A Combination of Phenotypic and Genotypic Characterization Strengthens Pyrenophora tritici-repentis Race Identification

ABSTRACT Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces multiple host-selective toxins (HSTs), including Ptr ToxA, Ptr ToxB, and Ptr ToxC. The specific complement of HSTs produced by a particular isolate determines its host cultivar specificity. Each unique specificity profile, represented by the differential induction of necrosis or chlorosis on a standard set of wheat differentials, defines a unique race. Eight races of P. tritici-repentis have been formally published, although additional races are under investigation. Although visual assessment of disease phenotype is often used in race designation of P. tritici-repentis, our results suggest that it has the potential to be misleading. Inoculation of the P. tritici-repentis isolates SO3 and PT82 on the current wheat differential set indicated classification as race 2 and race 8, respectively; however, genetic characterization revealed that these isolates do not possess the associated HSTs expected for these race assignments. Despite sharing disease phenotypes similar to known races, SO3 and PT82 were genotypically distinct from these previously characterized races of P. tritici-repentis. To ensure detection of the breadth of physiological variation among the isolates of P. tritici-repentis, our results indicate that race classification, where possible, should include both phenotypic and genotypic analyses and eventual expansion of the differential set.     

Virulence assessment of Australian Pyrenophora tritici-repentis isolates

The virulence of 57 Australian isolates of Pyrenophora tritici-repentis (Ptr), a necrotrophic fungal pathogen responsible for the major wheat disease tan spot, was assessed through plant infection assays. Isolates collected from the northern, southern, and western wheat-cropping regions of Australia were evaluated against 16 Australian bread wheat cultivars under controlled growth conditions. Following infection, the wheat panel displayed varying disease symptoms ranging from tiny necrotic specks to spreading chlorotic and necrotic lesions. Analysis of variance indicated that the wheat cultivar exhibited a greater effect on the disease response, explaining 62.7% of the variation, in comparison to the isolate (10.4%). The interaction between the cultivar and the isolate was statistically significant and was attributed to 9.8% of the total variation. All Ptr isolates examined were able to cause disease, but did not display a clear distinction in virulence on the wheat panel investigated, instead showing subtle differences in aggressiveness. Based on the disease responses, there was no obvious pattern between isolate aggressiveness and cropping region. Some cultivars, such as Hydra, exhibited an effective level of resistance in relation to the panel of isolates tested. All 57 Ptr isolates were found to possess the ToxA effector gene and lack the ToxB effector gene. The gene expression level of ToxA was up-regulated at 3 days postinfection in both ToxA-sensitive and -insensitive cultivars, independent of ToxA–Tsn1 recognition.     

Pathogenic and molecular variability among isolates of Pyrenophora tritici-repentis, causal agent of tan spot of wheat in Argentina

Tan spot, caused by Pyrenophora tritici-repentis, is a common disease of wheat (Triticum aestivum) responsible for economic losses in some wheat growing areas worldwide. In this study the pathogenic and genetic diversity of 51 P. tritici-repentis isolates collected from different ecological regions of Argentina were analyzed. Virulence tests were conducted on 10 selected wheat cultivars: Buck Halcón, Chris, Gabo, Glenlea, Klein Dragón, Klein Sendero, Max, ND 495, ProInta Guazú and ProInta Imperial. Data revealed significant differences between all main factors evaluated and the interactions for 19 of the isolates analyzed. Based on the reaction type of each isolate/cultivar combination, 48 different pathogenic patterns were detected. The molecular analysis using Inter-Simple Sequence Repeats (ISSR) revealed the existence of 36 different haplotypes among 37 isolates of P. tritici-repentis originally selected for this study. These results indicate that P. tritici-repentis on wheat in Argentina is a heterogeneous fungus, implying that screening wheat germoplasm for resistance for tan spot disease requires a wide range of pathogen isolates.     

Temperature Dependent Seed Transmission of Stagonospora nodorum in Wheat

The transmission of Stagonospora nodorum from four naturally infected winter wheat seedlots was quantified in controlled environment germination chambers at 9, 13, 17, 21, and 25 °C. Seedlings were harvested when the second leaf began to emerge. Coleoptiles and first seedling leaves were examined for the presence of lesions caused by S. nodorum. First leaves were incubated on Bannon's medium for 2 weeks, after which they were examined for pycnidia of S. nodorum. Transmission to the coleoptile occurred at all temperatures, but decreased from 100% to 72% as temperature increased from 9 to 25 °C. Transmission to the first leaf was less, dropping from 37% to 2% as temperature increased from 9 to 25 °C. At least 44% of infected first leaves were symptomless at all temperatures, with 96% of infected leaves showing no symptoms at 25 °C. Transmission to seedling leaves occurred over a broad temperature range. Under the high densities at which wheat is sown, a significant number of infected seedlings per unit area may originate from relatively low initial seed infection levels and transmission efficiencies.     

The Frequencies and Spatial Distribution of Mating Types in Stagonospora nodorum Are Consistent with Recurring Sexual Reproduction

ABSTRACT To test the hypothesis that Stagonospora nodorum undergoes regular cycles of sexual recombination, a total of 1,207 isolates sampled from 18 fields in 12 geographical regions in six countries on five continents were analyzed for mating type frequency and distribution using polymerase chain reaction amplification of the mating type locus. Restriction fragment length polymorphism and random amplified polymorphic DNA fingerprints were used to clone-correct the data sets. Both mating types were often found on the smallest spatial scales tested, including within the same lesion, the same leaf, and the same 1-m(2) plot. In only one case out of the 18 fields tested was there a significant departure from the expected 1:1 ratio. Combining this result with previous data on the population structure of S. nodorum, we conclude that this pathogen undergoes regular cycles of sexual recombination in all regions we examined.     

Infection of resistant and susceptible cultivars of wheat by Pyrenophora tritici-repentis

Conidial germination, appressorial formation. penetration of epidermal walls, formation of intracellular vesicles and growth of intracellular hyphae in epidermal cells occurred within 12 h of inoculation. Hyphae then grew slowly between mesophyll cells for the next 12 h. Some papillae formed beneath appressoria and most infected epidermal cells retained stain by 24 h after inoculation, indicating major changes in cellular physiology. Slight differences between cultivars in some of these events were not related to resistance.
On the second day. intercellular hyphae emerged more extensively from the infection sites into the mesophyll of the susceptible cultivar Banks, and formed significantly larger mycelia than in the resistant cultivar BH1146 by 3-5 days from inoculation. Rapid intercellular growth then continued in the susceptible cultivar but not in the resistant cultivar. Necrotic lesions expanded faster in the susceptible cultivar from day 3. By day 10. most lesions in this cultivar were large and light brown with a conspicuous chlorotic margin but those in the resistant cultivar were small and dark brown with inconspicuous chlorosis.     

Structural and Physical Properties of a Necrosis-Inducing Toxin from Pyrenophora tritici-repentis

ABSTRACT Cultivar-specific toxic metabolites of Pyrenophora tritici-repentis are involved in the appearance of necrotic and chlorotic foliar lesions characteristic of tan spot. A P. tritici-repentis necrosis-inducing toxin, Ptr necrosis toxin, was purified from isolate 86-124, sequenced by gas-phase amino acid microsequencing, and characterized by circular dichroism (CD) spectroscopy and isoelectric focusing. The purified protein had a similar amino acid composition and molecular weight as previously reported. Analysis of the CD spectrum from 178 to 250 nm indicated a protein consisting of 13% alpha-helix, 36% antiparallel beta-sheet, 25% turns, and 25% other structures. The Ptr necrosis toxin from isolate 86-124 has an isoelectric point near pH 10. Using overlapping proteolytic fragments obtained from the toxin, a sequence of 101 continuous amino acids was obtained, but the amino terminus was blocked and 9 to 16 amino acids could not be sequenced. Secondary structure prediction based on the amino acid sequence indicated a beta-sheet protein with little alpha-helix, which is in agreement with the structure determined by CD spectroscopy. Sequence analysis indicated the presence of a possible membrane adhesion site and several possible phosphorylation sites that may be involved in phytotoxicity.     

Molecular characterization of a Stagonospora nodorum lipase gene LIP1

A triglyceride lipase gene (LIP1) was cloned from Stagonospora nodorum, the causal agent of wheat glume blotch. LIP1 encodes a 561 amino acid preproprotein with a predicted N‐terminal signal peptide. Its expression was up‐regulated during plant infection and in culture media supplemented with saturated fatty glycerides. The recombinant Lip1 protein possessed lipolytic activity against a broad range of lipid substrates. When applied to wheat leaves, recombinant Lip1 decreased the hydrophobicity of the leaf surface, probably by liquefaction of epicuticular wax. Pretreatment of wheat leaves with Lip1 decreased the rate of conidial adhesion from 69·5% to 22·7% and from 58·9% to 28·4% in two independent assays based on different protocols. LIP1 replacement strains showed decreased lipolytic activity on culture media relative to the wild‐type strain, and adhesion of the conidia to the wheat leaf surface was impaired in the gene replacement strains. In two experiments, adhesion rates were 54·3% and 41·6% in the LIP1 replacement strains, as opposed to 77·7% and 66·6%, respectively, in the wild‐type. Collectively, the data demonstrate that the secreted lipase Lip1 is important for the adhesion of S. nodorum infection to wheat leaves.     

Reaction of Ptr ToxA-Insensitive Wheat Mutants to Pyrenophora tritici-repentis Race 1

ABSTRACT The host-selective toxin Ptr ToxA is produced by races 1 and 2 of Pyrenophora tritici-repentis, causal agent of tan spot of wheat. Ptr ToxA has been causally associated with pathogenicity by the race 2 phenotype in this system. However, the role of toxin in disease caused by race 1, the most prevalent form of the fungus in the central and northern Great Plains of North America, has not been rigorously investigated. Three independent wheat lines harboring mutations for insensitivity to Ptr ToxA were derived from ethylmethane sulfonate treatment of the hard red spring wheat cv. Kulm, possessing the single dominant gene for toxin sensitivity. Each of the three mutants was insensitive to Ptr ToxA in bioassays based on necrosis development and electrolyte leakage. Each mutant was crossed to each of the other mutants and to the wild-type Kulm. Segregation data indicate that each mutant line harbors a single recessive mutation for toxin insensitivity that maps to or near the same locus, possibly the toxin-sensitivity gene. Each toxin-insensitive mutant line was susceptible to two isolates of race 1 of P. tritici-repentis. F(2) and F(3) generations derived from crosses between Kulm and each mutant segregated for toxin reaction. However, segregation for fungal reaction was not evident, and all F(3) families were tan spot susceptible regardless of toxin reaction. Host insensitivity to Ptr ToxA is not necessarily equivalent to resistance to race 1. Ptr ToxA should not be used alone as a proxy for fungal inoculations by breeding programs aimed at developing tan spot-resistant wheat.     

Frequency of Phaeosphaeria nodorum, the Sexual Stage of Stagonospora nodorum, on Winter Wheat in North Carolina

ABSTRACT Ascocarps of Phaeosphaeria nodorum, which causes Stagonospora nodorum blotch (SNB) of wheat, have not been found by others in the eastern United States despite extensive searches. We sampled tissues from living wheat plants or wheat debris in Kinston, NC, each month except June from May to October 2003. Additional wheat samples were gathered in Kinston, Salisbury, and Plymouth, NC, in 2004 and 2005. For the 3 years, in all, 2,781 fruiting bodies were dissected from the wheat tissues and examined microscopically. Fruiting bodies were tallied as P. nodorum pycnidia or ascocarps, "unknown" (not containing spores, potentially P. nodorum or other fungi), or "other fungi." In the 2003 sample, asco-carps of P. nodorum were present each month after May at a frequency of 0.8 to 5.4%, and comprised a significantly higher percentage of fruiting bodies from wheat spikes than of those from lower stems and leaves. Ascocarps also were found at frequencies <10% in some wheat debris samples from 2004 and 2005. Analysis of the nucleotide sequences of internally transcribed spacer regions of 18 genetically distinct North Carolina isolates from 2003 suggested that all were P. nodorum, not the morphologically similar P. avenaria f. sp. triticea. Neither the 18 isolates from 2003 nor a set of 77 isolates derived from 2004 Kinston leaf samples gave reason to suspect a mating-type imbalance in the larger P. nodorum population (P >/= 0.4). We conclude that, in North Carolina, sexual reproduction plays a role in initiation of SNB epidemics and the creation of adaptively useful genetic variability, although its relative importance in structuring this population is uncertain.     

Studies on the Interaction Between Septoria tritici and Stagonospora nodorum in Wheat

Interactions between Stagonospora nodorum and Septoria tritici were studied. Results from a detached glume experiment indicated that the interaction may be isolate-dependent, as it was shown that the interaction between the two pathogens may be beneficial or antagonistic depending on the isolate of each pathogen present. The number of spores produced by both pathogens was significantly greater when an aggressive isolate of S. tritici was mixed with a non-aggressive isolate of S. nodorum, whereas the number of spores produced by both pathogens was significantly less when two non-aggressive isolates were mixed. There was a significant reduction in disease level when S. tritici was applied prior to S. nodorum, compared to vice versa in the growth chamber. Results from growth chamber and field studies showed that S. nodorum produced significantly more spores when both pathogens were present together. It is concluded that S. tritici has a stimulatory effect on spore production by S. nodorum. However, there was a reduction of S. tritici spores observed in the dual inoculation treatments, suggesting that S. nodorum inhibits S. tritici.     

Gene Flow and Sexual Reproduction in the Wheat Glume Blotch Pathogen Phaeosphaeria nodorum (Anamorph Stagonospora nodorum)

ABSTRACT Restriction fragment length polymorphisms (RFLPs) were used to characterize the genetic structures of three field populations of Phaeosphaeria nodorum from Texas, Oregon, and Switzerland. Data from seven nuclear RFLP loci were used to estimate gene diversity and genetic distances and to make indirect measures of gene flow between populations. Three of the seven RFLP loci differed significantly in allele frequencies across populations. On average, 96% of the total gene diversity was found within populations. There was little evidence for population subdivision, suggesting that gene flow was not restricted among populations. Based on an average population differentiation of 0.04, we estimated that the exchange of 11 migrants among populations per generation would be needed to account for the present level of population subdivision. Genotype diversity based on DNA fingerprints was at a maximum for the Swiss population, whereas populations in Texas and Oregon had lower genotype diversities. Many multilocus haplotypes were found in each population. Ninety-five percent of RFLP allele pairs were in gametic equilibrium. The data were consistent with random mating within each population.     

Mating Relationships between Isolates of Phaeosphaeria nodorum, (anamorph Stagonospora nodorum) from Geographical Locations

Mating types of Phaeosphaeria nodorum isolates (from North Africa, North America, Australia, Europe and Near East) were determined in laboratory conditions. Although both mating types were found, MAT1-1 and MAT1-2 were not evenly distributed among the isolates. Sexual compatibility pairings with standard mating type testers revealed that 56 of 101 isolates could be assigned to MAT1-1 and 5 to MAT1-2. Although the teleomorph occurred in different countries, the two mating types were observed only among isolates collected from France, Great Britain, Germany and Morocco. For Morocco, where no P. nodorum pseudothecia have been reported, this is the first report of the existence of the two opposite mating types. The remaining 40 isolates could not be designated to a specific mating type. However, in 17 crosses, pseudothecial initials or sterile pseudothecia were observed on wheat straw after two months. The implications of the predominance of one mating type are discussed.     

Genetic analysis of resistance to Pyrenophora tritici-repentis races 1 and 5 in tetraploid and hexaploid wheat

Tan spot of wheat, caused by the fungus Pyrenophora tritici-repentis, is a destructive disease worldwide that can lead to serious losses in quality and quantity of wheat grain production. Resistance to multiple races of P. tritici-repentis was identified in a wide range of genetically diverse genotypes, including three different species Triticum aestivum (AABBDD), T. spelta (AABBDD), and T. turgidum (AABB). The major objectives of this study were to determine the genetic control of resistance to P. tritici-repentis races 1 and 5 in 12 newly identified sources of resistance. The parents, F(1), F(2), and F(2:3) or F(2:5) families of each cross were analyzed for the allelism tests and/or inheritance studies. Plants were inoculated at the two-leaf stage under controlled environmental conditions and disease reaction was assessed based on lesion-type rating scale. A single recessive gene controlled resistance to necrosis caused by P. tritici-repentis race 1 in both tetraploid and hexaploid resistant genotypes. The lack of segregation in the inter- and intra-specific crosses between the resistant tetraploid and hexaploid genotypes indicated that they possess the same genes for resistance to tan necrosis and chlorosis induced by P. tritici-repentis race 1. A single dominant gene for chlorosis in hexaploid wheat and a single recessive gene for necrosis in tetraploid wheat, controlled resistance to P. tritici-repentis race 5.     

Quantitative trait loci for seedling and adult plant resistance to Stagonospora nodorum in wheat

Stagonospora nodorum blotch (SNB) caused by Stagonospora nodorum is a severe disease of wheat (Triticum aestivum) in many areas of the world. S. nodorum affects both seedling and adult plants causing necrosis of leaf and glume tissue, inhibiting photosynthetic capabilities, and reducing grain yield. The aims of this study were to evaluate disease response of 280 doubled haploid (DH) individuals derived from a cross between resistant (6HRWSN125) and susceptible (WAWHT2074) genotypes, compare quantitative trait loci (QTL) for seedling and adult plant resistance in two consecutive years, and assess the contribution of QTL on grain weight. Flag leaves and glumes of individuals from the DH population were inoculated with mixed isolates of S. nodorum at similar maturity time to provide accurate disease evaluation independent of morphological traits and identify true resistance for QTL analysis. Fungicide protected and inoculated plots were used to measure relative grain weight (RGW) as a yield-related trait under pathogen infection. The lack of similar QTL and little or no correlation in disease scores indicate different genes control seedling and adult plant disease and independent genes control flag leaf and glume resistance. This study consistently identified a QTL on chromosome 2DL for flag leaf resistance (QSnl.daw-2D) and 4BL for glume resistance (QSng.daw-4B) from the resistant parent, 6HRWSN125, explaining 4 to 19% of the phenotypic variation at each locus. A total of 5 QTL for RGW were consistently detected, where two were in the same marker interval for QSnl.daw-2D and QSng.daw-4B indicating the contribution of these QTL to yield related traits. Therefore, RGW measurement in QTL analysis could be used as a reliable indicator of grain yield affected by S. nodorum infection.     

Genetic Analysis of Sensitivity to a Pyrenophora tritici-repentis Necrosis-Inducing Toxin in Durum and Common Wheat

ABSTRACT The fungus Pyrenophora tritici-repentis produces a toxin (Ptr ToxA) that causes rapid cell necrosis in sensitive wheat genotypes. A single recessive gene (tsn1) on chromosome 5BL in common wheat confers insensitivity to this toxin. Our objectives were to analyze the allelic relationships of genotypes that have shown insensitivity to a P. tritici-repentis necrosis-inducing toxin, map the gene for insensitivity to the necrosis-inducing factor produced by P. tritici-repentis in a durum wheat population, and determine the reaction to P. tritici-repentis of aneuploid genotypes that do not contain the gene. Greenhouse-grown plants of seven populations from crosses of insensitive genotypes; an F(2) population of durum wheat; and 'Chinese Spring' aneuploid, substitution, and deletion lines were infiltrated with Ptr ToxA. All crosses involving insensitive genotypes failed to produce sensitive progeny, indicating that the same gene is present in these genotypes. The gene for insensitivity in the durum population was mapped to the same region on 5BL as in common wheat using restriction fragment length polymorphism markers. 'Chinese Spring', its homoeologous group 5 nullisomic-tetrasomic stocks, and 5BL deletion lines were insensitive to the toxin. Substitution of a 5B chromosome from sensitive genotypes into 'Chinese Spring' resulted in sensitivity. Therefore, insensitivity is not conferred by a gene product per se, but rather conferred by absence of a gene for sensitivity.     

Structural Characterisation of the Interaction between Triticum aestivum and the Dothideomycete Pathogen Stagonospora nodorum

The interaction between Stagonospora nodorum and a susceptible wheat cultivar was investigated using a range of microscopic techniques. Germination of pycnidiospores occurred approximately 3 h after making contact with the leaf surface and was followed by attempted penetration 8–12 h later. Penetration was observed through stomata and also directly through periclinal and anticlinal epidermal cell walls. Penetration down the anticlinal cell walls appeared to occur without a differentiated penetrating structure whilst structures identified as either lateral appressoria or hyphopodia were typically present when penetrating over a periclinal cell wall. Once inside the leaf, the fungus continued to grow for the next 4–5 days colonising all parts of the leaf except the vascular bundles. Only in the later phase of the infection was total host cell collapse apparent. Evidence of polyphenolic compounds was observed. The infection cycle was completed within 7 days as indicated by sporulation on the leaf surface. These results have allowed us to understand how the fungus physically interacts with the leaf and will help the overall understanding of the infection process.     

Quantitative Trait Loci Analysis and Mapping of Seedling Resistance to Stagonospora nodorum Leaf Blotch in Wheat

ABSTRACT Stagonospora nodorum leaf blotch is an economically important foliar disease in the major wheat-growing areas of the world. In related work, we identified a host-selective toxin (HST) produced by the S. nodorum isolate Sn2000 and determined the chromosomal location of the host gene (Snn1) conditioning sensitivity to the toxin using the International Triticeae Mapping Initiative mapping population and cytogenetic stocks. In this study, we used the same plant materials to identify quantitative trait loci (QTL) associated with resistance to fungal inoculations of Sn2000 and investigate the role of the toxin in causing disease. Disease reactions were scored at 5, 7, and 10 days postinoculation to evaluate changes in the degree of effectiveness of individual QTL. A major QTL was identified on the short arm of chromosome 1B, which coincided with the snn1 toxin-insensitivity gene. This locus explained 58% of the phenotypic variation for the 5-day reading but decreased to 27% for the 10-day reading, indicating that the toxin is most effective in the early stages of the interaction. In addition, relatively minor QTL were identified on chromosomes 3AS, 3DL, 4AL, 4BL, 5DL, 6AL, and 7BL, but not all minor QTL were significant for all readings and their effects varied. Multiple regression models explained from 68% of the phenotypic variation for the 5-day reading to 36% for the 10-day reading. The Chinese Spring nullisomic 1B tetrasomic 1D line and the Chinese Spring-Triticum dicoccoides disomic 1B chromosome substitution line, which were insensitive to SnTox1, were more resistant to the fungus than the rest of the nullisomictetrasomic and disomic chromosome substitution lines. Our results indicate that the toxin produced by isolate Sn2000 is a major virulence factor.