4
1
Pat3
(n = 6)
2
1
1
1
0
1
Pat5
Paa
(n = 3) (n = 15)
1
1
0
0
1
1
0
0
0
0
0
0
Pat, Phaeosphaeria avenaria f. sp. tritici.
sis methods parsimony, neighbor-joining and maximum
likelihood generated trees with similar topologies amongst
species. The topology and branch lengths of the parsimony
phylogenetic trees are shown in Figs. 1 and 2.
To elucidate phylogenetic relationships among 273 Phaeosphaeria species from wheat, Barley and wild grasses
and accurate species identifications, separate parsimony
trees were created using MAT1-1 and MAT1-2 gene sequences (Figs. 1 and 2). Fig. 1 shows the phylogenetic position of Phaeosphaeria isolates using MAT1-1 sequences.
The phylogenetic reconstruction revealed four highly supported clades corresponding to P. nodorum (containing 6
nucleotide haplotypes, 4 with synonymous mutations, 2
with non-synonymous mutations) (Table 4) and three formae speciales of P. avenaria including Pat5 (containing 2
nucleotide haplotypes, 1 with non-synonymous mutations),
Pat3 (containing 2 nucleotide haplotypes, 1 with nonsynonymous mutations) and Pat1. Fig. 2 shows the phylogenetic position of Phaeosphaeria isolates using MAT12 sequences. The tree contains six highly supported clades
corresponding to P. nodorum (containing 4 nucleotide
haplotypes, 4 with synonymous mutations, 1 with non-syn-
onymous mutations) and five P. avenaria formae specials
including Pat1, Pat3 (containing 2 nucleotide haplotypes, 1
with non-synonymous mutations), Pat5 (with 1 nucleotide
haplotype), P. avenaria f. sp. tritici 6 (Pat6), and Paa (with
01 nucleotide haplotype).
PCR-RFLP technique. We used a PCR-RFLP technique
to distinguish P. nodorum isolates from Pat1 isolates based
on fixed species polymorphisms. The 962-bp PCR products were amplified from genomic DNA with β-xylosidase
gene-specific primers and digested using ScaI enzyme.
PCR products from Pat1 isolates had a specific restriction
site and 695-bp and 267-bp fragments were produced.
There was no restriction site in PCR products of 221 P.
nodorum isolates, which produced 962-bp amplicons (Fig.
3).
Mating type distribution and fertility. The ratio of
MAT1-1 to MAT1-2 alleles and the results of chi-square
statistics for testing deviations from a 1:1 ratio of the two
mating types within fields are presented in Table 5. The
mating type ratio was not significantly different from 1:1
Fig. 3. PCR-RFLP assay used to differentiate between Phaeosphaeria nodorum and P. avenaria f. sp. tritici 1 (Pat1). The PCR products
from β-xylosidase gene amplifications were digested with enzyme ScaI. P. nodorum and Pat1 displayed different patterns of DNA fragments. The Pat1 isolate (lane 1) digestion using enzyme ScaI produced two bands of approximately 695 bp and 267 bp. Lane 2 is positive control. There was no cutting site for P. nodorum isolates (3-9) which produced 962 bp amplicons. PCR, polymerase chain reaction;
RFLP, restriction fragment length polymorphism.
86
Ghaderi et al.
Table 5. Measures of mating type ratios using the chi-square test in Iranian wheat fields from two sampling regions
Region
Kohgiluyeh va Boyer-Ahmad
Bushehr
Sample size
MAT1-1/MAT1-2
Chi-square value
16
8:8
0ns
P=1
18
25:23
(13w+12g:17w+6g)
0.0417ns
P=0 .83822
Wheat
Wild grass
30
Chi-square value for deviation from a 1:1 mating type ratio.
Significant at P < 0.05; ns, not significant.
Fig. 4. Ascocarp formation of Phaeosphaeria sp. on water agar
media supplied with sterilized wheat straws.
ratio for Kohgiluyeh va Boyer-Ahmad and Bushehr.
The mating ability of Phaeosphaeria species obtained in
this study was examined. None of isolates formed ascocarp
(pseudothecia) when grown alone. The pseudothecia were
obtained after 50 days of incubation on sterilized wheat
straws. Pseudothecia were recognized from pycnidia by the
absence of cirrhi. We could not observe mature pseudothecia containing asci and ascospores in any of the crosses in
laboratory conditions (Fig. 4).
Discussion
In this study, 67 Phaeosphaeria spp. were obtained from
symptomatic leaves and ears of wheat, barley and wild
grasses collected in Bushehr, Kohgiluyeh va Boyer-Ahmad
and Khuzestan Provinces in Iran. Based on morphological characteristics and molecular data, P. nodorum, Paa,
Pat5, Pat3, and Pat1 were identified. These included seven
new host records including A. tauschii, A. sativa and D.
glomerata for P. nodorum, barley for Pat1 and Pat3, C.
arvensis and A. tauschii for Paa. Phaeosphaeria species
complex have reported to have the ability to infect several
grass hosts (McDonald et al., 2012; Solomon et al., 2006).
However, the host range for Phaeosphaeria species is yet
unknown. Pat3 is reported in this study for the first time
from Iran.
P. nodorum isolates were wildly distributed in the
sampled areas. McDonald et al. (2012) observed the same
trend in the distribution of Phaeosphaeria species in global
scale. Ghaderi et al. (2010) studied the diversity of Phaeosphaeria species associated with poaceous plants in Iran
and identified P. nodorum, Paa, and Pat5. In another study,
Ghaderi and Razavi (2018) reported Phaeosphaeria dactylidis from wild grasses. Species richness of Phaeosphaeria
in Iran is consistent with the hypothesis of the origin in the
Fertile Crescent.
Since morphological characters between Phaeosphaeria
species often overlap and cultural characteristics are in
many cases variable (Bennett et al., 2003; Cunfer, 2000;
Shoemaker and Babcock, 1989), species relationships and
taxonomy need further molecular characterization. We used
molecular phylogeny of mating type genes to elucidate
relationships among the Phaeosphaeria species. Mat1-1
and Mat1-2 sequences of 67 isolates collected in this study
and 206 isolates which were collected in our previous studies (Ghaderi et al., 2017, 2020) were generated (Tables 1
and 2). The obtained sequences were combined with some
sequences of Mat1-1 and Mat1-2 published previously by
McDonald et al. (2012), and were used in the phylogenetic
analyses to infer relationships between Phaeosphaeria species. In the resulting trees, all clades were separated with
high bootstrap support. Isolates were grouped as P. nodorum clade, and four clades corresponding to different formae speciales including Paa, Pat5, Pat3, and Pat1. According to Turgeon (1998) within species variations is low for
MAT genes while between-species variation is high making
them a useful region to test the biological and phylogenetic
species concepts for outcrossing fungi. Turgeon (1998)
suggested MAT sequences are more useful in phylogenetic
resolution than ITS rDNA and GPD sequence regions.
Ueng et al. (2003) studied the potential use of mating type
genes in phylogeny and molecular classification of Pha-
Phaeosphaeria Species Mating Type Genes
eosphaeria species. They observed that phylogenetic relationships in cereal Phaeosphaeria isolates based on mating
type gene sequences were consistent with those based on
RFLP fingerprints and rDNA ITS sequences. Bennett et al.
(2003) observed between-species MAT variations in the genus Phaeosphaeria. They suggested MAT genes as a reliable diagnostic procedure to elucidate species relationships
in the Phaeosphaeria species pathogenic to cereal crops. In
addition to MAT phylogeny, we successfully used a PCRRFLP technique developed by McDonald et al. (2012) to
distinguish P. nodorum isolates from Pat1 isolates based on
fixed species polymorphisms and 221 P. nodorum isolates
were identified.
Both mating types were present in all sampling regions
from Iran. Mating type ratio for Khuzestan was not calculated because sample size was small. Mating type ratio of
the sampled areas in Bushehr was not significantly different from 1:1. In Kohgiluyeh va Boyer-Ahmad, the ratio
of mating types for 16 isolates obtained from wild grasses
in this study was 1:1. However, when we added the data
of 62 isolates which were previously obtained from wheat
in Ghaderi et al. (2020) (45 MAT1-1 vs. 17 MAT1-2), the
overall mating types ratio showed a significantly skewed
distribution in this area. A possible explanation for this
skewed distribution is that part of our sampling have been
done within a pycnidial clone. However, obtaining a robust estimate of the mating type distribution requires large
number of isolates and samplings that are more extensive
(Solomon et al., 2004). The ratio of mating types was not
deviated significantly from 1:1 when considering our entire
dataset of Iranian Phaeosphaeria population in Tables 1
and 2 (The chi-square statistic = 0.3091 and P = 0.578213).
However, MAT1-1 isolates were predominant. The same
results were obtained by Solomon et al. (2004) in populations of P. nodorum in Western Australia. They observed
that the ratio of mating type alleles was not significantly
different from equal proportions while MAT1-1 isolates
were predominant. They tested different hypotheses regarding the predominance of MAT1-1 alleles including greater
virulence and higher asexual fitness of MAT1-1 strains.
None of these hypotheses could explain the prevalence of
MAT1-1 strains.
We did not observe sexual structures in the sampled areas. One possible explanation would be the dry springs in
the sampling years, which have substantially decreased the
frequency of ascocarps. Another explanation would be that
we have collected plant materials at wrong timepoint in the
disease cycle. Mutations in other genes and sex barriers
such as female sterility have also been proposed as possible
explanations for inability to find the teleomorphs (Bennet
87
et al., 2003; Sommerhalder et al., 2006).
Mating type ratios have been explored for Phaeosphaeria species to study these pathogens biology in order to
reach evidence on the extent of sexual reproduction in
populations. Halama (2002) observed that MAT1-1 alleles
predominated in all of the populations sampled from different parts of the world. Bennett et al. (2003) observed
skewed distribution of mating types in one population of
P. nodorum and equal distribution in another population
from a different field in New York, USA. Sommerhalder et al. (2006) tested a comprehensive collection of P.
nodorum isolates from six countries on five continents and
reported that this pathogen has even distribution of both
mating types among all field populations. Vergnes et al.
(2006) examined Central Asia populations of P. nodorum
and reported the presence of both mating types in Kazakh
and Russian origins while no MAT1-2 isolates were found
in Tajikistan population. Mating type ratios data would
be used to infer interesting information about population
genetics, epidemiology, and control strategies of Phaeosphaeria species.
Our observations of the presence of both mating types
and a 1:1 mating type ratio for our entire data set indicate
that the Iranian Phaeosphaeria population have the opportunity to interact and undergo regular sexual reproduction
resulting high genetic diversity. This hypothesis is consistent with McDonald et al. (2012) and Ghaderi et al. (2020)
who showed that Iranian populations of Phaeosphaeria
species had high levels of genetic diversity. It is likely that
the main primary inoculum of Phaeosphaeria diseases
is airborne ascospores in years with favorable climatic
conditions. In the years with high sexual reproduction,
clean seed, and crop rotation techniques are not preventive
enough. The presence of a mixed reproductive system in
these pathogens should be considered in plant breeding and
fungicide screening programs. Quantitative resistance via
polygenic control would be useful to overcome the possible break up of co-adapted gene complexes in the sexual
reproducing periods.
Conflicts of Interest
No potential conflict of interest relevant to this article was
reported.
Acknowledgments
The authors would like to acknowledge the financial support of Yasouj University, Iran, under grant number of
d99/89/627.
88
Ghaderi et al.
References
Adhikari, T. B., Ali, S., Burlakoti, R. R., Singh, P. K., Mergoum,
M. and Goodwin, S. B. 2008. Genetic structure of Phaeosphaeria nodorum populations in the north-central and midwestern United States. Phytopathology 98:101-107.
Bennett, R. S., Yun, S.-H., Lee, T. Y., Turgeon, B. G., Arseniuk, E.,
Cunfer, B. M. and Bergstrom, G. C. 2003. Identity and conservation of mating type genes in geographically diverse isolates of Phaeosphaeria nodorum. Fungal Genet. Biol. 40:2537.
Cowger, C. and Silva-Rojas, H. V. 2006. Frequency of Phaeosphaeria nodorum, the sexual stage of Stagonospora nodorum,
on winter wheat in North Carolina. Phytopathology 96:860866.
Cunfer, B. M. 2000. Stagonospora and Septoria diseases of barley, oat, and rye. Can. J. Plant Pathol. 22:332-348.
Croll, D., Crous, P. W., Pereira, D., Mordecai, E. A., McDonald,
B. A. and Brunner, P. C. 2021. Genome-scale phylogenies
reveal relationships among Parastagonospora species infecting domesticated and wild grasses. Persoonia 46:116-128.
Ghaderi, F. and Razavi, M. 2018. Identification of the species
Parastagonospora dactylidis on poaceous plants in Iran. Mycol. Iran. 5:35-41.
Ghaderi, F., Sharifnabi, B. and Javan-Nikkhah, M. 2017. Introduction of some species of Parastagonospora on poaceous
plants in Iran. Rostaniha 18:150-165.
Ghaderi, F., Sharifnabi, B., Javan-Nikkhah, M., Brunner, P. C.
and McDonald, B. A. 2020. SnToxA, SnTox1, and SnTox3
originated in Parastagonospora nodorum in the Fertile Crescent. Plant Pathol. 69:1482-1491.
Halama, P. 2002. Mating relationships between isolates of Phaeosphaeria nodorum (anamorph Stagonospora nodorum)
from geographical locations. Eur. J. Plant Pathol. 108:593596.
Halama, P. and Lacoste, L. 1992. Étude des conditions optimales
permettant la pycniogenèse de Phaeosphaeria (Leptosphaeria) nodorum (Müll) Hedj agent de la septoriose du blé [Study
of the optimal conditions allowing the pycniogenesis of Phaeosphaeria (Leptosphaeria) nodorum (Müll) Hedj agent of
wheat septoria]. Agronomie 12:705-710 (in French).
Keller, S. M., McDermott, J. M., Pettway, R. E., Wolfe, M. S. and
McDonald, B. A. 1997a. Gene flow and sexual reproduction
in the wheat glume blotch pathogen Phaeosphaeria nodorum (Anamorph Stagonospora nodorum). Phytopathology
87:353-358.
Keller, S. M., Wolfe, M. S., McDermott, J. M. and McDonald,
B. A. 1997b. High genetic similarity among populations of
Phaeosphaeria nodorum across wheat cultivars and regions
in Switzerland. Phytopathology 87:1134-1139.
Librado, P. and Rozas, J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics
25:1451-1452.
Malkus, A., Chang, P.-F. L., Zuzga, S. M., Chung, K.-R., Shao, J.,
Cunfer, B. M., Arseniuk, E. and Ueng, P. P. 2006. RNA polymerase II gene (RPB2) encoding the second largest protein
subunit in Phaeosphaeria nodorum and P. avenaria. Mycol.
Res. 110:1152-1164.
Malkus, A., Reszka, E., Chang, C. J., Arseniuk, E., Chang, P. F.
and Ueng, P. P. 2005. Sequence diversity of β-tubulin (tubA)
gene in Phaeosphaeria nodorum and P. avenaria. FEMS Microbiol. Lett. 249:49-56.
McDonald, B. A., Miles, J., Nelson, L. R. and Pettway, R. E.
1994. Genetic variability in nuclear DNA in field populations
of Stagonospora nodorum. Phytopathology 84:250-255.
McDonald, M. C., Razavi, M., Friesen, T. L., Brunner, P. C. and
McDonald, B. A. 2012. Phylogenetic and population genetic
analyses of Phaeosphaeria nodorum and its close relatives
indicate cryptic species and an origin in the Fertile Crescent.
Fungal Genet. Biol. 49: 882-895.
Murray, M. G. and Thompson, W. F. 1980. Rapid isolation
of high molecular weight plant DNA. Nucleic Acids Res.
8:4321-4325.
Notteghem, J. L. and Silué, D. 1992. Distribution of mating type
alleles in Magnaporthe grisea populations pathogenic on rice.
Phytopathology 82:421-424.
Posada, D. 2008. jModelTest: phylogenetic model averaging.
Mol. Biol. Evol. 25:1253-1256.
Quaedvlieg, W., Verkley, G. J., Shin, H. D., Barreto, R. W., Alfenas, A. C., Swart, W. J., Groenewald, J. Z. and Crous, P. W.
2013. Sizing up Septoria. Stud. Mycol. 75:307-390.
Reszka, E., Chung, K. R., Tekauz, A., Malkus, A., Arseniuk, E.,
Krupinsky, J. M., Tsang, H. and Ueng, P. P. 2005. Presence
of β-glucosidase (bgl1) gene in Phaeosphaeria nodorum and
Phaeosphaeria avenaria f.sp. triticea. Can. J. Bot. 83:10011014.
Shaw, D. E. 1957. Studies on Leptosphaeria avenaria f. sp. triticea on cereals and grasses. Can. J. Bot. 35:113-118.
Shipton, W. A., Boyd, W. J. R., Rosielle, A. A. and Shearer, B.
I. 1971. The common Septoria diseases of wheat. Bot. Rev.
37:231-262.
Shoemaker, R. A. and Babcock, C. E. 1989. Phaeosphaeria. Can.
J. Bot. 67:1500-1599.
Solomon, P. S., Lowe, R. G., Tan, K. C., Waters, O. D. and Oliver, R. P. 2006. Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat. Mol. Plant Pathol. 7:147-156.
Solomon, P. S., Parker, K., Loughman, R. and Oliver, R. P. 2004.
Both mating types of Phaeosphaeria (anamorph Stagonospora) nodorum are present in Western Australia. Eur. J. Plant
Pathol. 110:763-766.
Sommerhalder, R. J., McDonald, B. A. and Zhan, J. 2006. The
frequencies and spatial distribution of mating types in Stagonospora nodorum are consistent with recurring sexual
reproduction. Phytopathology 96:234-239.
Swofford, D. L. 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sinauer Associates, Sunderland, MA, USA.
Phaeosphaeria Species Mating Type Genes
Turgeon, B. G. 1998. Application of mating type gene technology to problems in fungal biology. Annu. Rev. Phytopathol.
36:115-137.
Ueng, P. P. and Chen, W. 1994. Genetic differentiation between
Phaeosphaeria nodorum and P. avenaria using restriction
fragment length polymorphisms. Phytopathology 84:800806.
Ueng, P. P., Dai, Q., Cui, K.-R., Czembor, P. C., Cunfer, B. M.,
Tsang, H., Arseniuk, E. and Bergstrom, G. C. 2003. Sequence
diversity of mating-type genes in Phaeosphaeria avenaria.
Curr. Genet. 43:121-130.
Ueng, P. P., Subramaniam, K., Chen, W., Arseniuk, E., Wang, L.,
89
Cheung, A. M., Hoffmann, G. M. and Bergstrom, G. C. 1998.
Intraspecific genetic variation of Stagonospora avenae and its
differentiation from S. nodorum. Mycol. Res. 102:607-614.
Vergnes, D. M., Zhanarbekova, A., Renard, M.-E., Duveiller, E.
and Maraite, H. 2006. Mating types of Phaeosphaeria nodorum (anamorph Stagonospora nodorum) from Central Asia. J.
Phytopathol. 154: 317-319.
Wang, C.-L., Malkus, A., Zuzga, S. M., Chang, P.-F. L., Cunfer,
B. M., Arseniuk, E. and Ueng, P. P. 2007. Diversity of the
trifunctional histidine biosynthesis gene (his) in cereal Phaeosphaeria species. Genome 50:595-609.