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.     

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.     

Population genetic structure of four regional populations of the barley pathogen Pyrenophora teres f. maculata in Iran is characterized by high genetic diversity and sexual recombination

The leaf spot form of the barley disease net blotch, caused by the fungus Pyrenophora teres f. maculata (PTM), is an increasingly important foliar disease of barley. Studies of population genetic structure and reproductive mode are necessary to make predictions of the evolutionary potential of the pathogen. Sources of resistance to PTM have been found in Iranian landraces, which may have the potential to improve plant breeding efforts. However, little is known about the population genetic structure of this fungus in Iran. In this study, we analysed the frequency of the mating type genes to assess the potential for sexual mating of PTM collected from four provinces—Khuzestan, Hamadan, Golestan, and East Azerbaijan—and we investigated the population genetic structure using seven simple sequence repeat markers. High genotype diversity, linkage equilibrium, and equal ratios of mating types frequencies in the PTM populations at Khuzestan and Hamadan support the occurrence of sexual reproduction in these populations, while in Golestan and East Azerbaijan populations, significant gametic disequilibrium and relatively low genotype diversity suggest a higher incidence of clonality or different demographic histories. Unequal mating type frequencies in Golestan confirm a predominance of asexual reproduction. Finally, we found significant evidence for strong population structure with most of the genetic variation represented within regional populations (89%). Overall, our study provides evidence for high genetic variation in Iranian PTM populations, which may be the basis for rapid adaptive evolution in this pathosystem. This highlights the need for integrated efforts to control the disease.     

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.     

Effects of temperature and leaf wetness on Pyrenophora teres growing on barley cv. Sonja

Conidia of Pyrenophora teres germinated only in the presence of liquid water and at temperatures above 2°C. The speed with which germination occurred was inversely proportional to temperature measured from a base of 2°C, up to the maximum temperature tested of 21°C. Once conidia on leaves had been wetted, about 40% of all infections that would eventually occur were established within 100°C-hours. Subsequent lesion extension was rapid, with area doubling times of about 1 day between 10 and 20°C.
If conidia germinated, up to 80% formed successful infections on young, susceptible leaves. On older leaves fewer spores germinated and the proportion that then infected was smaller.
The latent period, defined as the time before which sporulation did not occur under any wetness conditions, ranged from about 25 days at 5°C to 11 days at 20°C under dry conditions. Under continuously wet conditions it was about 20% shorter at all temperatures. Its inverse had a curvilinear relation to temperature.
Spores were produced after one to several days of humidity above 95%. The precise period decreased with increasing temperature, but at 25°C spores never appeared. The drier a dead leaf was, the longer the pathogen in it look to produce spores.     

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.     

Pyrenophora teres growth and severity of net blotch on barley under salt stress

European Journal of Plant Pathology - Pyrenophora teres causes one of the most destructive barley diseases in semi-arid production areas worldwide. With the burgeoning issue of soil salinization,...     

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.     

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.     

Effects of Pyrenophora teres on dry matter production and yield components of winter barley

Inoculation of winter barley plants in a glasshouse with Pyrenophora teres at three different growth stages (GS 11, 13 or 30) greatly decreased root and shoot dry matter production and the size of healthy leaves produced subsequently, but there was no significant yield loss with a single inoculation at GS 11 or 30. Inoculation at GS 13, GS 31 or GS 39 decreased grain yield by 12,17 and 20% respectively, and also straw yield. Larger yield losses resulted from repeated inoculations on five successive occasions (GS 11, 13, 30, 31 and 39) which caused much disease on all leaves throughout the life of the plant. All components of yield measured were decreased by the cumulative effect of successive inoculations; ear number by 15%), grain sites per ear by 20%, grain yield by 48% and straw yield by 32%.     

Correlation between sensitivity of barley to Pyrenophora teres toxins and susceptibility to the fungus

Detached leaves of 25 barleys, ranging from highly susceptible to highly resistant to Pyrenophora teres f. teres and Pyrenophora teres f. maculata, were tested for their reaction to three phytotoxins isolated from cultures of the fungus: toxin A [ L, L - N -(2-amino-2-carboxyethyl)aspartic acid], toxin C (aspergillomarasmine A) and toxin B (anhydroaspergillomarasmine A). 0.75 m M toxin A caused mainly dark yellow chlorotic symptoms but little necrosis, whereas leaves treated with 0.25 m M toxin C developed distinct necrotic symptoms and zones of light yellow chlorosis. Toxin B is only weakly toxic, and toxin B and control solutions containing aspartic acid in the concentration of 0.75 m M did not cause any symptoms. The best differentiation between the barleys was obtained by scoring chlorosis after 120 h, and the optimal toxin concentrations for this differentiation were 0.75 m M toxin A and 0.25 m M toxin C, respectively. Results with different toxin concentrations inducing distinct variation in symptom expression indicate that the two toxins have different potencies as phytotoxins. The reaction of the barleys to toxins A and C correlated well with their reaction to infection by P. teres f. teres and P. teres f. maculata, suggesting that toxins A and C may be used to select resistant barley lines in the early stages of a breeding programme.     

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.     

The genetic diversity of Icelandic populations of two barley leaf pathogens, Rhynchosporium commune and Pyrenophora teres

Twelve species of fungi have been found growing on barley leaves in Icelandic fields. The study presented here on the population structure of two of these species, the pathogens Rhynchosporium commune and Pyrenophora teres f. teres, reveals high levels of genetic diversity, low levels of migration, and a significant differentiation from other European populations, despite the short history of continuous barley cultivation in Iceland. The gene diversity for R. commune in Iceland was 0.55 compared to a range of 0.43?C0.73 for six European populations. The gene diversity for P. teres was higher in Iceland than in populations from Russia and Finland. The two mating types were found to overlap in distribution for both fungi making sexual reproduction a possibility, supported by the few clones and the gametic equilibrium within the Icelandic populations. When the high levels of diversity, low levels of migration, and the genetic differentiation observed between Icelandic and Scandinavian populations are put into context with the short history of barley cultivation in Iceland, it raises questions regarding the origin of the Icelandic fungal populations. It also underlines the importance of proper analysis of pathogens prior to starting resistance breeding projects. The findings are an addition to the ongoing analysis of the global diversity of barley fungal pathogens in general and an important input into future barley breeding projects in Iceland in particular.     

Effect of barley yellow dwarf virus on susceptibility of barley cultivars to net blotch (Pyrenophora teres) and leaf blotch (Rhynchosporium secalis)

The effect of barley yellow dwarf virus (BYDV) on the development of net blotch (Pyrenophora eves) and leaf blotch (Rhynchosporium secalis) was examined on seven barley cuitivars. Seedlings were infected with BYDV at the two-leaf stage (G.S. 12). Their susceptibility to three isolates of p teres and isolates of two races (U.K. 1, U.K. 2) of R. secalis was examined at the four-leaf stsge (G.S. 14) and when plants were more mature (G.S. 33/38). At G.S. 14 numbers of lesions produced by P. teres and R. secalis were reduced, on average, by 37 and 72% respectively, and at G.S. 33/38 by 61 and 74%.     

Assessment of the reaction of some spring barley cultivars to Pyrenophora teres using whole plants, detached leaves and toxin bioassay

Twenty-one spring barley cultivars were tested for their reaction to Pyrenophora teres using three methods of assessment: inoculation of plants grown outdoors, a detached-leaf inoculation assay, and bioassay with P. teres toxins produced in vitro. Dram was the most resistant of the cultivars tested; a number of other cultivars including Goldmarker, Egmont and Georgie were also moderately resistant. Akka and Havila were highly susceptible. Good correlation was obtained between results from the three test methods.     

Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance

Resistance to QoI fungicides in Pyrenophora teres (Dreschsler) and P. tritici-repentis (Died.) Dreschsler was detected in 2003 in France and in Sweden and Denmark respectively. Molecular analysis revealed the presence of the F129L mutation in resistant isolates of both pathogens. In 2004, the frequency of the F129L mutation in populations of both pathogens further increased. The G143A mutation was also detected in a few isolates of P. tritici-repentis from Denmark and Germany. In 2005, the F129L mutation in P. teres increased in frequency and geographical distribution in France and the UK but remained below 2% in Germany, Switzerland, Belgium and Ireland. In P. tritici-repentis, both mutations were found in a significant proportion of the isolates from Sweden, Denmark and Germany. The G143A mutation conferred a significantly higher level of resistance (higher EC50 values) to Qo inhibitors (QoIs) than did the F129L mutation. In greenhouse trials, resistant isolates with G143A were not well controlled on plants sprayed with recommended field rates, whereas satisfactory control of isolates with F129L was achieved. For the F129L mutation, three different single nucleotide polymorphisms (SNPs), TTA, TTG and CTC, can code for L (leucine) in P. teres, whereas only the CTC codon was detected in P. tritici-repentis isolates. In two out of 250 isolates of P. tritici-repentis from 2005, a mutation at position 137 (G137R) was detected at very low frequency. This mutation conferred similar resistance levels to F129L. The structure of the cytochrome b gene of P. tritici-repentis is significantly different from that of P. teres: an intron directly after amino acid position 143 was detected in P. teres which is not present in P. tritici-repentis. This gene structure suggests that resistance based on the G143A mutation may not occur in P. teres because it is lethal. No G143A isolates were found in any P. teres populations. Although different mutations may evolve in P. tritici-repentis, the G143A mutation will have the strongest impact on field performance of QoI fungicides.     

Relative tolerances of wild and cultivated barley to infection by Blumeria graminis f.sp. hordei (Syn. Erysiphe graminis f.sp. hordei). II—the effects of infection on photosynthesis and respiration

The relative levels of tolerance of two wild barley lines (Hordeum spontaneum), B19909 and I-17-40, and one cultivated barley (Hordeum vulgare), cv. Prisma, to Blumeria graminis f.sp. hordei were determined by comparing the effects of different levels of infection on the photosynthesis and respiration rates of the third leaf. Infection caused the early onset of senescence in all three lines, and in particular in cv. Prisma, and was accompanied by decreases in gross and net photosynthesis rates, increases in respiration rates, and loss of chlorophyll. The onset of senescence occurred at approximately the same time in infected leaves of the two wild lines, but once triggered, photosynthesis rates and chlorophyll levels declined more rapidly in I-17-40 than in B19909. A burst of respiratory activity accompanied the onset of senescence, and this was greatest in cv. Prisma. Conidial production was higher in B19909, indicating a higher level of tolerance in this line.     

Effects of Sterol Biosynthesis Inhibitor Fungicides on Growth and Sterol Composition of Ustilago maydis,Botrytis cinerea and Pyrenophora teres

The effects of the sterol biosynthesis inhibitor (SBI) fungicides fenarimol, fenpropimorph, imazalil, prochloraz, propiconazole and triadimenol on growth and sterol composition of Ustilago maydis, Botrytis cinerea and Pyrenophora teres, grown from spores or sporidia in liquid culture, were determined. Growth of U. maydis was only slightly inhibited by SBI fungicides at concentrations which caused considerable changes in both sterol content and composition. Conversely, in B. cinerea and P. teres, growth was strongly inhibited under conditions where ergosterol was still the predominant sterol, suggesting that, in these two fungi, growth may be more sensitive to SBI fungicides than overall sterol production. Demethylase inhibitor fungicides behaved as a homogeneous group in their effects on growth and on sterol profiles of the three fungi studied.