Development of apothecia of the eyespot pathogen Tapesia on cereal crop stubble residue in England

A reservoir of infection of Tapesia yallundae may exist after harvest in bases of cereal stems due to the presence of apothecia capable of discharging infective ascospores. Apothecia of T. yallundae developed in a seasonal pattern on winter barley inoculated with the pathogen, with maximum numbers of apothecia produced on stubble 5–7 months after harvest. A similar pattern of development was observed on infected winter wheat. However, the peak in numbers of mature apothecia was observed 2 months later than in winter barley. Apothecia capable of discharging ascospores were present for up to 6 months on stubble. Apothecia of T. acuformis were not detected on spring or winter barley, or spring or winter wheat stubble, despite inoculation of growing crops with isolates of compatible mating type.     

Observations of apothecia of Tapesia yallundae and the cultural phenotypes of their progeny

Apothecia of Tapesia yallundae, the perfect state of Pseudocercosporella herpotrichoides, were found in March 1989 on wheat and barley stubble at nine of 37 naturally infested sites and also at one inoculated site in Germany. Apothecia were found on about 1% of stems collected from the naturally infested sites and 5% of stems from the inoculated site, and were first found in the second week of March, coinciding with the onset of an early spring. Apothecia were not found on either green plants or second-year stubble. In total, 143 single-spore isolates were obtained from the apothecia and described according to a range of morphological characteristics. The majority were ascribed to P. herpotrichoides var. herpotrichoides. This is the first isolation of this variety from T. yallundae apothecia in Germany. Even single-conidial isolates from one apothecium showed much variation, some being like var. herpotrichoides, some like var. acuformis and some intermediate.     

Population Genetic Structure of Tapesia acuformis in Washington State

ABSTRACT Eyespot of wheat is caused by Tapesia yallundae and T. acuformis. Historically, T. yallundae has been considered the more important causal agent of the disease in Washington state and consists of a large homogeneous population with a genetic structure consistent with both sexual and asexual reproduction. T. acuformis has increased significantly in Washington in the past 10 years and apothecia were found recently under natural field conditions, indicating that T. acuformis may have a more important role in eyespot of wheat than previously was thought. To determine the genetic structure of T. acuformis in Washington, 141 single conidial isolates were sampled from four subpopulations in the eastern wheat-growing region of the state. Isolates were scored for mating type and six amplified fragment length polymorphism markers. All markers segregated in a 1:1 ratio and were determined to be unlinked based on genetic analysis of 24 progeny from an in vitro cross. No significant differences in allele frequencies (0.127 < P < 0.809) were found among individual loci across the four subpopulations and over all loci based on contingency table analysis of the log-likelihood ratio statistic G(2). Likewise, no overall differences between subpopulations were detected using the population differentiation statistic theta (theta = -0.004, P = 0.537). Random mating could not be rejected within each subpopulation or for the combined data using clone-corrected data sets based on (i) 1:1 ratio of mating-type, (ii) multilocus gametic disequilibrium analyses (index of association), (iii) phylogenetic analyses (parsimony tree length permutation test), and (iv) genotypic diversity analyses. T. acuformis has a genetic structure similar to that of sympatric populations of T. yallundae in Washington, with both sexual and asexual reproduction contributing to the structuring of this species.     

Changes in populations of the eyespot fungi Tapesia yallundae and T. acuformis under different fungicide regimes in successive crops of winter wheat, 1984–2000

Effects of regular treatments with the fungicides carbendazim and prochloraz applied to whole plots divided into subplots with different initial population mixtures of carbendazim-sensitive or carbendazim-resistant Tapesia yallundae or T. acuformis were studied in successive crops of winter wheat from 1984/85 to 1999/2000. In unsprayed and carbendazim-sprayed whole plots, a stable coexistence of about 50% each of T. yallundae and T. acuformis developed within five seasons, but in whole plots sprayed with prochloraz or prochloraz plus carbendazim, the proportion of T. acuformis increased to > 80%. A discrete time difference equation model was derived from knowledge of the biology of eyespot and competition theory to describe the population changes. The model was fitted to the data from treatments where coexistence occurred [subplots in unsprayed (1985–92) and carbendazim-sprayed (1985–89) whole plots], using nonlinear least squares regression. The optimized value of the resource overlap coefficient was small, suggesting niche differences between the two species. Populations were nearly 100% carbendazim-resistant in carbendazim-sprayed whole plots by July 1985 (one season) and in whole plots sprayed with prochloraz plus carbendazim by July 1986 (two seasons). In prochloraz-sprayed whole plots, the proportion of carbendazim-resistant isolates decreased more rapidly than in unsprayed whole plots in the 1980s, but by July 1992 a shift in populations in unsprayed and prochloraz-sprayed whole plots towards predominantly carbendazim-resistant strains had occurred.     

Changes in fungicide sensitivity and relative species abundance in Oculimacula yallundae and O. acuformis populations (eyespot disease of cereals) in Western Europe

Changing fungicide sensitivities in populations of Oculimacula yallundae and O. acuformis , the species responsible for cereal eyespot in Western Europe, were determined over a 17 year period between 1984 and 2000. The data were collected by Aventis Crop Science as part of their long-term survey to monitor changes in sensitivity to prochloraz and the methyl benzimidazole carbamate (MBC) fungicides in eyespot populations. The results show evidence for reduced sensitivity to both fungicides over the period of the survey. The decline in MBC sensitivity is in agreement with reports of practical resistance (a detectable loss of disease control in the field) to this fungicide which were widely reported from the mid 1980s onward. Prochloraz sensitivity was more complex, with the emergence of a higher resistance category of isolates in the late 1980s and early 1990s which then decreased in frequency towards the end of the survey. This may be partly explained by the introduction and increased use of cyprodinil in the mid 1990s. Although all trends were similar across Europe, differences were observed between the two eyespot species. A higher frequency of O. yallundae isolates showed decreased sensitivity to MBC, whereas decreased sensitivity to prochloraz was at a higher frequency in O. acuformis populations. The relative abundance of the two eyespot species was influenced by their differential levels of fungicide sensitivity, with the ratio increasing toward the species with the highest level of resistance to the prevailing fungicide.     

Differentiation and quantification of the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis using a PCR assay

Isolates of Tapesia yallundae and Tapesia acuformis were subjected to Random Amplified Polymorphic DNA (RAPD) assay. Amplification products common to isolates of either species were cloned and primers were generated from each sequence for use in conventional PCR. The primer pair derived from a T. yallundae specific RAPD marker amplified a product only from DNA of T. yallundae isolates and not from DNA of a range of other fungal species associated with the stem base disease complex of cereals. Similarly, the primer pair generated from a T. acuformis -specific RAPD marker amplifed a product only from DNA of T. acuformis isolates. Quantitative assays were developed for both species of Tapesia from these primer pairs, using competitive PCR . Competitive PCR was used to determine the level of colonization of seedlings by each species in glasshouse- and field-inoculated cereal hosts and results compared to those for conventional seedling disease assessment.     

The role of organic vs. conventional farming practice,soil management and preceding crop on the incidence of stem-base pathogens on wheat

Journal of Plant Diseases and Protection - A stem-base disease complex of winter wheat includes several causal agents, such as eyespot (Oculimacula yallundae and O. acuformis), sharp eyespot...     

First record of Tapesia yallundae as the teleomorph of Pseudocercosporella herpotrichoides var. acuformis, and its occurrence in the field in the Federal Republic of Germany

Tapesia yallundae is reported for the first time in the Federal Republic of Germany. Apothecia were found on wheat and rye stubble at two sites in the north of the country and one in the centre, in April 1988. Isolates of Pseudocercosporella herpotrichoides var. acuformis were obtained from germinating single ascospores, identified on the basis of colony morphology, mode of conidial production and pathogenicity to wheat and rye.     

Development of apothecia ofTapesia yallundae in contrasting populations selected by fungicides

Apothecia of the eyespot fungus,Tapesia yallundae, were found on 0–18% of straws in plots of wheat stubble in February–March 1994. The fungicides carbendazim, prochloraz or carbendazim plus prochloraz had been applied repeatedly to the same plots in each of the previous 9 years in which successive wheat crops had been grown. The factors most strongly correlated with the incidence of apothecia were the incidence and severity of eyespot in the preceding wheat crop and the frequency of carbendazim-resistant W-type fungus in populations recovered from that wheat crop. Plots treated with carbendazim, which had previously had more disease and more resistance to carbendazim in the pathogen population relative to untreated plots, therefore yielded most apothecia. Plots treated with prochloraz, which had selected for predominantly R-type fungus and decreased eyespot, yielded few apothecia. Single-ascospore isolates were all of the W-type and were more frequently carbendazim-sensitive than expected, except those from plots treated only with carbendazim. None showed decreased sensitivity to prochloraz. The implications of applying fungicides regularly for controlling eyespot on the capability of the eyespot fungus for genetic variation through sexual reproduction are discussed.     

Incidence of apothecia of Tapesia yallundae at setaside sites in England and sensitivity of the ascospore offspring to the fungicides benomyl and prochloraz

A total of 45 field sites in England were surveyed once for the presence of apothecia of Tapesia yallundae from 1992 to 1994. Apothecia were found at 21 locations and were mainly present on less than 3% of stems. However, apothecia were found on 1532% of stems at four sites. Analysis of the growth characteristics of ascospore isolates from seven sites showed that most produced colonies characteristic of the W-type of T. yallundae , with only one site yielding the R-type, Most ascospore isolates were resistant to the fungicide benomyl and effectively all remained sensitive to prochloraz. The results of the study are discussed in relation to the fungicidal control and epidemiology of T. yallundae , and the risk of spread of disease from set-aside sites.     

Development of Oculimacula yallundae and O. acuformis (eyespot) lesions on stems of winter wheat in relation to thermal time in the UK

Relationships between development of eyespot, caused by Oculimacula yallundae (OY) or O. acuformis (OA) on stems of winter wheat (cv. Avalon), and thermal time (°C days after sowing) were investigated in field experiments in 1985/86, 1986/87 and 1987/88 (two experiments). In all experiments, the incidence and severity of stem eyespot (uninoculated plots, OY- and OA-inoculated plots) were linearly related to accumulated thermal time after sowing. There were ca. 600–800°C days from the time of the first sample when eyespot lesions were recorded on stems to the time when maximum eyespot incidence or severity was recorded. Relationships between stem eyespot incidence or severity and thermal time differed between seasons, with more severe eyespot in 1986/87 and 1987/88 than 1985/86. Both the severity and volume of stem lesions were initially greater in OY-inoculated plots than OA-inoculated plots in spring but differences were less by harvest in all seasons. The percentage of plants with stems colonized by OY or OA over all plots (including uninoculated) showed a consistent pattern in 1986/87 and 1987/88 (two experiments), with the percentage colonized by OY greater initially and the percentage colonized by OA gradually increasing with time towards harvest.     

Occurrence of Tapesia yallundae apothecia on field and laboratory-inoculated material and evidence for recombination between isolates

Tapesia yallundae was observed in early July on wheat straws, which had been inoculated with isolates of Pseudocercosporella herpotrichoides (W-type) 9 months previously and placed in plots of winter wheat, to act as inoculum in a resistance screen. A perfect state was not observed on straws inoculated with R-type isolates in the same experiment. One hundred and thirty-four single-ascospore isolates were obtained and observations on morphology in culture, pathogenicity to wheat and rye seedlings, resistance to benomyl and the pattern of restriction fragment length polymorphisms demonstrated that all isolates were W-type, although some grew at half the expected rate on agar, and at least two isolates were involved in the production of apothecia. Apothecia were produced in vitro on straws inoculated with single isolates of P. herpotrichoides only when they were incubated in contact with other straws similarly inoculated with certain other isolates. No apothecia were produced on straws with only isolate present, suggesting the need for at least two strains, possibly representing different mating types, for apothecium formation. Apothecia of T. yallundae were produced in vitro on straw pieces which had been inoculated individually with W-type isolates, 14-16 weeks after straws of different isolates were mixed. Evidence for genetic recombination between isolates was obtained when single-ascopore colonies were analysed for resistance to benomyl in culture and for restriction-length polymorphisms (RFLPs).     

Visual disease and PCR assessment of stem base diseases in winter wheat

Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four-week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum , F. culmorum , both varieties of Microdochium nivale , both eyespot-causing species of Tapesia and Rhizoctonia cerealis . PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R-type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var . majus , accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.     

Comparison of isoenzyme and DNA markers for differentiating W-, R- and C-pathotypes of Pseudocercosporella herpotrichoides

Biochemical differences between the wheat, rye and couch-grass pathotypes of the eyespot pathogen, Pseudocercosporella herpotrichoides, have been identified by comparing isoenzyme profiles and DNA markers from several isolates. The wheat (W) and rye (R) pathotypes were clearly differentiated by both techniques, with isoenzyme profiling also separating the couch-grass isolates from the W- and R-type isolates. The isoenzyme profiles separated the P. herpotrichoides pathotypes from the two related species, P. anguioides and P. aestiva. The isoenzyme profiles of P. anguioides were closer to those of the R-type isolates of P. herpotrichoides than the W-type isolates, whereas the banding pattern of P. aestiva was very different. The isoenzyme profiles of the two German isolates, originally obtained from Nirenberg's laboratory, P. herpotrichoides var. herpotrichoides and var. acuformis, were similar to those of the wheat and rye pathotypes, respectively, isolated from UK-infected material.     

Isolation of R-type progeny of Tapesia yallundae from apothecia on wheat stubble in England

Apothecia of Tapesia yallundae were collected from a set-aside straw stubble site in Lincolnshire in March 1993. Single ascospore isolates were obtained which produced colonies with morphologies and growth rates characteristic of the R-pathotype of Pseudocercosporella herpotrichoides. Isolates were confirmed to be R-type by the use of randomly amplified polymorphic DNA (RAPD) markers. These observations confirm that T. yallundae is the teleomorph of the R-type of P. herpotrichoides and represent the first detection of the sexual stage of the R-type in the UK.     

Differential seedling resistance to the eyespot pathogens,Oculimacula yallundae and Oculimacula acuformis,conferred by Pch2 in wheat and among accessions of Triticum monococcum

Eyespot is an economically important stem‐base disease of wheat caused by two fungal species: Oculimacula yallundae and Oculimacula acuformis. This study investigated the efficacy of two sources of resistance, viz. the genes Pch1, introgressed into hexaploid wheat from Aegilops ventricosa, and Pch2, identified in wheat cv. Cappelle Desprez, against O. yallundae and O. acuformis separately. In a series of seedling bioassays Pch1 was found to be highly effective against both species. Although Pch2 was found to confer resistance against both pathogen species, it was significantly less effective against penetration from O. yallundae than O. acuformis. Furthermore, a quantitative trait locus (QTL) analysis was not able to locate any resistance to O. yallundae on chromosome 7A of Cappelle Desprez. This has important implications for the use of Pch2 in commercial cultivars as it is necessary to have genes that confer resistance to both pathogens for effective eyespot control. In addition, a set of 22 T. monococcum accessions was screened for resistance to both O. yallundae and O. acuformis to identify potentially novel resistances and to assess the accessions for evidence of differential resistance to the eyespot species. Significant differences in resistance to the two pathogens were identified in four of these lines, providing evidence for differential resistance in T. monococcum. This study demonstrates that future screening for novel sources of eyespot resistance should investigate both pathogen species.     

Incidence of eyespot in winter wheat and quantification of the fungi Oculimacula acuformis and O. yallundae in Lithuania

Journal of Plant Diseases and Protection - Eyespot, caused by Oculimacula acuformis and O. yallundae, is one of the most damaging diseases in areas, including Lithuania, where mild and damp autumn...     

A New Source of Resistance to Tapesia yallundae Associated with a Homoeologous Group 4 Chromosome in Thinopyrum ponticum

ABSTRACT Wheat (Thinopyrum ponticum line SS767; PI 611939) with 42 chromosomes previously was identified as a new source of eyespot resistance. Individual plants of SS767 were tested for reaction to Tapesia yallundae, the major pathogen of eyespot in the Pacific Northwest region of the United States. Resistance of this line was similar to the resistant winter wheat cv. Madsen (carrying gene Pch1 for eyespot resistance). Polymerase chain reaction analysis with primers specific for the J or E genomes revealed that SS767 contains Thinopyrum chromatin. Cytological and Cbanding analyses demonstrated that SS767 is a chromosome substitution line in which wheat chromosome 4D is replaced by a homoeologous group 4 chromosome of Thinopyrum ponticum. Genomic in situ hybridization using St genomic DNA from Pseudoroegneria strigosa as a probe, which can differentiate chromosomes from different genomes of Thinopyrum, indicated that this chromosome belongs to the J genome. Molecular analysis of an F(2) population segregating for chromosome 4J and resistance to eyespot confirmed that eyespot resistance in line SS767 is associated with chromosome 4J of Thinopyrum ponticum. This is the first report of genetic control of resistance to eyespot derived from Thinopyrum ponticum. This source of resistance provides a new opportunity to improve wheat resistance to eyespot by adding to the diversity of resistance sources available.     

Detection and quantification of Pratylenchus thornei in DNA extracted from soil using real-time PCR

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.     

Oculimacula yallundae lifestyle revisited: relationships between the timing of eyespot symptom appearance, the development of the pathogen and the responses of infected partially resistant wheat plants

Possible relationships between the timing of eyespot symptom appearance, the development of Oculimacula yallundae and the responses of infected wheat plants were investigated. By comparing, for 45 days after inoculation (d.a.i.), eyespot- and mock-inoculated coleoptiles and leaf sheaths of a wheat line carrying the Pch1 partial resistance gene, this study showed that cell death, used as a criterion for symptom appearance, occurred on the first leaf sheath around 30 d.a.i. Monitoring of O. yallundae development by optical and scanning electron microscopy revealed that the fungus colonized the coleoptile within 30 d.a.i. It then attacked the first leaf sheath and underwent morphological and growth modifications. For 30 d.a.i., infected plants responded by local accumulations of H₂O₂ and callose in coleoptile epidermal cells and by up-regulation of pathogenesis-related (PR) genes. From 30 d.a.i., natural browning occurred in the first leaf sheath epidermis and the expression level of PR genes, determined by quantitative PCR, significantly increased in infected plants. These results show that the wheat/ O. yallundae interaction is characterized by two distinct phases: the asymptomatic phase and the symptomatic phase. The possibility that O. yallundae should be considered hemibiotrophic rather than necrotrophic is discussed.