Identification of Molecular Markers Linked to a Pyrenophora teres Avirulence Gene

ABSTRACT Genetic control of avirulence in the net blotch pathogen, Pyrenophora teres, was investigated. To establish an appropriate study system, a collection of 10 net form (P. teres f. teres) and spot form (P. teres f. maculata) isolates were evaluated on a set of eight barley lines to identify two isolates with differential virulence on an individual host line. Two net form isolates, WRS 1906, exhibiting avirulence on the cv. Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for reaction on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (chi(2) = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism markers closely linked to the avirulence gene (Avr(Heartland)). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model and represents an initial step toward map-based cloning of this gene.     

Variation in the resistance of barley cultivars and in the pathogenicity of Drechslera teres f. sp. maculata and D. teres f. sp. teres isolates from France

Isolates of Drechslera teres that cause net or spot-type symptoms on barley ( Hordeum vulgare ) were collected in 1986 and 1987 from fields in different regions of France. Variations in pathogenicity were evaluated using 12 barley cultivars. The Middle-Eastern cultivars Arrivate and 79-S10-10 were resistant to all isolates except R5 and S5. The Ethiopian cultivar C1 5791, previously reported to be resistant, was susceptible to the R5 and S5 biotypes. There was a high correlation coefficient between the classification of cultivars for resistance to D. teres f. sp. teres and D. teres f. sp. maculata. A method for conserving the virulence of the isolates on straw is evaluated. The virulence level of the isolates was the same after 4 years of storage using this method.     

Proteinaceous Metabolites from Pyrenophora teres Contribute to Symptom Development of Barley Net Blotch

ABSTRACT Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), induces a combination of necrosis and extensive chlorosis in susceptible barley cultivars. Cell-free filtrates from both net and spot forms of P. teres; P. teres f. sp. teres, and P. teres f. sp. maculata were found to contain phytotoxic low molecular weight compounds (LMWCs) and proteinaceous metabolites which appear to be responsible for different components of the symptoms induced by the two forms of the pathogen in a susceptible cultivar of barley (cv. Sloop). Proteins induced only brown necrotic spots or lesions similar to those induced by the pathogens 72 h after inoculation. In contrast, LMWCs induced general chlorosis seen 240 h after inoculation but not the localized necrosis. Neither hydrolyzed or heat- or protease-treated proteinaceous metabolites induced the symptoms. This is the first report of the involvement of proteins produced by P. teres in symptom development during net blotch disease of barley.     

Genetic variation of Pyrenophora teres f. teres isolates in Western Australia and emergence of a Cyp51A fungicide resistance mutation

Genome-wide, unlinked, simple sequence repeat markers were used to examine genetic variation and relationships within Pyrenophora teres f. teres, a common pathogen of barley, in Western Australia. Despite the region's geographic isolation, the isolates showed relatively high allelic variation compared to similar studies, averaging 7.11 alleles per locus. Principal component, Bayesian clustering and distance differentiation parameters provided evidence for both regional genotypic subdivision together with juxtaposing of isolates possessing different genetic backgrounds. Genotyping of fungicide resistant Cyp51A isolates indicated a single mutation event occurred followed by recombination and long-distance regional dispersal over hundreds of kilometres. Selection of recently emergent favourable alleles such as the Cyp51A mutation and a cultivar virulence may provide an explanation, at least in part, for juxtaposed genotypes. Factors affecting genotypic composition and the movement of new genotypes are discussed in the context of grower practices and pathogen epidemiology, together with the implications for resistance breeding.     

Pathogenic variation in populations of Drechslera teres f. teres and D. teres f. maculata and differences in host cultivar responses

The current study examined the variability in the pathogenicity of populations of Drechslera teres f. teres and D. teres f. maculata (the net and spot forms of D. teres) from Ireland and northern Europe. A population of progeny isolates from a mating of net and spot forms was also examined. Significant variation in virulence was found both between and among net form and spot form isolates (p<0.001). In the Irish population, significant differences were found between the net and spot forms, with the spot form isolates more virulent (p<0.05). Progeny isolates were significantly more virulent than net form or spot form populations (p<0.001). Significant differences were found in cultivar reactions, with cv. Botnia most susceptible to both forms of the pathogen (p<0.001). Cultivar Boreal 94145, although quantitatively resistant, was found to be very susceptible to both forms of the pathogen and to progeny isolates. Cultivars CI 5791, CI 2330 and CI 9819 were all less susceptible to infection by both forms, but were more susceptible to spot form isolates. Significant correlations were found between whole plants and detached leaf experiments for the net form isolates only (p<0.001). This study illustrates the importance of including both net form and spot form isolates in resistance studies and the need for a clearer understanding for the genetic basis of resistance to the net and spot forms. It also highlights the limitations of using a detached leaf assay for screening of net blotch of barley.     

Virulence profile and genetic structure of a North Dakota population of Pyrenophora teres f. teres, the causal agent of net form net blotch of barley

A Pyrenophora teres f. teres population in North Dakota was analyzed for virulence variation and genetic diversity using 75 monospore isolates that were collected across a 4-year period (2004 to 2007) from two North Dakota State University agricultural experiment stations at Fargo and Langdon. Pathogenicity tests by inoculation onto 22 barley differential lines at seedling stage revealed 49 pathotypes, indicating a wide range of pathogenic diversity. Two-way analysis of variance of disease ratings revealed a significant difference in the virulence among isolates and in the resistance among barley lines, as well as in the interactions between the two. 'CI5791', 'Algerian', and 'Heartland' were three barley lines showing a high level of seedling resistance to all North Dakota isolates tested; however, many previously reported resistance genes have been overcome. Forty multilocus genotypes were identified from this set of isolates by genotyping at 13 simple-sequence repeat loci. High percentages of clonal cultures were detected in the samplings from 2005 and 2007 in Fargo and 2005 in Langdon. Using a clone-corrected sample set, the mean gene diversity (h) was estimated to be 0.58, approximately the same for both locations. The calculated Wright's F(ST) value is small (0.11) but was significantly >0, indicating a significant differentiation between the Fargo and Langdon populations. In the gametic disequilibrium test, only 3 of 78 possible pairwise comparisons over all isolates showed significant (P < 0.05) nonrandom association, suggesting a random mating mode. Our results suggest that the populations from the two locations are derived from a common source and undergo frequent recombination. This research provides important information for barley breeders regarding development and deployment of cultivars with resistance to net form net blotch in this region.     

AFLP-based PCR markers that differentiate spot and net forms of Pyrenophora teres

Specific polymerase chain reaction (PCR) primers were developed from amplified fragment length polymorphism (AFLP) fragments of Pyrenophora teres , the causal agent of net blotch on barley leaves. The primers were designed specifically to amplify DNA from P. teres f. teres (net form) and allow its differentiation from P. teres f. maculata (spot form), which is morphologically very similar to P. teres f. teres in culture. The PCR amplification was carried out successfully from DNA extracted from fungal mycelium. The PCR assay was validated with 60 samples of Pyrenophora species. The amplification with four designed PCR primer pairs provided P. teres form-specific products. No cross-reaction was observed with DNA of several other species, such as P. tritici-repentis , P. graminea and Helminthosporium sativum .     

Genetic structure of a Pyrenophora teres f. teres population over time in an Australian barley field as revealed by Diversity Arrays Technology markers

Net form of net blotch caused by Pyrenophora teres f. teres (Ptt) is a major foliar disease of barley (Hordeum vulgare) worldwide. Knowledge of the evolution of Ptt pathogen populations is important for development of durable host-plant resistance. This study was conducted to investigate changes in genetic structure of a Ptt population within a barley field during three cropping years. The susceptible barley cultivar Henley was inoculated with Ptt isolate NB050. Leaf samples were collected during the years 2013–15 and 174 single spore Ptt isolates stored. Genotyping using Diversity Arrays Technology markers identified that 25% of isolates were clones of the inoculated isolate and 75% of isolates were multilocus genotypes (MLGs) differing from the original inoculated genotype. The novel genotypes probably originated from a combination of windborne spores from neighbouring fields, infected seed and sexual recombination in the field. The rapid change in the genotypic composition of the Ptt population in this study suggests adaptive potential of novel genotypes and demonstrates the need for barley breeders to use multiple sources of host-plant resistance to safeguard against resistance being overcome.     

Inheritance and molecular mapping of barley genes conferring resistance to wheat stripe rust

ABSTRACT Most barley cultivars are resistant to stripe rust of wheat that is caused by Puccinia striiformis f. sp. tritici. The barley cv. Steptoe is susceptible to all identified races of P. striiformis f. sp. hordei (PSH), the barley stripe rust pathogen, but is resistant to most P. striiformis f. sp. tritici races. To determine inheritance of the Steptoe resistance to P. striiformis f. sp. tritici, a cross was made between Steptoe and Russell, a barley cultivar susceptible to some P. striiformis f. sp. tritici races and all tested P. striiformis f. sp. hordei races. Seedlings of parents and F(1), BC(1), F(2), and F(3) progeny from the barley cross were tested with P. striiformis f. sp. tritici races PST-41 and PST-45 under controlled greenhouse conditions. Genetic analyses of infection type data showed that Steptoe had one dominant gene and one recessive gene (provisionally designated as RpstS1 and rpstS2, respectively) for resistance to races PST-41 and PST-45. Genomic DNA was extracted from the parents and 150 F(2) plants that were tested for rust reaction and grown for seed of F(3) lines. The infection type data and polymorphic markers identified using the resistance gene analog polymorphism (RGAP) technique were analyzed with the Mapmaker computer program to map the resistance genes. The dominant resistance gene in Steptoe for resistance to P. striiformis f. sp. tritici races was mapped on barley chromosome 4H using a linked microsatellite marker, HVM68. A linkage group for the dominant gene was constructed with 12 RGAP markers and the microsatellite marker. The results show that resistance in barley to the wheat stripe rust pathogen is qualitatively inherited. These genes might provide useful resistance against wheat stripe rust when introgressed into wheat from barley.     

Development of an international standard set of barley differential genotypes for Pyrenophora teres f. teres

International comparison of virulence profiles of Pyrenophora teres f . teres (Ptt), the cause of barley net blotch, is seriously restricted by inconsistencies in differential testers used among researchers. This paper reports an attempt to develop an appropriate set of differentials to standardize characterization of Ptt populations globally. Fourteen barley genotypes (Canadian Lake Shore (CLS), Harbin, c-8755, c-20019, Manchurian, Tifang, CI 9825, CI 5791, CI 9819, Beecher, CI 9214, Skiff, Prior and Corvette) were selected from among genotypes previously used as Ptt differentials. Three cultivars (Pirkka, Haruna Nijo and Harrington) were included to identify a universally susceptible control. Genotypes were inoculated with approximately 1000 Ptt isolates from Russia, Europe, Australia and Canada. The mean reaction frequency of genotypes ranged from highly resistant (CI 9819, CI 5791, c-8755 and CI 9825) to highly susceptible (Harrington, Haruna Nijo and Pirkka). The best differential abilities were demonstrated by Harbin, CLS, c-20019, Manchurian and Prior. Application of cluster analyses identified genotypes with similar reaction patterns, which supported a reduction of genotypes in the set. When combined with an algorithm comparing the ability of individual genotypes to discriminate among Ptt isolates, a further reduction of genotypes was justified. A new, concise set of barley genotypes for differentiating virulences in Ptt was formulated. It is proposed that these genotypes be adopted as the standard, international differential set to characterize and identify the virulence properties of Ptt populations across environments. The new Ptt differential set consists of the genotypes c-8755, c-20019, CI 5791, CI 9825, CLS, Harbin, Prior, Skiff and Harrington.     

Single Cystosorus Isolate Production and Restriction Fragment Length Polymorphism Characterization of the Obligate Biotroph Spongospora subterranea f. sp. subterranea

ABSTRACT Spongospora subterranea f. sp. subterranea causes powdery scab in potatoes and is distributed worldwide. Genetic studies of this pathogen have been hampered due, in part, to its obligate parasitism and the lack of molecular markers for this pathogen. In this investigation, a single cystosorus inoculation technique was developed to produce large amounts of S. subterranea f. sp. subterranea plasmodia or zoosporangia in eastern black nightshade (Solanum ptycanthum) roots from which DNA was extracted. Cryopreservation of zoosporangia was used for long-term storage of the isolates. S. subterranea f. sp. subterranea-specific restriction fragment length polymorphism (RFLP) markers were developed from randomly amplified polymorphic DNA (RAPD) fragments. Cystosori of S. subterranea f. sp. subterranea were used for RAPD assays and putative pathogen-specific RAPD fragments were cloned and sequenced. The fragments were screened for specificity by Southern hybridization and subsequent DNA sequence BLAST search. Four polymorphic S. subterranea f. sp. subterranea-specific probes containing repetitive elements, and one containing single copy DNA were identified. These RFLP probes were then used to analyze 24 single cystosorus isolates derived from eight geographic locations in the United States and Canada. Genetic variation was recorded among, but not within, geographic locations. Cluster analysis separated the isolates into two major groups: group I included isolates originating from western North America, with the exception of those from Colorado, and group II included isolates originating from eastern North America and from Colorado. The techniques developed in this study, i.e., production of single cystosorus isolates of S. subterranea f. sp. subterranea and development of RFLP markers for this pathogen, provide methods to further study the genetic structure of S. subterranea f. sp. subterranea.     

Virulence and Molecular Diversity in Cochliobolus sativus

ABSTRACT Spot blotch, caused by the fungal pathogen Cochliobolus sativus, is an important disease of barley in many production areas of the world. To assess genetic diversity in this pathogen, a worldwide collection of C. sativus isolates was evaluated for virulence on barley and DNA polymorphism. Three pathotypes (0, 1, and 2) were identified among the 22 isolates tested in this study and the 36 isolates characterized previously on three barley differentials (ND5883, Bowman, and NDB112) that differ in their resistance to C. sativus. Pathotype 2, which exhibits high virulence on cv. Bowman, was only found in North Dakota, whereas the other two pathotypes occurred in many other regions of the world. Genetic diversity of the 58 C. sativus isolates, together with isolates of three related pathogenic Cochliobolus spp. (C. heterostrophus, C. carbonum, and C. victoriae) was analyzed using amplified fragment length polymorphism (AFLP) markers. A total of 577 polymorphic AFLP markers were recorded among the 70 isolates of the four Cochliobolus spp. using eight primer combinations. Cluster analysis revealed distinct groups corresponding to the four different species, except in one case where race 0 of C. carbonum was placed in an outgroup that may belong to a different species. In C. sativus, 95 polymorphic AFLP markers were detected with the eight primer pairs used, and each isolate exhibited a unique AFLP pattern. Allelic diversity in the pathotype 2 group was lower (0.10) than in the pathotype 0 (0.23) and pathotype 1 (0.15) groups, indicating that pathotype 2 may have arisen more recently. Cluster analysis did not reveal a close correlation between pathotypes and AFLP groups, although two AFLP markers unique to pathotype 2 isolates were identified. This low correlation suggests that genetic exchange may have occurred through parasexual recombination in the fungal population. Some isolates collected from different regions of the world were clustered into the same AFLP group, suggesting that migration of the fungal pathogen around these regions has occurred.     

Effects of inoculation of barley inflorescences with Drechslera teres upon the location of seed-borne inoculum and its transmission to seedlings as modified by temperature and soil moisture

An investigation of the location of Drechslera teres on barley (cv. Thibaut) seeds showed that, when artificial contamination was carried out at the time of flowering, all parts of the caryopsis. including the embryo, were infected by the two forms (f. teres and f. maculata ) of the pathogen. An assessment was made of the possible role of abiotic factors, including the effects of temperature and soil moisture, on the expression of the primary symptoms caused by the two forms of the pathogen. The most severe damage was observed in dry soils (pF 3-4) at temperatures off. 12°C. On young barley seedlings, the seed-borne inoculum caused coleoptile symptoms in the case of the form maculata , whereas the form teres induced mostly foliar symptoms. Foliar necroses may result from systemic invasion by the pathogen located in the seed.     

Genetic variability within Phaeoisariopsis griseola from Central America and its implications for resistance breeding of common bean

The genetic and virulence variability of 112 isolates of Phaeoisariopsis griseola , collected from various locations in Central America, were studied using seven random amplified polymorphic DNA (RAPD) primers and 12 common-bean differential genotypes. Broad molecular diversity ( H  = 0·92) among isolates was found using RAPD markers. Fifty pathotypes were identified on 12 differential bean genotypes, 29 of which were represented by only one isolate. Only 18 pathotypes were found in two or more countries. Pathotype 63-63 was the most virulent and caused leaf spots on all 12 common-bean differential genotypes. Comparison of virulence phenotypes and RAPD profiles to known Andean P. griseola isolates confirmed that all isolates belonged to the Mesoamerican group. Pairwise comparison between individual RAPD loci showed that the majority were in gametic phase linkage disequilibrium, revealing that P. griseola maintains a genetic structure that is consistent with asexual reproduction. The molecular and virulence diversities of P. griseola isolates from Central America imply that using single resistance genes to manage angular leaf spot is inadequate and stacking resistance genes may be necessary to manage the disease effectively.     

Genotypic diversity in barley powdery mildew populations in northern France

Diversity in populations of Erysiphe graminis ( Blumeria graminis ) f.sp. hordei was studied with virulence and molecular markers. Isolates were sampled in two locations in northern France from a winter barley cultivar (Plaisant) and a spring cultivar (Caruso). Only a few pathotypes (determined by virulence markers) were common. The rest of the population was diverse. Diversity within common pathotypes, estimated by five RAPD and two SCAR markers, was generally high, except for one pathotype, which was frequent on Plaisant. This pathotype carried only one virulence, Va22, out of the 11 virulences tested. It appeared as a clonal lineage, which had occurred previously, at least in 1992, in northern France, demonstrating survival of asexual lineages in populations that often reproduce sexually.     

Variation in Pathogenicity and Genotype Among Single-Zoospore Strains of Aphanomyces euteiches

ABSTRACT The role of asexual reproduction in the production of pathogenic and genotypic variation in Aphanomyces euteiches was investigated. Variation was studied among three groups of 18 single-zoospore progeny of A. euteiches derived from each of three single-zoospore parental strains. Pathogenicity was assessed by evaluating disease severity (DS) on roots of five pea lines possessing different levels of resistance to Aphanomyces root rot and of a susceptible cultivar of snap bean and alfalfa. None of the single-zoospore progeny incited significantly higher DS levels than their parental strain on any of the seven hosts; however, 3 or 4 of the 18 progeny in each group incited significantly lower DS than their parental strains. The host range of the progeny either decreased or remained the same as compared with parental strains. Genotypic variation was assessed with randomly amplified polymorphic DNA (RAPD) analysis. Polymorphic RAPD markers that distinguished parental and progeny strains were detected within two of the three groups of strains with two of four RAPD primers used. Of 76 total RAPD markers that were detected among all strains in all groups, four (5%) were polymorphic. The polymorphic markers were not associated with the pathogenic variation.     

Comparison of Genetic and Virulence Diversity of Melampsora larici-populina Populations on Wild and Cultivated Poplar and Influence of the Alternate Host

ABSTRACT The aims of this study were, first, to compare the genetic and virulence diversity between populations of the rust fungus Melampsora larici-populina on wild and cultivated poplar stands and, second, to investigate the influence of the presence of the alternate host of the pathogen, larch, on which its sexual reproduction occurs, on these diversities. Nine French M. larici-populina populations collected from poplar trees in autumn and four populations collected from larch trees during the following spring were analyzed using both virulence factors and neutral markers. In all, 30 pathotypes were identified within the 13 populations studied. The pathotypic structure clearly distinguished the cultivated stands with high richness and complexity from the wild stands with low richness and complexity. High linkage disequilibria between virulences indicated preferential virulence associations, probably due to selection by the host. In all, 19 random amplified polymorphic DNA (RAPD) markers were used, which revealed a very high genetic diversity in the 743 isolates analyzed. The nine populations from poplar appeared moderately differentiated, indicating long-distance gene flow, and no isolation by distance was found. Linkage disequilibria between RAPD markers generally were low, indicating frequent recombination, but they were not lower in populations located near larch, probably due to long-distance dispersal.     

Attempted somatic hybridization of Puccinia striiformis f. sp. tritici and P. striiformis f. sp. hordei

Attempts were made to produce somatic hybrids between isolates of Puccinia striiformis f. sp. tritici and f. sp. hordei. A mixed infection was produced on a common susceptible barley host, Fong Tien, using white-spored isolates of P. striiformis f. sp. tritici and yellow-spored isolates off. sp. hordei. Selection was made for non-parental spore colour on selective wheat and barley hosts, and variants thus isolated were analysed for virulence markers, and for isozyme and double-stranded RNA (dsRNA) markers, all of which clearly differentiated the parental isolates. Two white-spored (non-parental) isolates were found on the selective barley host which otherwise resembled the parental f. sp. hordei isolate in virulence, isozyme and dsRNA markers. The most likely explanation of the origin of these isolates is mutation to white spore colour in the f. sp. hordei isolate.     

Diversity and Complexity of Erysiphe graminis f.sp. hordei Collected from Barley Cultivar Mixtures or Barley Plots Treated with a Resistance Elicitor

Powdery mildew populations were analysed to determine the effects of a resistance elicitor and cultivar mixtures on genetic complexity and diversity. Isolations were made from a range of spring barley monocultures and mixtures in a field trial, and characterised for virulence and RAPD profile. In a second trial, isolates were taken from a single mixture from untreated and resistance elicitor-treated areas and from the components of the mixture in monoculture. The mildew population was not only highly heterogeneous for virulence characteristics, but also proved heterogeneous within pathotypes for molecular markers, indicating the major impact of sexual recombination on population structure and the lack of clonal dominance. Various diversity measurements were compared and the value of dissimilarity measurement for revealing genetic distance within a population was highlighted. There was a trend towards increasing complexity as the season progressed, but there was no consistent relationship between cultivar or mixture, disease control treatment, fertiliser treatment, replicate or position in trial, and pathogen genotype. Whilst the resistance elicitor did reduce mildew by 78% in the first trial, and there was no interaction with fertiliser level in its expression, control was substantially less in the second trial. There were no differences between mildew isolates from elicitor and control treatments. It was felt that more effective and consistent resistance elicitors need to be developed before it can be stated that they are unlikely to be eroded by selecting resistant or adapted mildew genotypes.     

Genetic variability of Colletotrichum lindemuthianum in wild populations of common bean

The genetic structure of wild populations of Colletotrichum lindemuthianum was evaluated for virulence and molecular markers. Forty-five isolates were collected from five wild common bean populations located in their South-Andean centre of origin. The five pathogen populations were monomorphic in their ITS regions, but 45 polymorphic markers were identified using RAPDs. Polymorphism for virulence was also observed; 15 pathotypes were characterized on an international set of 12 differentials. A molecular variance analysis ( AMOVA ) showed that a very large part of the total genetic variation was within populations. Statistical analysis showed that there was a weak though significant differentiation among the five populations for the RAPD and virulence markers. A positive and significant correlation was found between geographic distance and the distances from RAPD and virulence data, suggesting migration between adjacent populations along the Argentinian transect. Our results suggest that the Andean wild isolates of C. lindemuthianum do not reflect all the putative diversity found in the isolates from cultivated common bean.