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Genetic structure of Pyrenophora teres net and spot populations as revealed by microsatellite analysis A-SVOBODOV a,*, Vera MINAR IKOVA b, Leona LEISOV A b c, Pavel MATUSINSKY , Martina HUDCOVICOVA c c, Jozef GUBIS Katarına ONDREICKOV A 507, Prague 6 e Ruzyne 161 06, Department of Molecular Biology, Crop Research Institute Prague, Drnovska Czech Republic b Agrotest fyto, ltd., Havlıckova 2787, Kromerız 767 07, Czech Republic c cesta 122, Piestany 921 68, Slovak Republic Plant Production Research Centre Piestany, Bratislavska a
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abstract
Article history:
The population structure of the fungal pathogen Pyrenophora teres, collected mainly from
Received 17 July 2013
different regions of the Czech and Slovak Republics, was examined using a microsatellite
Received in revised form 8 November 2013
analyses (SSR). Among 305 P. teres f. teres (PTT) and 82 P. teres f. maculata (PTM) isolates ^ ¼ 0.12 and h ^ ¼ 0.13, respecthat were collected, the overall gene diversity was similar (h
Accepted 18 November 2013
tively). A high level of genetic differentiation (FST ¼ 0.46; P < 0.001) indicated the existence
Available online 3 December 2013
of population structure. Nine clusters that were found using a Bayesian approach represent
Corresponding Editor:
the genetic structure of the studied P. teres populations. Two clusters consisted of PTM pop-
Brenda Diana Wingfield
ulations; PTT populations formed another seven clusters. An exact test of population differentiation confirmed the results that were generated by Structure. There was no
Keywords:
difference between naturally infected populations over time, and genetic distance did
Drechslera teres
not correlate with geographical distance. The facts that all individuals had unique multilo-
Hordeum vulgare
cus genotypes and that the hypothesis of random mating could not be rejected in several
PTM
populations or subpopulations serve as evidence that a mixed mating system plays a role
PTT
in the P. teres life cycle. Despite the fact that the genetic differentiation value between PTT
SSR
and PTM (FST ¼ 0.30; P < 0.001) is lower than it is between the populations within each form (FST ¼ 0.40 (PTT); FST ¼ 0.35 (PTM); P < 0.001) and that individuals with mixed PTT and PTM genomes were found, the two forms of P. teres form genetically separate populations. Therefore, it can be assumed that these populations have most likely undergone speciation. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Pyrenophora teres Drechs. (anamorph Drechslera teres (Sacc.) Shoem.) is a heterothallic ascomycete that causes foliar disease in barley and is common throughout the major barleygrowing regions of the world. Under favourable conditions,
this fungus may become a serious threat to grain yield and malt quality. Yield loss can reach 40 %, as described by Steffenson et al. (1991). The fungus occurs in two forms: P. teres f. teres (PTT) and P. teres f. maculata (PTM), which produce different leaf symptoms (Smedeg ard-Petersen 1971). Despite the difficulty in
* Corresponding author. Tel.: þ4220 702 087 672; Fax: þ420 233 311 591. -Svobodova ). E-mail address:
[email protected] (L. Leisova 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.11.008
Genetic structure of PTT and PTM populations
distinguishing between the two forms morphologically in culture, they differ genetically on DNA level detected using molecular markers and a next generation sequencing approach so markedly that they have been proposed to be distinct species (Rau et al. 2007; Serenius et al. 2007; Ellwood et al. 2012). Regardless of their classifications, it has been suggested that limited gene flow may occur between the two forms (Campbell et al. 2002; Leisova et al. 2005a). The PTT has been regarded as the more prevalent, but recently, there have been reports of epidemics caused by PTM, particularly in Australia and Canada (Tekauz 1990; McLean et al. 2010). In the Czech Republic, both forms occur (Leisova et al. 2005a). Winter barley is infected at a higher level by a spot form of the pathogen (PTM), while spring barley is more sensitive to the classical net form (PTT) (Minarikova & Polisenska 1999). PTT and PTM can hybridise in the laboratory (Smedeg ardPetersen 1971; Campbell et al. 1999). The hybrid progeny were found to be genetically stable after cycles of inoculation, re-isolation and backcrossing, but not in all laboratories (Smedeg ard-Petersen 1977; Campbell & Crous 2003; Serenius et al. 2005; Mironenko & Afanasenko 2011). In the nature, PTTPTM hybrids occurrence has not been proven yet. Moreover, phylogenetic studies based on the sequence of intergenic regions have suggested that the two forms of P. teres diverged in the middle Pleistocene. Therefore, they are genetically isolated and should be treated separately when studying pathogen virulence and host resistance (Ellwood et al. 2012). Pyrenophora teres has a mixed reproductive system, with one generation of sexual reproduction occurring on the infected barley debris remaining on the surface after harvest. Pyrenophora teres has a heterothallic nature (McDonald 1963). Therefore, successful mating requires strains with opposite mating types (Rau et al. 2005), as well as specific environmental conditions (Shipton et al. 1973). After initial colonisation, the fungus produces a large number of conidia, which are dispersed by strong wind or rain to other plants and new barley fields. During the growing season, several secondary cycles can occur, causing high disease severity on susceptible plants if environmental conditions are favourable (Shipton et al. 1973). No significant life cycle differences have been found between PTT and PTM (McLean et al. 2009). The P. teres population structure is affected largely by the relative importance of the two stages in the fungus life cycle and by the potential gene flow. Several papers have been published evaluating the variability of P. teres populations using the molecular biological methods RAPD, AFLP, mating type genes, and SSR (Peever & Milgroom 1994; Peltonen et al. 1996; Jonsson et al. 2000; Campbell et al. 2002; Rau et al. 2003; Serenius et al. 2007; Bogacki et al. 2010). Most of these studies have demonstrated a high level of variability within the fungus populations, even on a small scale in terms of sampling area, compared with other fungi (Campbell et al. 2002). While the genetic differentiation was found to be low among those fields that were located close to each other (e.g., within 20 km) (Peever & Milgroom 1994; Jonsson et al. 2000; Serenius et al. 2007), the differentiation among populations that were separated by longer distances was higher, indicating limited gene flow (Serenius et al. 2007). All studies have shown that PTT and PTM isolates are separated into two genetically
181
divergent groups and that the genetic variability is slightly higher among the PTT population (Rau et al. 2003; Serenius et al. 2005). Microsatellites or simple-sequence repeats (SSR) consist of a single sequence motif no more than six bases long that is tandemly repeated. They have been developed into one of the most popular classes of genetic markers due to their high reproducibility, multiallelic nature, codominant mode of inheritance, abundance and wide genome coverage. They have been detected within the genomes of every organism € tterer 1999). The abundance of repetitive (Goldstein & Schlo DNA found in the fungus P. teres has been used to develop microsatellite markers (Keiper et al. 2007) that have already been used in P. teres population studies (Bogacki et al. 2010) and for genetic linkage mapping (Ellwood et al. 2010). As both forms of P. teres occur commonly in the Czech Republic, the aim of this study was to evaluate the genetic structure both within and among several PTT and PTM populations based on microsatellite analyses. The second objective was to assess the prevalence of sexual reproduction in field populations, even between the two forms of P. teres. For this purpose, special attention was given a) to isolates from the Tovacov region, where two intermediate isolates had been found (Leisova et al. 2005a), and b) to isolates from an artificially inoculated field assay.
Material and methods Fungal isolates A total of 508 isolates of Pyrenophora teres were collected from several localities within the Czech Republic, the Slovak Republic, Canada, Germany, Norway, and Hungary. Additionally, three isolates of Pyrenophora graminea, three isolates of Pyrenophora tritici-repentis, and two isolates of Pyrenophora flavispora were added into the collection. In total, 516 isolates were analysed (Table 1). For more detailed study, a set of 387 P. teres isolates from several localities was chosen (Table 2). In this study, we considered a population to be a group of PTT or PTM isolates that were collected from a plot or a field (mostly from the same barley variety) in the same year. Typically, only populations with more than five PTT or PTM isolates were chosen. In total, 34 populations were analysed, focussing on two regions from which 12 and 10 populations were taken. The first region was Tovacov, where two isolates were hypothesised to be hybrids between PTT and PTM. Tovacov is located in the central part of Moravia within the main region for malting barley growth. Both forms of P. teres occur regularly in the same field there, even in the same lesion. The second region was Prague, where a field trial was performed. Two winter barley varieties (Okal and Merlot) and four spring barley varieties (Forum, Heris, Beate, and Prestige) were grown in 3 years on the same field in plots of 4 m2. In May 2006, the plants in all of the plots were inoculated with a mixture of three isolates of P. teres: H602 (PTT from Drnholec 1998, MAT 1), H605 (PTT from Bodorova 1986, MAT 1) and H615 (PTM from Luzany 2005, MAT 2). In autumn 2006, harvested infectious straw was placed on the plots with sowed winter barley. This
-Svobodova et al. L. Leisova
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Table 1 e P teres isolates used in this study. Region
Localities
Years of collections
Na
PTTb
PTMc
nPd
%Pe
Experimental field, Kompost Tovacov, Lobodice, U dubu, U Moravy, U prejezdu, U sterkovny, Za prejezdem, Troubky, Zarici Luzany Velky Beranov, Zhor
2007, 2008 2002, 2005, 2006, 2009, 2011
139 155
102 98
37 57
184 211
67 77
2004, 2005 2001,
33 15
8 12
25 3
145 125
53 45
2007 2006
23 28
16 27
7 1
129 138
47 50
2002, 2008, 2010 2001, 2002, 2010 1996, 1999, 2000, 2002, 2003
23 17 27
18 12 13
5 5 14
124 124 147
45 45 53
2002, 2001, 1986, 2010 2000,
11 6 18
6 4 15
5 2 3
92 93 139
33 34 50
13 508
3 339
10 169
125 266
45 96
Name of the region
1 2
Prague Tovacov region
3 4
West of Bohemia Vysocina region
5 6
South of Bohemia Slovak Republic
7 8 9
Nord Bohemia Central Bohemia Olomouc region
10 11 12
South Moravia Nord Moravia Kromeriz region
Libejovice, Tabor Bodorova, Borovce, Maly Saris, Pstrusa, Sladkovicovo Chrastava, Zatec Stupice, Caslav Domaninek, Hradec nad Svitavou, Lostice, Cervenka Chrlice, Strelice, Drnholec Puste Jakartice, Rymarov Kromeriz, Rataje, Uhersky Ostroh
13
Other countries
Canada, Hungary, Germany, Norway
a b c d e
2001, 1989, 2003, 1996, 1986,
2002, 2003, 1997, 1999, 2004 2002, 2003, 2002, 2004,
2003 2002 1989, 2000, 2002, 2005, 2001, 2006
n: number of isolates. Number of Pyrenophora teres f. teres isolates. Number of Pyrenophora teres f. maculata isolates. nP: number of polymorphic loci. %P: percentage of polymorphic loci.
placement was repeated in autumn 2007, during which the barley sowing seed from a previous harvest was used. In 2007 and 2008, symptomatic leaves were collected and kept in paper bags. All of the isolates were derived from single conidia taken from leaf tissue. The leaves were surface-sterilised, placed in a wet chamber for 48 h and incubated at 20e23 C. Individual spores were then isolated using a sterile needle under a binocular magnifying glass, transferred onto potato dextrose agar (PDA) in Petri dishes and incubated at room temperature for a week. The fungal mycelia for the DNA extraction were produced by cultivating colonies on cellophane membranes that were placed directly on PDA.
DNA extraction DNA was extracted from mycelia according to an optimised protocol using extraction buffer (0.35 M sorbitol, 0.1 M Tris, 5 mM EDTA, pH 7.5), lysis buffer (2 M NaCl, 0.2 M Tris, 50 mM EDTA, 2 % CTAB, pH 7.5) and a 5 % solution of CTAB
in a high concentration of NaCl (min. 0.5 M). The DNA was precipitated by one volume of absolute ethanol and diluted in an appropriate volume of TE buffer. The DNA was run in 0.8 % agarose gels to verify the quality and the concentration. l HindIII (Fermentas, Vilnius, Lithuania) was used to determine the size and the concentration of DNA.
Specific PCR Two diagnostic PCR tests, described by Leisova et al. (2005b) and Williams et al. (2001), were used to verify the classification of each isolate as either PTT or PTM. The amplification was performed in a 15-ml reaction mixture containing 1 Mg-free buffer (Biotools), 2 mM MgCl2, 0.33 mM of each dNTP (Invitrogen), 0.33 mM of each primer (PTT, PTM, PTM-d, and PTT-h, Applied Biosystems), 1U Tth polymerase (Biotools) and 100 ng DNA template. The PCR was performed in a Sensoquest Labcycler (Goettingen, Germany) under the following conditions: an initial denaturing step of 95 C for 5 min, followed by 35 cycles of 30 s at 95 C, 30 s at 55 C (annealing temperature for the PTT
Table 2 e Determination of the form of P. teres isolates.
PTMa PTTb Total
Original determination
Determination þ markers þ microsatellite analysis
205 303 508
116 271 387
a Number of Pyrenophora teres f. teres isolates. b Number of Pyrenophora teres f. maculata isolates.
Differences from original determination based on: Markers þ microsatellite analysis
Markers only
Microsatellite analysis only
þ19 þ75
þ12 0
þ1 þ14
121
Total
148 360 508
Genetic structure of PTT and PTM populations
and PTM primers), or 30 s at 50 C (annealing temperature for the PTM-d primer) or 30 s at 62 C (annealing temperature for the PTT-h primer) 40 s at 72 C and 72 C for 5 min. The amplification products were separated electrophoretically in a 1.5 % agarose gel, stained with ethidium bromide and visualised under UV light. The mating type was examined using PCR with specific primers that were developed by Rau et al. (2005). The amplification was performed in a 15-ml reaction mixture containing a 1 buffer with 2.5 mM MgCl2 (Qiagen), 0.33 mM of each dNTP (Invitrogen), 0.33 mM of each primer (MAT1 and MAT2, Applied Biosystems), 1U Taq polymerase (Qiagen) and 100 ng DNA template. The PCR was performed in a Sensoquest Labcycler (Goettingen, Germany) under the following conditions: an initial denaturing step of 95 C for 5 min, followed by 35 cycles of 30 s at 95 C, 30 s at 55 C, 1 min at 72 C, and 72 C for 5 min. The amplification products were separated electrophoretically in 1.5 % agarose gel that was stained with ethidium bromide and visualised under UV light.
Microsatellite analyses To study the Pyrenophora teres population, a set of 23 microsatellite loci were chosen from those reported by Keiper et al. (2007). The PCRs with fluorescently labelled primers (6-fam, vic, ned and pet) were performed in a reaction volume of 15 ml, which consisted of a 1 Mg-free buffer (Biotools), 2 mM MgCl2, 0.33 mM of each dNTP (Invitrogen), 0.33 mM of each primer (Life Technology), 1U Tth polymerase (Biotools) and 50 ng DNA template. The PCR was performed in a Sensoquest Labcycler (Goettingen, Germany) under the following conditions: an initial denaturing step of 95 C for 5 min, followed by 35 cycles of 30 s at 95 C, 30 s at 55 C, 40 s at 72 C and 72 C for 5 min. The analysis of the PCR products was performed using capillary electrophoresis on the sequencer ABI PRISM 3130 (Applied Biosystems). A multiplexed configuration of four reactions was used in one analysis. The internal size standard LIZ500 (Applied Biosystems) was used. The electrophoretograms were processed using the GeneMapper software.
Data analysis A matrix of distances between all of the isolates was calculated using the Jaccard dissimilarity coefficient in the DARwin software (http://darwin.cirad.fr/darwin; Perrier & Jacquemoud-Collet 2006). For clustering, an unweighted neighbour-joining method was used. The support for the phenogram branches was obtained using 2000 bootstrap resamplings. The diversity statistics for each population included the percentage of polymorphic loci, the average diversity of the ^ (Nei 1973), the loci using the Nei’s unbiased gene diversity h Shannon Information index (Shannon & Weaver 1949; Lewontin 1972) and Nei’s (1978) unbiased genetic distance (D) estimates. All of these statistics were calculated using the POPGENE software, version 1.31 (Yeh et al. 1999). The divergence statistics were estimated using the hierarchical analysis of molecular variance (AMOVA), which uses haploid molecular variance data rather than marker
183
frequency (Excoffier et al. 1992). The AMOVA was performed using Arlequin version 3.1 (Excoffier et al. 2005) and was used to partition the total genetic variation into three specific hierarchical levels: among the isolates collected within localities, among the different localities within forms and between the two forms of Pyrenophora teres. Regarding population changes in the time study, another three specific hierarchical levels were evaluated: among the isolates collected within plots, among the different plots within years and between years. The significance levels for the resultant molecular variance components were computed by 1023 nonparametric permutation procedures (Excoffier et al. 1992). The degree of population subdivision was measured by Wright’s fixation index (Fst). An exact test for population differentiation was calculated using the Tools for Population Genetic Analyses (TFPGA; version 1.3; Miller 1997) with 10 000 permutation steps. Another approach to studying the population structure analysis is based on Bayesian statistics. Structure version 2.3.4 (Pritchard et al. 2000) was used to determine the genetic architecture of the P. teres populations. Ten independent runs of 1e20 groups (K ¼ 1e20) were performed using 10 000 Markov chain iterations after a burn-in period of 10 000 iterations. The number (K) of clusters into which the sample data (X) were fitted with posterior probability Pr (XjK) was estimated using a model with admixture and correlated allele frequency (Falush et al. 2003). The optimal value of K was estimated based on ln(K) and on the DK calculation, which considers the rate of change in the lnP(D) values among successive K runs to account for patterns of dispersal that are not homogeneous among populations (Evanno et al. 2005). The index of association (IA) used to test for multilocus linkage disequilibrium (i.e., the non-random association of alleles among loci) was calculated using Multilocus version 1.3b (Agapow & Burt 2001) within each population and across all populations. IA is a generalised measure of linkage disequilibrium (Maynard Smith et al. 1993). Moreover, an alternative standardisation for the covariances (rD) was inferred using the index of association that was modified to remove the dependency of the number of loci analysed. Tests of significance using 1000 randomisations of the dataset were performed to test the null hypothesis that alleles are randomly associated.
Results The original classification of Pyrenophora teres isolates as either PTT or PTM was tested by two diagnostic PCR assays that were developed by Williams et al. (2001) and Leisova et al. (2005a). Both assays produced the same results. In 106 cases, the form identified was opposite to the original determination (Table 2). In 15 cases, the results were in agreement with the original determination, but the isolates clustered into contrary clusters (see below; Table 2; Fig 1). The PTT and PTM isolates occurred together in the same field at the same time in the localities of Prague Kompost, Tovacov Lobodice and Tovacov U Moravy. In Tovacov U Moravy, the PTT and PTM even occurred in the same lesion.
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-Svobodova et al. L. Leisova
Fig 1 e Neighbour-joining cluster analysis of 516 diverse Pyrenophora spp. isolates based on 276 alleles of 23 microsatellite loci. The numbers on the branches indicate bootstrap values (expressed in percentages; based on 2000 replications). Colours represent Pyrenophora species and forms: orange e Pyrenophora teres f. teres; green e Pyrenophora teres f. maculata; red e isolates ambiguously determined (Table 2); violet e Pyrenophora graminea, P. flavispora, and P. tritici-repentis (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
A total of 285 alleles were detected analysing 23 microsatellite loci across all 516 Pyrenophora spp. isolates. After removing the alleles with correlation coefficients greater than 0.75, 276 alleles were used for further analyses. The number of alleles per locus overall isolates ranged from 6 (PT-02 [AGAC22] and PT-18 [TCAC3]) to 23 (PT-11 [AGTG23] and PT-17 [AGTG15]), with a mean number of alleles per locus of 12.0. The percentage of polymorphic loci ranged from 33 % for the South Moravia region
to 77 % for the Tovacov region, with an average of 49.5 % across all P. teres regions. Ten alleles were specific to species other than P. teres. Two loci (PT-04 [AGTG4] and PT-15 [AGTG7]) were specific to either PTT or PTM; all of the other loci were common between the PTT and PTM populations. Neither the PTT nor PTM population group was monomorphic at any locus. A cluster analysis showed five main clusters with bootstrap values lower than 50 % (Fig 1). In one cluster, mainly PTM
Population 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Total 26 27 28 29 30 31 32 33 34 Total Total
Form PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTT PTM PTM PTM PTM PTM PTM PTM PTM PTM PTM PTT/PTM
Locality e host plant variety
Year of sampling
na
MAT1b
MAT2c
MAT1 þ MAT2d
%Pe
^f h
Ig
Prague e Kompost Prague e Beate Prague e Beate Prague e Okal Prague e Prestige Prague e Prestige Prague e Forum Tovacov Tovacov Tovacov Lobodice Tovacov U dubu Tovacov U Moravy Tovacov U prejezdu Tovacov U sterkovny Tovacov Troubky Bodorova (SR) e Krystal Maly Saris (SR) Pstrusa (SR) Zatec e Advent Libejovice e Prestige Luzany Domaninek e Amulet Stupice e Advent, Blanik Chrastava e Prestige, Jersey Chrlice e Nordus
2007 2007 2008 2007 2007 2008 2008 2002 2005 2009 2005 2006 2006 2006 2011 1986 2006 2006 2010 2007 2005 2002 2010 2008 2002
Prague e Kompost Prague e Kompost Prague e Heris Tovacov Tovacov Lobodice Tovacov U Moravy Tovacov Za prejezdem Luzany Edmonton (CAN)
2007 2008 2007 2002 2009 2006 2006 2005 2006
19 15 17 8 20 20 2 9 14 17 7 18 31 8 6 5 9 7 13 13 13 14 7 9 5 306 15 6 16 2 11 17 5 5 5 82 388
19 13 9 8 3 20 0 8 5 1 0 5 0 8 6 5 9 6 1 13 8 14 7 6 5 179 2 0 12 2 8 17 3 2 2 48 227
0 0 3 0 16 0 2 1 9 14 7 12 30 0 0 0 0 1 12 0 5 0 0 3 0 115 13 6 0 0 3 0 2 3 3 30 145
0 2 5 0 1 0 0 0 0 2 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 4 0 0 0 0 0 0 4 16
18 28 14 20 39 24 2 28 37 32 17 29 30 14 11 14 21 20 30 27 28 30 17 18 17 79 32 20 21 6 29 24 23 22 26 63 88
0.05 0.07 0.04 0.07 0.10 0.08 0.01 0.08 0.09 0.07 0.06 0.07 0.06 0.05 0.04 0.05 0.06 0.06 0.08 0.07 0.08 0.07 0.06 0.06 0.06 0.12 0.09 0.07 0.07 0.03 0.09 0.07 0.09 0.09 0.11 0.13 0.14
0.08 0.12 0.06 0.10 0.16 0.12 0.02 0.13 0.14 0.12 0.09 0.11 0.09 0.07 0.06 0.08 0.10 0.10 0.13 0.11 0.13 0.12 0.09 0.09 0.10 0.20 0.14 0.11 0.10 0.04 0.14 0.11 0.13 0.13 0.16 0.21 0.23
I Ai
rDj
Pk
0.87 3.20 0.43 0.48 2.39 5.95 e 4.05 3.86 3.42 2.29 2.02 1.45 0.94 1.60 1.51 0.32 1.10 1.87 4.31 3.08 2.60 0.91 0.47 11.64
0.018 0.044 0.012 0.009 0.023 0.095 e 0.053 0.039 0.041 0.051 0.026 0.020 0.025 0.040 0.135 0.006 0.021 0.023 0.060 0.045 0.032 0.020 0.009 0.142