Genetics of host-pathogen interactions in the <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> (net form) – barley (<Emphasis Type="Italic">Hordeum vulgare)</Emphasis> pathosystem

The genetics of host-pathogen interactions in the Hordeum vulgare – P. teres f. teres pathosystem was studied in twelve resistant barley accessions, i.e. CI 9825, CI 9819, Diamond, CI 4922, CI 5401, Harbin, c-8755, c-21849, c-8721 c-23874, c-19979, c-15811. F₂ analyses of crosses with susceptible genotypes employing various isolates (from Europe, USA, Canada, and Australia) revealed that resistance is mostly isolate-specific and controlled by one or two genes. Segregation in ascospore progeny from two crosses between isolates of different origin revealed that avirulence in P. teres is also determined by one or two genes. An epistatic effect of suppressor genes on avirulence genes is proposed for the genetics of virulence to Diamond, Harbin, CI 5401 and c-8721 in the fungal crosses D (181-6 × A80) and F (H-22 × 92-178/9). Segregation in F₂ of crosses of three new sources of resistance (c-23874, c-19979, c-15811) to the susceptible cv. Pirkka was studied in laboratory and greenhouse tests by using seven P. teres isolates, i.e. 181-6, d8-3, d8-4, d9-1, d9-4, F4 and F74. In addition, virulence to these barley accessions of ascospore progeny from crosses of the same isolates was studied. Based on these studies it was concluded that depending on the isolate used, resistance of c-23874 is determined at least by two genes and in c-19979 and c-15811 by three genes. The results of this parallel analyses of genetics of resistance and genetics of virulence allows the postulation of a gene–for–gene interaction in the P. teres – H. vulgare pathosystem.     

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.     

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.     

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.     

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.     

Identification of Molecular Genetic Markers in Pyrenophora teres f. teres Associated with Low Virulence on 'Harbin' Barley

ABSTRACT Two isolates of the barley net blotch pathogen (Pyrenophora teres f. teres), one possessing high virulence (0-1) and the other possessing low virulence (15A) on the barley cultivar Harbin, were crossed and the progeny of the mating were isolated. Conidia from cultures of the parent and progeny isolates were used as inoculum to determine the inheritance of virulence in the pathogen. Of the 82 progeny tested, 42 exhibited high virulence and 40 exhibited low virulence on 'Harbin' barley. The data support a model in which a single, major gene controls virulence in P. teres f. teres on this barley cultivar (1:1 ratio; chi(2) = 0.05, P = 0.83). Preparations of DNA were made from parental and progeny isolates, and the DNA was subjected to the random amplified polymorphic DNA (RAPD) technique in a search for molecular genetic markers associated with the virulence phenotype. Five RAPD markers were obtained that were associated in coupling with low virulence. The data indicate that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres.     

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 .     

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.     

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.     

Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres

A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.     

Identification of resistance to Cercospora leaf spot of cowpea

Twelve selected cowpea cultivars were screened for resistance to Cercospora leaf spot (CLS) disease caused by Pseudocercospora cruenta and Cercospora apii s. lat. under artificial epiphytotic conditions in a replicated field trial, with the objective of developing a quantitative measure of disease resistance. CLS incidence, leaf spotting score, lesion density, lesion size, proportion of nodes infected, diseased leaf area, conidia number mg⁻¹ and fascicle density were assessed in 12 cowpea genotypes at crop maturity. Proportion of nodes infected and leaf spotting score were best able to quantitatively differentiate between the levels of resistance, and allow the exploitation of quantitative resistance to the disease. Both lesion density and lesion size were important in determining the final leaf spotting score but the former was epidemiologically more important than the latter, indicated by its correlation to most of the CLS symptom measures. There was differential resistance to the P. cruenta and C. apii s. lat. among the cowpea varieties screened. Among the cowpea lines screened, resistance to P. cruenta was more common than resistance to C. apii s. lat. Nevertheless, P. cruenta was considered the more aggressive and epidemiologically more important than C. apii s. lat. on the varieties tested evidenced by the strong correlation of P. cruenta incidence with acropetal spread of CLS, intensity of leaf spotting, conidia number mg⁻¹ and fascicle density. The highly susceptible varieties namely VRB7, Los Banos Bush Sitao no.1 and CB27 were susceptible to both Cercospora pathogens. The cowpea variety VRB-10 was completely resistant to both pathogens and is a useful source of resistance in CLS breeding programmes.     

Resistance in Australian barley (Hordeum vulgare) germplasm to the exotic pathogen Puccinia striiformis f. sp. hordei,causal agent of stripe rust

Eighty‐eight Australian and 10 international barley cultivars were assessed for resistance to the barley stripe (yellow) rust pathogen, Puccinia striiformis f. sp. hordei (Psh). All cultivars were tested for seedling resistance to two UK‐derived isolates of Psh (11.01 and 83.39) that were shown to differ in virulence based on responses on 16 differential barley genotypes. The 98 barley cultivars differed substantially in stripe rust response; 45% were susceptible to Psh 11.01, 53% to Psh 83.39 and 44% to both isolates. The observed diverse infection types (ITs) suggest the presence of both known and uncharacterized resistance. However, further multipathotype tests are required for accurate gene postulation. The Yerong × Franklin (Y×F) doubled haploid (DH) population was phenotypically assessed as seedlings using both Psh isolates. Yerong and Franklin were immune and highly resistant, respectively, to both isolates used in this study. Marker‐trait and QTL mapping identified a major effect on the long arm of chromosome 7H contributed by Franklin in response to all isolates. The resistance of Yerong was mapped to 113·96 and 169·38 cM on chromosome 5HL in response to Psh 11.01 and 83.39, respectively. The Psh resistance sources identified in this study can be used for further genetic analysis and introgression for varietal improvement.     

甘肃陇南感病小檗在小麦条锈病发生中起提供(初始)菌源作用的直接证据

陇南是中国小麦条锈菌易变区、小麦条锈病的常发流行区和防治的关键地区.明确陇南小麦条锈菌转主寄主小檗在小麦条锈病发生中的作用,对阐释该地区小麦条锈菌新小种产生的来源和指导小麦条锈病的综合防控具有重要意义.本研究从陇南春季自然受锈菌侵染的堆花小檗及其邻近的小麦上分离获得小麦条锈菌菌系,19个来自发病小檗的单夏孢子堆菌系在中...     

Genetic and pathogenic diversity within Ascochyta rabiei(Pass.) Lab. populations in Pakistan causing blight of chickpea (Cicer arietinum L.)

Pathogenic and genetic diversity in Ascochyta rabiei populations in Pakistan were evaluated. Biological pathotyping of 130 A. rabiei isolates (obtained from hierarchically collected samples) was conducted on a set of three chickpea differentials, i.e. ILC 1929 (susceptible), ILC 482 (tolerant) and ILC 3279 (resistant), under controlled conditions. Disease severity data were recorded 12 days after inoculation. Statistical analysis grouped the isolates into three pathotype classes. Four isolates belonged to pathotype I (least aggressive), 79 isolates to pathotype II (medium aggressive) and 47 isolates to pathotype-III (highly aggressive).Genetic analysis was performed using RAPDs and oligonucleotide fingerprinting, where Hinf I-digested DNA was hybridized to the³²P-endlabeled oligonucleotide probes (CAA)₅, (GAA)₅, (GA)₈, (CA)₈and (GATA)₄. Dendrograms produced by cluster analysis discriminated 46 genotypes in the A. rabiei population of Pakistan. Genetic distances and relatedness between isolates were calculated. At a genetic distance of 0.3, genotypes were divided into six distinct genotype groups A, B, C, D, E and F containing 16, 11, 2, 5, 5 and 7 isolates, respectively. Most of the genotypes were area specific or predominated in certain areas but did not belong to a distinct pathotype, while most of the aggressive isolates (pathotype III) occurred in Northern Punjab and in the North Western Frontier Province.     

Further Contributions to the Development of a Differential Set of Pea Cultivars (Pisum sativum) to Investigate the Virulence of Isolates of Aphanomyces euteiches

Common root rot (Aphanomyces euteiches Drechs.) has become a very destructive disease in the French pea crops since 1993. For an accurate investigation of the virulence variability among French A. euteiches populations and between French and foreign populations, a new set of differential pea genotypes was developed. Thirty-three American and European pea lines, displaying different levels of resistance, were screened in a growth chamber against two French isolates. Symptoms (disease severity from 0 to 5, evaluating symptom surface on roots and epicotyl) and percentage of top fresh weight (inoculated/uninoculated top fresh weight ratio) were measured. From this screening 12 relatively resistant lines, from various genetic backgrounds, were identified along with a highly susceptible control. This set of 13 genotypes was inoculated under controlled conditions with 14 isolates from France, Sweden, USA, Canada and New Zealand, to investigate genotype–isolate interactions. Root symptoms were rated (disease severity), and a susceptibility/resistance threshold was established at disease severity = 1. Significant quantitative interactions were observed, and five 'resistance patterns' were identified, leading to a set of six pea genotypes: Baccara (susceptible), Capella, MN313, 902131, 552 and PI180693. Fields trials of this set in 1999 and 2000 gave the same resistance rankings than in growth chamber conditions. This set will allow more accurate assessments of the variability in virulence/aggressiveness of A. euteiches isolates from France and foreign countries, and further investigations of the epidemiological and genetic basis of pea–A. euteiches interactions.     

Notes on the Occurrence of Pathogenic Races of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>Found in Hiroshima Prefecture in 1999

The pathogenic race of 59 cultures of Xanthomonas oryzae pv. oryzae, a pathogen of bacterial leaf blight of rice, isolated from six locations in the inland mountainous area of Hiroshima Prefecture in 1999, were determined by a set of traditional differentials. Four races—I, II, V and VII—were found across the area; however, we noticed the composition of the races as well as the dominant race in each location different. All races were avirulent on differential cultivar Te-tep. Races V and VII were new to Hiroshima. The rice cultivars infected with bacterial leaf blight in Hiroshima are thought to be grouped into the Kinmaze group, which does not have any resistance genes. Apparently, a variety of races occurred unexpectedly on the cultivars contrary to stabilizing selection theory. Received 25 February 2000/ Accepted in revised form 13 July 2000     

Susceptibility of German spring barley cultivars to loose smut populations from different European origins

Forty-two registered spring barley cultivars from the German official list were tested under natural infection conditions for susceptibility to loose smut (Ustilago tritici f.sp. hordei) during two test cycles at two locations. Only cv. Steffi was found to be resistant to the local loose smut population. Cultivar Sigrid showed lowest susceptibility because of flowering inside the leaf sheath. Less than 1% infection at all sites showed up in cvs Auriga, Jacinta and Hendrix. Twenty-one cultivars had an infection rate of less than 2%. Cultivar Danuta displayed the highest susceptibility with an average of 12.6%. Another 23 spring barley accessions with expected loose smut resistance were inoculated artificially with loose smut populations obtained from 11 locations in Germany and neighbouring countries. Only Jet with the resistance Un3/6, CDC Freedom with Un8, CIho9973 with quantitative resistance, as well as Lino and GangTuoQuingKeHao1 remained disease-free. In addition to these, another eight accessions in this test group are recommended to become part of a differential tester set to distinguish origins of loose smut. Statistical analysis showed that for scoring of cultivars more importance has to be given to the number of locations for infestation than to the number of test locations to determine the degree of attack. In view of the existing inspection limits for production of certified seed in European countries, the currently registered German barley cultivars put organic seed producers and breeders at high risk in respect to loose smut infection, if the number of generations for multiplication under organic farming increases.     

Low diversity and fast evolution in the population of Puccinia triticina causing durum wheat leaf rust in France from 1999 to 2009, as revealed by an adapted differential set

No internationally agreed differential set is available for characterization of virulences in populations of Puccinia triticina causing wheat leaf rust on durum wheat. In a first step, 73 potentially differential host genotypes were tested with 96 durum leaf rust isolates collected in France. A differential set, adapted to the local epidemiological context and useful for comparison with international studies was selected, including French commercial cultivars, Thatcher lines with Lr genes, and international cultivars. In the second step, a sample of 310 isolates collected in France from 1999 to 2009 was characterized on this set. Diversity was very low, as only five pathotypes were distinguished. Genotyping of a subset of 76 isolates according to 20 SSR markers confirmed this low diversity, with 73 isolates belonging to a single dominant genotype. Population was strongly shaped by cultivars, and the findings explain the successive breakdown of resistance sources deployed in French durum wheat cultivars. The gene Lr14a, suggested to be an efficient source of resistance in several European and American countries, was overcome by pathotypes frequent in France since 2000. Postulation of resistance genes in the commercial cultivars led to a proposed simplified version of the differential set. This study, providing new information about leaf rust resistance genes present in the French durum wheat germplasm, highlights the need to diversify sources of resistance to P. triticina in this germplasm. The results are also discussed in terms of relatedness and intercontinental migration of P. triticina on durum wheat.     

A standard set of host differentials and unified nomenclature for an international collection of Diplocarpon rosae races

Black spot of rose is distributed throughout the world and is the most serious disease of roses (Rosa spp.) in the outdoor landscape. Resistance breeding has been frustrated by the occurrence of races of the causal pathogen Diplocarpon rosae. Races from Germany, North America and the UK have been characterized and maintained in a pathogenic state. However, these races were characterized using independent sets of host genotypes and are referenced using different nomenclatures. In the present study, a total of 15 D. rosae isolates from these locations, as well as Belgium and Italy, were inoculated to a common set of 15 rose cultivars in replicated, detached leaf trials. Baby Love^TM (cv. Scrivluv) was resistant to all isolates except for one originating from the UK. The rose cultivars Mrs Doreen Pike (Ausdor) and Hansa were resistant to all isolates except for one originating from Minnesota, USA. No rose genotype was universally susceptible. A total of 11 pathogenic races were differentiated based on their unique host ranges and were assigned an international race nomenclature. Nine cultivars are proposed as the first standard set of differential genotypes for characterization of D. rosae races.