Content uploaded by Anke Martin
Author content
All content in this area was uploaded by Anke Martin on Dec 18, 2018
Content may be subject to copyright.
Investigating hybridisation between the forms of Pyrenophora
teres based on Australian barley field experiments and cultural
collections
B. Poudel &M. S. McLean &G. J. Platz &J. A. McIlroy &
M. W. Sutherland &A. Martin
Accepted: 15 August 2018
#Koninklijke Nederlandse Planteziektenkundige Vereniging 2018
Abstract Pyrenophora teres f. teres (Ptt)andP. teres f.
maculata (Ptm) cause net and spot form of net blotch of
barley (Hordeum vulgare L.), respectively. Both patho-
gens co-exist in barley fields and each can reproduce
sexually, resulting in hybridisation and potential gener-
ation of novel virulences that could overcome barley
host resistances. In this study, three field experiments
were conducted during three successive years to inves-
tigate the occurrence of hybridisation. Susceptible bar-
ley was sown and inoculated with Ptt and Ptm.Form-
specific PCR markers were used to analyse 822 conidia
and 223 ascospores sampled from infected leaf tissue
and 317 P. teres isolates collected across Australia dur-
ing 1976–2015. None of the isolates were hybrids.
Investigation of ascospores indicated that hybridisation
had taken place within the forms, demonstrating prefer-
ence for recombination within forms. Possible contribu-
tions of reproductive barriers have been appraised but
further investigation is required to explore the rare
hybridisation between the forms.
Keywords Sexual hybridisation .Pyrenophora teres .
Reproductive isolation .Pre- and post- mating barriers .
Form-specific markers .Inter-form hybrids
Introduction
Net form of net blotch (NFNB) caused by Pyrenophora
teres f. teres and spot form of net blotch (SFNB) caused
by P. teres f. maculata are economically important foliar
diseases of barley (Hordeum vulgare L.)globally.These
pathogens are morphologically similar but genetically
distinct and co-exist in the same field (McLean et al.
2009; Liu et al. 2011). They are stubble borne and
reproduce both asexually and sexually. The asexual
stage consists of genetically identical conidia that form
clonal genotypes. The sexual stage produces
pseudothecia which contain asci with ascospores. Two
opposite mating types (MAT1–1and MAT1–2) are re-
quired for sexual reproduction that will fuse to produce
recombinant genotypes, which increases the genetic
variation of the offspring relative to the parents
(McDonald 1963;McLeanetal.2010; Liu et al.
2011). Sexual reproduction within each form (i.e. Ptt x
Ptt and Ptm xPtm) has been frequently reported in the
field across the world (Rau et al. 2003; Serenius et al.
2007;Lehmensieketal.2010).
Sexual reproduction between Ptt andPtm is induc-
ible in the laboratory (Smedegård-Petersen 1971;
Eur J Plant Pathol
https://doi.org/10.1007/s10658-018-1574-9
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10658-018-1574-9) contains
supplementary material, which is available to authorized users.
B. Poudel :M. W. Sutherland :A. Martin (*)
Centre for Crop Health, University of Southern Queensland, West
Street, Toowoomba, QLD 4350, Australia
e-mail: Anke.Martin@usq.edu.au
M. S. McLean
Agriculture Victoria, Horsham, VIC 3401, Australia
G. J. Platz :J. A. McIlroy
Department of Agriculture and Fisheries, Hermitage Research
Facility, Warwick, QLD 4370, Australia
Campbell et al. 1999;Jalli2011). The resulting inter-
form hybrids have unique virulence patterns compared
to the parental isolates, with some hybrids being
highly virulent (Jalli 2011). In the laboratory, hy-
brids retain their virulence, fertility and genetic
stability over time (Campbell and Crous 2003).
This implies that if stable hybridisations between
the two forms occurs in the field, the resulting
hybrids could potentially overcome host resistance.
Several international phylogenetic studies have
shown that Ptt xPtm hybrids are rare or absent in the
field (Rau et al. 2003; Serenius et al. 2005; Bakonyi and
Justesen 2007; Akhavan et al. 2015). Based on the
divergence of the mating type genes (Rau et al. 2007)
and orthologous intergenic regions (Ellwood et al.
2012), the two forms of P. teres were suggested to be
genetically isolated and should be treated separately
when studying pathogen virulence and hosts. Neverthe-
less, the existence of Ptt xPtm hybrids in the field has
been reported in a study conducted in South Africa and
in the Czech Republic, where three isolates shared
unique Ptt and Ptm alleles (Campbell et al. 2002;
Leisova et al. 2005). In a recent study conducted in
Australia, one hybrid (WAC10721) was identified
among 60 Ptm isolates (McLean et al. 2014).
The occasional occurrence of hybridisation in the
field is of concern as it might be sufficient to introduce
new pathotypes into field populations. The recently-
identified hybrid from an Australian field suggests that
hybridisation could occur under field conditions but are
not reported frequently due to the infrequent use of
genetic markers or small samples sizes being evaluated
by molecular analysis. During regular disease diagnosis,
Ptt and Ptm pathogens are identified by the symptoms
they induce on barley leaves, but symptoms of hybrids
can resemble those of either of the parents, such that
hybrids fail to be detected by visual inspection of infect-
ed plants. Furthermore, in the studies where molecular
markers have been used to determine the presence of
hybrids, only low sample numbers were evaluated
(Serenius et al. 2007;Lehmensieketal.2010; McLean
et al. 2014). If hybrids occur at a low frequency, quite
large numbers of isolates need to be sampled to identify
hybrids, especially at sites where both Ptt and Ptm are
found to be present.
This study aimed to detect the occurrence and esti-
mate the frequency of hybridisation between Ptt and
Ptm in the field. For this purpose, field experiments
were established across three successive years at three
sites in Australia to facilitate hybridisation. In addition,
molecular characterisation was performed using DNA
of P. teres isolates collected during 1976–2015 from
different barley growing regions in Australia.
Methods
Fieldexperimentlocations
Three field experiments were established to investigate
the occurrence of Ptt xPtm hybrids in the field: two sites
were located at the Hermitage Research Facility (HRF;
28
o
12’40.0^S150
o
06’06.0″E) Queensland Department
of Agriculture and Fisheries, near Warwick, Queensland
and one at Longerenong Agricultural College (36°40′
23.0^S142°17′37.3″E), Agriculture Victoria, near
Horsham, Victoria. Monthly temperature and rainfall
data for Hermitage and Horsham were obtained from
nearby Australian Bureau of Meteorology stations, 2016
(Supplementary Table 1). The maximum average daily
temperature between April–November ranged from 17
to 32 °C and 13 to 29 °C and the minimum average daily
temperature ranged from 0 to 16 °C and 1 to 13 °C, at
Hermitage and Horsham, respectively. The total rainfall
measured each year throughout the growing season was
in the range of 165–515 mm at Hermitage and 155–
390 mm at Horsham.
Hermitage research facility site 1 The experiment was
conducted in 2013, 2014 and 2015. A field area of
0.05 ha containing a black Vertosol soil was mainly
rain-fed except for May and July 2013 when the field
was irrigated for 2 h/day for four consecutive days at a
rate of 4 mm/h. The land was fertilised using 140 to
170 kg/ha urea prior to sowing. Weeds were managed
by using Hotshot (0.7 l/ha), Roundup CT® (2 l/ha),
Starane (0.7 l/ha) or 2-methyl-4-chlorophenoxyacetic
acid (MCPA; 0.6 l/ha) as required.
Each year, barley cultivar Henley (susceptible to both
Ptt and Ptm) was planted at a rate of 60 kg/ha during
June in 2013 and July in 2014 and 2015. The site was
inoculated with straw (barley cv. Shepherd, susceptible
to both Ptt and Ptm) that was infected with Ptt isolate
NB050 (MAT 1–1)andPtm isolate SNB320 (MAT 1–2)
4–6 weeks after sowing. For inoculum used to produce a
source of infested straw, isolate NB050 and SNB320
were placed in separate Potato Dextrose Agar (PDA)
plates for five days under 12 h light at 19 °C. Five plugs
Eur J Plant Pathol
of mycelium were suspended in 100 ml of Potato Dex-
trose Broth which was shaken for 4 days to increase
mycelium growth. The mycelium collected from 37
bottles of each isolate was blended together and a total
volume of 8.5 l of inoculum was sprayed onto the barley
cv. Shepherd via knapsack sprayer in a glass house.
Infected stubble was retained during summer to facili-
tate infection in the following season.
Hermitage research facility site 2 The experiment was
conducted in 2014, 2015 and 2016. The field comprised
a 0.3 ha area, contained black Vertosol soil and was rain-
fed. Urea was applied at 140 to 150 kg/ha prior to
sowing and Roundup CT® (2 l /ha), Gran Am
(160 kg/ha), Starane Advanced (~0.7 l /ha) and MCPA
(0.6 l/ha) were applied for weed management.
The site was planted with a 50:50 mix of Ptt and Ptm
susceptible barley cvs Grimmett and Oxford at 60 kg/ha
each year. In the first-year field experiment, seeds were
planted in June 2014. In August, irrigation was applied
for an hour prior to inoculation. The field was inoculated
with mycelium of Ptt isolate NB053 (MAT1–2)andPtm
isolate SNB74 (MAT1–1). Inoculum was produced in
the same method as described above and sprayed in five
different spots within the field via a knapsack sprayer.
The inoculated areas were covered with large plastic
tubs (1.8 m × 1.2 m) for 12 h to create a moist environ-
ment. For the second and third year, seeds were planted
in July and the infected straw from the previous year
was applied as the source of the inoculum. The field was
left uncultivated during the summer season.
Longerenong site The experiment was conducted in
2013 and 2014. A field area of 0.14 ha area had grey
Vertosol soil and was rain-fed. The site was fertilised
with 100 kg/ha Urea and 70 kg/ha Mono ammonium
phosphate at sowing. Weeds were managed by using
Glyphosate (450 g/L), triallate (500 g/ l), liquid hydro-
carbon (471 g/ l), prosulfocarb (800 g/ l), S-metolachlor
(120 g/ l), pinoxaden (100 g/ l), cloquintocet-mexyl
(25 g/ l), MCPA (280 g/ l), bromoxynil (140 g/ l),
dicamba (40 g/ l), iodosulfuron-methyl-sodium (50 g/
Kg), trifluralin (480 g/l) as required.
Each year, barley cv. Bass (susceptible to both forms)
was planted at 60 kg/ha in June 2013 and Ju-
ly 2014. Stubble residue naturally infected with
Ptt and Ptm was spread over the area 4–6weeks
after sowing to generate infection. The field was
left uncultivated during summer.
Collection of leaf samples and stubble
Leaf samples were collected arbitrarily throughout the
fields during September to November each year at all
sites. First year samples were collected to confirm the
presence of the original parental isolates in the field and
second and third year samples were used to investigate
the presence of hybrids. In the second year, stubble
samples were collected during October and November
from all the three sites. In addition, stubble samples
were collected from the HRF experiment at Sites 1 and
2inJuly2015and2016,respectively.
From the HRF and Longerenong field sites, 1045
P. teres isolates were collected from infected barley leaf
samples during 2013 to 2016. These included 285 co-
nidia collected from HRF experiment site 1, 403 conidia
collected from HRF experiment site 2, and 134 conidia
collected during 2013 and 2014 from Longerenong. We
also obtained six ascospores from pseudothecia on a
piece of stubble collected from HRF site 1 and 217
ascospores obtained from pseudothecia on a single piece
of stubble collected from HRF site 2.
Collection of conidia from leaf samples
Infected leaf samples were cut into 3-cm pieces and
surface sterilised in 70% ethanol for 10s, followed by
5% bleach for 30 s and rinsed thrice with distilled water.
The samples were placed on moist filter paper inside
petri plates and kept on a window sill under natural light
conditions at approximately 22 °C. Conidia emerged
within 2–7 days (observed through a dissecting micro-
scope) and single conidia were transferred to PDA plates
using a glass needle.
Collection of ascospores from stubble
Stems containing mature pseudothecia were cut longi-
tudinally into two halves and were soaked in sterile
water for 2 h. To collect ascospores, the stem was fixed
to the lid of a petri plate using Vaseline White Petroleum
Jelly and the lid was placed on top of a plate containing
2% water agar. The plate was incubated at 15 °C with a
12 h light: 12 h dark photoperiod until the ascospores
were ejected onto the water agar plate. Plates were
checked daily up to 5 days and single ascospores were
transferred to a PDA plate with a glass needle.
Eur J Plant Pathol
Collection of Pyrenophora teres cultures
from Australian barley growing regions
Three hundred and nineteen freeze-dried cultures or leaf
samples of Ptt and Ptm were sourced from long-term
storage at Agriculture Victoria and HRF. These were
collected from different barley growing regions of Aus-
tralia during 1976 and 2014. This set also included
isolates obtained from 25 infected leaf samples collected
in 2015 in Western Australia. Isolates are listed in Fig. 1
and Supplementary Table 2.
DNA extraction
Mycelium was harvested after growing single-spore
derived cultures for 10 days on PDA at 25 °C in the
dark. DNA extraction was carried out using the Wizard
Genomic DNA Extraction Kit (Promega Corporation)
as per the manufacturer’s instruction. Extracted DNA
was quantified using an Implen NanoPhotometer (Inte-
grated Sciences).
Identification of hybrids
Twelve primer pairs, six specific to Ptt (PttQ1, PttQ2,
PttQ3, PttQ4, PttQ5, and PttQ6 and six to Ptm (PtmQ7,
PtmQ8, PtmQ9, PtmQ10, PtmQ11, and PtmQ12) were
used (Poudel et al. 2017). These markers can distinguish
between the two form of P. teres and their hybrids.
Hybrids can be identified based on the presence of at
least one Ptt and one Ptm specific marker. All 12
markers were amplified across each of the samples.
PCR amplification was carried out as described earlier
by Poudel et al. (2017).
Mating type markers
The DNA of isolates collected from field experiment
sites was amplified using the primer sequences of mat-
ing type markers MAT1–1and MAT1–2for Ptt and Ptm
(Lu et al. 2010) to determine the distribution of mating
types of Ptt and Ptm within the field. The PCR reaction
was carried out with few modifications. The reaction
mixture consisted of 1× buffer, 1.5 mM MgCl
2
,100μM
of each dNTP, 0.5 U GoTaq® FlexiDNA Polymerase
(Promega Corporation) with 5 μMofeachprimerand
20 ng of DNA in a total volume of 10 μL. The poly-
merase chain reaction (PCR) cycle was carried out for
7 min at 95 °C, followed by 35 cycles at 94 °C for the
30 s, 55 °C for 30 s and 72 °C for 30 s and one final
cycle at 72 °C for 10 min. PCR products were visualised
on a 1% agarose gel electrophoresis following staining
Fig. 1 Location of Pyrenophora
teres collections from Australian
barley growing regions from
1976 to 2015. Base layer of the
Australian map obtained from
Naturalearth.com
Eur J Plant Pathol
with Ethidium bromide. Chi-square tests were conduct-
ed to determine if the observed mating type ratio for
each of the populations of P. t e r e s from each year
departed significantly from the null hypothesis of a 1:1
mating type ratio. A 0.05 Type I error rate was applied to
accept or reject the null hypothesis of a statistically
equal mating type ratio of 1:1. Chi-square test was
performed using R software (v 3.4.0). Chi-square tests
were not conducted on populations with sample sizes of
less than 10 because of a lack of statistical power.
Results
Molecular characterisation of isolates from Hermitage
research facilities site 1
In total, 285 isolates were collected during the three
years of field experiments (Table 1). In the first year of
the field experiment, form-specific markers identified
23 Ptt and 2 Ptm isolates. All six Ptt-specific markers
were present in the Ptt isolates and none of the Ptm-
specific markers amplified on these samples, whereas
only the six Ptm-specific markers were present in the
Ptm isolates. Of 23 Ptt isolates collected, 10 had MAT1–
1and 13 had MAT1–2 loci while the two Ptm isolates
had MAT1–2loci(Table1). The chi-square test indicat-
ed a significant departure from the 1:1 mating type ratio
for years 2014 and 2015 of the Ptt population (Table 1).
Of the 80 isolates sampled in the second year, all
were Ptt isolates. Forty-nine of the isolates had the
PttMAT1–1mating type and 31 had PttMAT1–2.After
sowing in 2015, stubble infected with SNB320 was
dispersed into the field to increase the Ptm inoculum.
However, only 13 isolates of a total of 180 isolates
collected from this site were Ptm isolates. Among the
180 isolates screened, mating type markers identified
102 as PttMAT1–1,65asPttMAT1–2,5asPtmMAT1–1
and8asPtmMAT1–2. None of the collected isolates
amplified bands with both the Ptt and Ptm-specififc
markers and thus none of the isolates were hybrids.
Stubble collected in second year during November
did not have mature pseudothecia and thus ascospores
could not be obtained. From the third year (i.e. Ju-
ly 2015) stubble collection, six ascospores were collect-
ed that identified as Ptt isolates using the form-specific
markers. Mating type markers analysis indicated that
two ascospores had the PttMAT1–1locus and four had
the PttMAT1–2locus.
Molecular characterisation of isolates from Hermitage
research facilities site 2
The HRF site 2 was inoculated with mycelium of Ptt
isolate NB053 (MAT1–2)andPtm isolate SNB74
(MAT1–1). These isolates were chosen as they had
previously produced hybrids when crossed in vitro and
some of those hybrids were virulent across a number of
Tabl e 1 Distribution of mating type loci in Pyrenophora teres f. teres and P.teres f. maculata isolates. Isolates were obtained from leaf
samples collected at the Hermitage Reseach Facility (HRF) and Longerenong Agriculture College field sites from 2013 to 2016
Experiment site Year Pyrenophora teres f. teres Pyrenophora teres f. maculata
MAT1–1MAT1–2To tal χ2P MAT1–1MAT1–2Total χ2P
HRF Site 1 2013 10 13 23 0.391 0.531 0 2 2 ––
2014 49 31 80 4.050 0.044* 0 0 0 ––
2015 102 65 167 8.198 0.004* 5 8 13 0.692 0.405
All Years 161 109 270 5 10 15
HRF Site 2 2014 12 13 25 0.040 0.841 6 3 9 ––
2015 98 55 153 12.085 0.001* 24 22 46 0.086 0.768
2016 87 68 155 2.329 0.127 8 7 15 0.066 0.796
All Years 197 136 333 38 32 70
Longerenong 2013 14 11 25 0.360 0.549 12 4 16 4.000 0.046*
2014 5 3 8 –– 46 39 85 0.576 0.447
All Years 19 14 33 58 43 101
*Denotes mating-type frequencies that are significantly different from a 1:1 ratio at p=0.05
Eur J Plant Pathol
barley genotypes tested (ElMor 2016). During the three
years of field trials, 403 single conidia isolates were
obtained (Table 1). In the first year, 25 Ptt and 9 Ptm
isolates were identified (Table 1) of which 12 had the
PttMAT1–1,13hadthePttMAT1–2, six had the
PtmMAT1–1, and three PtmMAT1–2loci, respectively.
A significant departure from the 1:1 mating type ratio
was observed for the Ptt population in 2015 (Table 1).
In the second year, 153 Ptt and 46 Ptm isolates were
identified. In the third year, 155 isolates were Ptt while
15 were Ptm isolates. MAT1–1and MAT1–2loci of both
Ptt and Ptm forms were identified in the second and
third year of the field experiment (Table 1). There were
no hybrids in the collection.
Stubble collected in second year (i.e. November
2014) contained immature pseudothecia and produced
no ascospores. Of the stubble collected in third year (i.e.
July 2016), 217 ascospores were obtained. Genotyping
of these ascospores using the form-specific makers in-
dicated that they were all Ptm isolates. Of the 217 Ptm
ascospores collected, 119 and 98 had the PtmMAT1–1
and PtmMAT1–2loci, respectively.
Molecular characterisation of isolates
from Longerenong field site
In total, 134 isolates were collected. In the first year
proportions of Ptt and Ptm isolates were similar and a
significant departure from 1:1 mating type ratio was
observed (Table 1). In the second year, eight Ptt isolates
and 85 Ptm isolates were collected. Both MAT1–1and
MAT1–
2were found equally proportioned (Table 1).
There were no hybrids in the collection.
Stubble collected in second year (i.e October
2014) did not have mature pseudothecia and pro-
ducednoascospores.
Molecular characterisation of Australian P. teres
Of 317 isolatescollected from barley growing regions in
Australia, the markers confirmed 110 isolates as Ptt and
207 isolates as Ptm (Supplementary Table 2). Four
isolates (Ptt13–170, Ptt13–174, Ptt13–178 and WA18)
previously identified as Ptt according to lesion pheno-
type were identified as Ptm using the form-specific
markers.Four isolates previously identified as Ptm
(Ptm13–226, WA19, WA20 and WA35) were identified
as Ptt. No hybrids were identified in this set.
Discussion
This is the first field-based study designed to specifically
investigate sexual hybridisation between Ptt and Ptm.
Form-specific markers previously designed by our
group (Poudel et al. 2017)wereusedtoidentifyhybrids.
The six Ptt-specific markers were present in all Ptt
isolates and all six Ptm-specific markers were present
in all of the Ptm isolates. The Ptt-specific markers did
not amplify on the Ptt isolates and vice versa. None of
the isolates had bands of both form-specific makers.
Thus our results identified no hybrids despite establish-
ing ideal conditions for it to occur. Isolates other than
those used in the inoculation of the field trials were also
present in the field. This presented the opportunity for
both within and between form hybridisation to occur at
these sites. Results from this study indicate that sexual
hybridisation within the two forms was far more likely
than hybridisation between the two forms. As similar
ratios of both mating types within the forms are normal-
ly present in a field situation (Rau et al. 2005; Serenius
et al. 2005;McLeanetal.2014), sexual preference for
within form recombination would greatly reduce the
likelihood of hybridisation between the two forms.
While a few studies have reported sexual
hybridisation between the two forms of P. teres under
field conditions (Campbell et al. 2002; Leisova et al.
2005;McLeanetal.2014), others have indicated that
hybridisations are rare or absent (Rau et al. 2003;
Bakonyi and Justesen 2007; Serenius et al. 2007;
Akhavan et al. 2015). In our study, no hybrids were
identified in the samples collected from the field exper-
iment sites. In addition, 317 isolates collected from
barley growing regions in Australia during 1976–2016
underwent molecular analysis to ensure that no hybrids
were overlooked due to lesion appearance being the
same as those of the parents. The marker results mostly
concurred with phenotypic identifications, except for
eight isolates in which the molecular analysis suggested
that they had been misidentified when originally col-
lected and catalogued. None of the isolates analysed
were hybrids. Our results confirm that hybridisation
between the two forms of P. teres is rare under field
conditions and supports phylogenetic evidence for ge-
netic isolation between the two forms (Rau et al. 2007;
Ellwood et al. 2012).
The field assessments suggest that Ptt and Ptm are
reproductively isolated. Reproductive isolation provides
a barrier to genetic exchange between two divergent
Eur J Plant Pathol
populations (Giraud et al. 2008). The factors restricting
the hybridisation between two forms of P. teres under
field conditions have not been identified. The reproduc-
tive isolation inferred through this study could arise due
to pre- and post-mating barriers. Pre-mating reproduc-
tive barriers such as sexual selection and temporal dif-
ferences prevent the mating of two individuals while
post-mating reproductive barriers occur due to gametic
incompatibility causing unfit or non-viable hybrids
(Kohn 2005; Giraud et al. 2008) or as suggested by
Serenius et al. (2005) unsuccessful meiosis in crosses
of the two forms.
Sexual selection arises from competition or mate
preference that can lead to differential mating success
among individuals (Giraud et al. 2008). In our study, the
field was inoculated with only one Ptt and one Ptm
isolate and recombinant offspring within the same form
of P. teres were observed. Stubble collected from the
HRF field experiment sites 1 and 2, produced asci with
mature ascospores. Form-specific markers identified
these ascospores as Ptt or Ptm and mating type markers
confirmed that both MAT1–1or MAT1–2loci were
present, indicating that the collected ascospores were
recombinants of Ptt xPtt (HRF site 1), and Ptm xPtm
(HRF site 2). Recombinants from both sites were
derived either from crosses between inoculated isolates
and those dispersed from nearby fields or from crosses
between immigrants. The migrated isolates were con-
firmed when mating type markers from opposite mating
types of Ptt and Ptm isolates were identified in the first-
year field experiments. This indicated that hybridisation
within the same forms of isolates occurs readily under
field conditions. Moreover, Ptt and Ptm isolates mate
with the compatible mating type of the same form rather
than between the two forms. This could occur due to
pheromone production induced during the mating re-
sponse. Although the theory has not been established in
P. teres isolates, experiments conducted in Saccharomy-
ces cerevisiae have shown that preferences occur for the
partner that produces the highest level of pheromone
(Jackson and Hartwell 1990). To observe competition in
mating within and between forms and to understand
sexual selection and the mechanism involved, competi-
tion mating assays involving individuals of both Ptt and
Ptm of opposite mating types need to be conducted
under laboratory conditions and further investigated.
The other pre-mating factor limiting inter-form
hybridisation is temporal isolation, which occurs when
there is a difference in reproduction time (Giraud et al.
2008). Under laboratory conditions, hybridisation oc-
curs at 15 °C under 12 h day/night conditions. This
condition is maintained until hybrids are produced. In
the field, climatic conditions fluctuate. At our field sites,
the temperature recorded was 0–32 °C during the barley
growing seasons and moisture levels varied depending
on rainfall during that year. This may influence the life
cycle of Ptt and Ptmsuch that no overlapping reproduc-
tive cycle may occur between Ptt and Ptm, causing a
premating barrier for hybridisation between the two
forms. However, this does not seem to be the case in
our study as stubble samples collected in July from both
HRF field sites had mature pseudothecia which pro-
duced ascospores of each form of P. teres. This suggests
that sexual reproduction within each form had occurred
at a similar time.
Post-mating barriers could occur due to genetic in-
compatibilities between the two parental genomes, lead-
ing to unequal crossing over during meiosis causing loss
or gain of key genes (Kohn 2005; Stukenbrock 2013),
which may lead to unfit or inviable hybrids. However,
studies have shown that laboratory produced P. teres
hybrids retain their pathogenicity, fertility and genetic
stability over time (Campbell and Crous 2003; Jalli
2011). Two isolates, NB053 and SNB74S inoculated
at HRF site 2 were shown to have produced hybrid
progeny in vitro and the resulting hybrids had infected
most of the barley genotypes tested under glasshouse
conditions (ElMor 2016). This indicates that there is no
genetic incompatibility between the two forms and that
the hybrids are fit enough to induce infection and to be
virulent on some barley genotypes. Although hybrids
are viable and fertile under in vitro conditions, extrinsic
factors may be responsible for post-mating isolation
(Kohn 2005). Intermediate traits of hybrids could have
reduced fitness and may be out-competed by their par-
ents, when co-existing under the same environmental
conditions. Comparative whole genome sequence anal-
yses would help to identify allele combinations contrib-
uting to fitness in hybrids.
In conclusion, this was a unique study that facilitated
the potential formation of Ptt xPtm hybrids across three
successive years. Our results suggest that sexual repro-
duction between Ptt and Ptm is rare under field condi-
tions in Australian barley growing regions, while sexual
reproduction within forms of P. teres occurs frequently.
Sexual preference for the same form or low hybrid
fitness in parental habitat could contribute to reproduc-
tive isolation between the two forms of P. teres. Further
Eur J Plant Pathol
investigations are needed to provide insights into mech-
anisms conferring reproductive isolation. Although re-
productive barriers exist, these are occasionally
breached in the field, allowing formation of Ptt xPtm
hybrids that are fit under particular field conditions.
Therefore, conducting future experiments in confined,
more tightly managed field environments may shed
further light on the conditions which allow hybridisation
between Ptt and Ptm in the field.
Acknowledgements The authors would like to thank Ryan
Fowler (Department of Agriculture and Fisheries, Queensland,
Australia) and Dr. Sanjiv Gupta (Murdoch University, Western
Australia) for the isolates provided by them. The authors would
also like to thank Dr. Adam H. Sparks (Centre for Crop Health,
University of Southern Queensland) for providing the script of
Australian Map in R. This project (DAQ00187) was partly funded
by the Grains Research and Development Corporation, Australia.
Compliance with ethical standards This research does not
contain any research involving humans or animals.
Conflict of interest The authors declare no conflict of interest.
References
Akhavan, A., Turkington, T. K., Kebede, B., Tekauz, A., Kutcher,
H. R., Kirkham, C., et al. (2015). Prevalence of mating type
idiomorphs in Pyrenophora teres f. teres and P. teres f.
maculata populations from the Canadian prairies. Canadian
Journal of Plant Pathology, 37(1), 52–60. https://doi.
org/10.1080/07060661.2014.995710.
Bakonyi, J., & Justesen, A. F. (2007). Genetic relationship of
Pyrenophora graminea,P. teres f.maculataand P. teres f.
teres assessed by RAPD analysis. Journal of Phyto pathology,
155(2), 76–83. https://doi.org/10.1111/j. 143 9-
0434.2007.01192.x.
Campbell, G. F., & Crous, P. W. (2003). Genetic stability of net x
spot hybrid progeny of the barley pathogen Pyrenophora
teres .Australasian Plant Pathology, 32(2), 283–287.
https://doi.org/10.1071/Ap03016.
Campbell, G. F., Crous, P. W., & Lucas, J. A. (1999). Pyrenophora
teres f. maculata, the cause of Pyrenophora leaf spot of
barley in South Africa. Mycological Research, 103(3), 257–
267. https://doi.org/10.1017/S0953756298007114.
Campbell, G. F., Lucas, J. A., & Crous, P. W. (2002). Evidence of
recombination between net- and spot-type populations of
Pyrenophora teres as determined by RAPD analysis.
Mycological Research, 106(5), 602–608. https://doi.
org/10.1017/s0953756202005853.
Ellwood, S.R., Syme, R. A., Moffat, C. S., & Oliver, R. P. (2012).
Evolution of three Pyrenophora cereal pathogens: recent
divergence, speciation and evolution of non-coding DNA.
Fungal Genetics and Biology, 49(10), 825–829.
ElMor, I. (2016). Investigating the virulence of isolates produced
by sexual recombination between different Pyrenophora teres
isolates. Traditional Thesis, University of Southern
Queensland, Australia.
Giraud, T., Refregier, G., Le Gac, M., de Vienne, D. M., & Hood,
M. E. (2008). Speciation in fungi. Fungal Genetics and
Biology, 45(6), 791–802. https://doi.org/10.1016/j.
fgb.2008.02.001.
Jackson, C. L., & Hartwell, L. H. (1990). Courtship in
S. cerevisiae:Both cell types choose mating partners by
responding to the strongest pheromone signal. Cell, 63(5),
1039–1051. https://doi.org/10.1016/0092-8674(90)90507-B.
Jalli, M. (2011). Sexual reproduction and soil tillage effects on
virulence of Pyrenophora teres in Finland. Annals of Applied
Biology, 158(1), 95–105. https://doi.org/10.1111/j.1744-
7348.2010.00445.x.
Kohn, L. M. (2005). Mechanisms of fungal speciation. Annual
Review of Phytopathology, 43,279–308. https://doi.
org/10.1146/annurev.phyto.43.040204.135958.
Lehmensiek, A., Bester-van der Merwe, A. E., Sutherland, M. W.,
Platz, G., Kriel, W. M., Potgieter, G. F., et al. (2010).
Population structure of South African and Australian
Pyrenophora teres isolates. Plant Pathology, 59(3), 504–
515. https://doi.org/10.1111/j.1365-3059.2009.02231.x.
Leisova, L., Minarikova, V., Kucera, L., & Ovesna, J. (2005).
Genetic diversity of Pyrenophora teres isolates as detected
by AFLP analysis. Journal of Phytopathology, 153(10), 569–
578. https://doi.org/10.1111/j.1439-0434.2005.01019.x.
Liu, Z., Ellwood, S. R., Oliver, R. P., & Friesen, T. L. (2011).
Pyrenophora teres: profile of an increasingly damaging bar-
ley pathogen. Molecular Plant Pathology, 12(1), 1–19.
https://doi.org/10.1111/j.1364-3703.2010.00649.x.
Lu,S.,Platz,G.J.,Edwards,M.C.,&Friesen,T.L.(2010).
Mating type locus-specific polymerase chain reaction
markers for differentiation of Pyrenophora teres f. teres and
P. t e r e s f. maculata, the causal agents of barley net blotch.
Phytopathology, 100(12), 1298–1306. https://doi.
org/10.1094/PHYTO-05-10-0135.
McDonald, W. C. (1963). Heterothallism in Pyrenophora teres.
Phytopathology, 53(121), 771–773.
McLean, M. S., Howlett, B. J., & Hollaway, G. J. (2009).
Epidemiology and control of spot form of net blotch
(Pyrenophora teres f. maculata) of barley: a review. Crop
& Pasture Science, 60(4), 303–315. https://doi.org/10.1071
/Cp08173.
McLean, M. S., Keiper, F. J., & Hollaway, G. J. (2010). Genetic
and pathogenic diversity in Pyrenophora teres f. maculata in
barley crops of Victoria, Australia. Australasian Plant
Pathology, 39(4), 319–325. https://doi.org/10.1071
/Ap09097.
McLean,M.S.,Martin,A.,Gupta,S.,Sutherland,M.W.,
Hollaway,G.J.,&Platz,G.J.(2014).Validationofanew
spot form of net blotch differential set and evidence for
hybridisation between the spot and net forms of net blotch
in Australia. Australasian Plant Pathology, 43(3), 223–233.
https://doi.org/10.1007/s13313-014-0285-8.
Poudel, B., Ellwood, S. R., Testa, A. C., McLean, M., Sutherland,
M. W., & Martin, A. (2017). Rare Pyrenophora teres hybrid-
ization events revealed by development of sequence-specific
Eur J Plant Pathol
PCR markers. Phytopathology.https://doi.org/10.1094
/PHYTO-11-16-0396-R.
Rau, D., Brown, A. H. D., Brubaker, C. L., Attene, G., Balmas, V.,
Saba, E., et al. (2003). Population genetic structure of
Pyrenophora teres Drechs. the causal agent of net blotch in
Sardinian landraces of barley (Hordeum vulgare L.).
Theoretical and Applied Genetics, 106(5), 947–959.
https://doi.org/10.1007/s00122-002-1173-0.
Rau, D., Maier, F. J., Papa, R., Brown, A. H. D., Balmas, V., Saba,
E., Schaefer, W., & Attene, G. (2005). Isolation and charac-
terization of the mating-type locus of the barley pathogen
Pyrenophora teres and frequencies of mating-type
idiomorphs within and among fungal populations collected
from barley landraces. Genome, 48(5), 855–869.
Rau, D., Attene, G., Brown, A. H., Nanni, L., Maier, F. J., Balmas,
V., et al. (2007). Phylogeny and evolution of mating-type
genes from Pyrenophora teres, the causal agent of barley "net
blotch" disease. Current Genetics, 51(6), 377–392.
https://doi.org/10.1007/s00294-007-0126-1.
Serenius, M., Mironenko, N., & Manninen, O. (2005). Genetic
variation, occurrence of mating types and different forms of
Pyrenophora teres causing net blotch of barley in Finland.
Mycological Research, 109(7), 809–817. https://doi.
org/10.1017/s0953756205002856.
Serenius, M., Manninen, O., Wallwork, H., & Williams, K.
(2007). Genetic differentiation in Pyrenophora teres popula-
tions measured with AFLP markers. Mycological Research,
111(2), 213–223. https://doi.org/10.1016/j.
mycres.2006.11.009.
Smedegård-Petersen, V. (1971). Pyrenophora teres f. maculata f.
nov. and Pyrenophora teres f. teres on barley in Denmark. In
Yearbook of the Royal Veterinary and Agricultural University
(pp. 124-144). Copenhagen.
Stukenbrock, E. H. (2013). Evolution, selection and isolation: a
genomic view of speciation in fungal plant pathogens. New
Phytologist, 199(4), 895–907. http s://doi.org/10.1111
/nph.12374.
Eur J Plant Pathol
