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.     

Species and Mating-Type Distribution of Tapesia yallundae and T. acuformis and Occurrence of Apothecia in the U.S. Pacific Northwest

ABSTRACT Eyespot of wheat is caused by the discomycete fungi Tapesia yallundae and T. acuformis. T. yallundae is considered the most important causal agent of the disease in this region but no apothecia of either species have been found in the U.S. Pacific Northwest (PNW). Two compatible isolates of T. yallundae from the PNW were used to inoculate a field plot in the fall of 1998 and apothecia developed in the spring and fall of 2000 on standing wheat stubble. In the spring of 2000, wheat stubble from eight naturally infected fields was examined for the presence of apothecia of T. yallundae and T. acuformis. Apothecia of T. acuformis were found in two fields but no apothecia of T. yallundae were found. This is the first report of apothecia of the eyespot pathogens occurring in the PNW. Species and mating-type distribution of T. yallundae and T. acuformis in the PNW were determined from 817 isolates collected from diseased wheat over 3 years at spatial scales ranging from within fields to across states. In all, 460 isolates were identified as T. yallundae and 357 isolates were identified as T. acuformis with MAT1-1/MAT1-2 ratios not significantly different from 1:1 based on chi(2) tests at most scales tested. The apparent increase in frequency of T. acuformis from previous surveys may indicate a shift in the predominant species causing eyespot. The occurrence of apothecia under field conditions, along with the widespread distribution of mating types of both species, suggests that sexual reproduction may be occurring in both species.     

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.     

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).     

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.     

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.     

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 production and release of ascospores of Pyrenopeziza brassicae on oilseed rape

The results of observation for three seasons in oilseed rape crops of the production and release of ascopores of Pyrenopeziza brassicae , the cause of light leaf spot disease of brassicas, are presented. Large apothecia (1–2 mm in diameter) take at least 3 weeks to develop on leaf petioles after infected leaves die, but small apothecia (50–200 um) may form on leaf lamellae after about 15 days. Apothecia were found on all 12 oilseed rape varieties examined. Spore trapping experiments in infected crops show that ascospore release is associated with rain, but most ascospores are released after rainfall when the crop debris bearing apothecia are wet. Ascospores can be released for up to 5 days after rain. Laboratory measurements show that apothecia can continue to release ascospores for up to 3 weeks even when they are subjected to wet and dry cycles. The consequences of the widespread occurrence of the sexual stage of P. brassicae are discussed and possible cycles of disease and spore production in oilseed crops are suggested.     

Effects of Environmental Factors on Development of Pyrenopeziza brassicae (Light Leaf Spot) Apothecia on Oilseed Rape Debris

ABSTRACT The development of Pyrenopeziza brassicae (light leaf spot) apothecia was studied on petiole debris from artificially infected oilseed rape leaves incubated at temperatures from 6 to 22 degrees C under different wetness regimes and in 16 h light/8 h dark or continuous darkness. There was no significant difference between light treatments in numbers of apothecia that developed. Mature apothecia developed at temperatures from 5 to 18 degrees C but not at 22 degrees C. The rate of apothecial development decreased as temperature decreased from 18 to 5 degrees C; mature apothecia were first observed after 5 days at 18 degrees C and after 15 days at 6 degrees C. Models were fitted to estimates of the time (days) for 50% of the maximum number of apothecia to develop (t(1); model 1, t(1) = 7.6 + 55.8(0.839)(T)) and the time for 50% of the maximum number of apothecia to decay (t(2); model 2, t(2) = 24.2 + 387(0.730)(T)) at temperatures (T) from 6 to 18 degrees C. An interruption in wetness of the petiole debris for 4 days after 4, 7, or 10 days of wetness delayed the time to observation of the first mature apothecia for approximately 4 days and decreased the number of apothecia produced (by comparison with continuous wetness). A relationship was found between water content of pod debris and electrical resistance measured by a debris-wetness sensor. The differences between values of t(1) predicted by model 1 and observed values of t(1) were 1 to 9 days. Model 2 did not predict t(2); apothecia decayed more quickly under natural conditions than predicted by model 2.     

High genetic diversity, phenotypic uniformity, and evidence of outcrossing in sclerotinia sclerotiorum in the columbia basin of washington state

ABSTRACT Sclerotinia sclerotiorum, the causal agent of potato stem rot, is prevalent and poorly managed on potatoes in the Columbia Basin of Washington. Because of the ubiquitous nature of the fungus and high crop diversity within the Columbia Basin, understanding the population structure and the potential for outcrossing of the pathogen would be helpful in developing disease management strategies. The population structure of S. sclerotiorum in the Columbia Basin from potato was examined using microsatellite markers and mycelial compatibility. Analysis of molecular variance revealed that 92% of the variability among 167 isolates was found within subpopulations, with limited, yet statistically significant impact of the collection date, but not the year or location of collection. Linkage disequilibrium and index of association analyses noted a potential for outcrossing in two locations, which was substantiated by the discovery of recombinant ascospores in three field-generated apothecia from the 12 apothecia examined. Microsatellite haplotypes were not correlated with mycelial compatibility groups. This high haplotypic diversity did not seem to impact pathologically important phenotypes. Greenhouse inoculations of potato plants exhibited no significant differences in aggressiveness on potato stems. Moreover, in vitro studies of response to fungicides and temperature stimuli yielded no significant differences among studied isolates. These findings illustrate the potential for outcrossing in warm temperate regions of North America, where a diversity of crops are planted simultaneously and in neighboring fields. This study also indicates that the unsatisfactory management of potato stem rot is likely not directly attributable to genetic factors, but to gaps in agricultural practices.     

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.     

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.     

Mating type distribution and genetic structure are consistent with sexual recombination in the Swedish population of Phaeosphaeria nodorum

Mating type ratios and SSR marker analysis were used to study the genetic structure of Phaeosphaeria nodorum , the causal agent of glume blotch in wheat. The study was based on leaf collections in five fields located in different regions in Sweden. In total 302 isolates of P. nodorum were obtained from 203 sampling sites (including eight ascospore isolates). Three strong indications of sexual recombination were found: (i) the two mating types were present at a 1:1 ratio; (ii) the genetic structure was diverse, with many unique genotypes, and 69 of the 93 genotypes were only found once; and (iii) random association of alleles indicated that genetic recombination was frequent. However, asexual reproduction could not be excluded since identical genotypes were found within the fields. The fungal population had experienced a demographic bottleneck, as indicated by a low ratio of number of alleles to microsatellite size range ( M -value) of 0·5.     

In vitro evaluation of fungal isolates for their ability to influence leaf rheology,production of pseudothecia,and ascospores of Venturia inaequalis

As part of an ongoing research project on biological control of apple scab, this study presents a novel approach for the in vitro selection of potential antagonists of the saprophytic phase of Venturia inaequalis. A collection of forty-two fungal isolates were tested for their in vitro ability to degrade apple leaf tissue, inhibit pseudothecia, and ascospore production. The inhibition of ascospore production cannot always be linked reliably with leaf degradation or the evaluation of pseudothecia production. Consequently, ascospore production was retained as the most useful screening parameter. Six isolates proved to significantly reduce the ascospore production of Venturia inaequalis. Two were as effective as Athelia bombacina, a previously reported antagonist of pseudothecia formation and inhibited over 98% of the ascospore production. These new organisms are now available for future field tests. Future selections from a large collection of fungal and bacterial saprophytes can now be based on a reliable and simple in vitro screening method.     

Occurrence of Tapesia yallundae, teleomorph of Pseudocercosporella herpotrichoides, on unharvested wheat culms in England

Apothecia of Tapesia yallundae are reported for the first time in the UK. They were observed on decaying stems and leaf sheaths of unharvested wheat near Bristol in January 1989. Isolates from single ascospores produced cultures typical of the W-type form of the anamorph, Pseudocercosporella herpotrichoides . Pathogenicity on wheat was confirmed by inoculation and re-isolation from glasshouse-grown wheat seedlings.     

Characterization of Sclerotinia minor populations in Texas peanut fields

In the summer of 2004 an epidemic of sclerotinia blight of peanut, a disease caused by Sclerotinia minor, occurred in Texas in fields where the disease was never previously detected. The disease was observed on many plants within one of the fields (>3000 disease foci), although most foci were <1 m. It is hypothesized that these observations were inconsistent with the recent introduction of a monocyclic pathogen, even if disease developed under conducive environmental conditions. The pattern of disease is most suggestive of the presence of foliar (ascospore) infections, although air temperature was above the known limits for apothecia development if the pathogen had arrived in the field in 2004 peanut seed. To further examine this epidemic, 232 isolates were collected, across a variety of spatial scales spanning this field and other Texas peanut fields, and evaluated for aggressiveness, fungicide sensitivity and genotypic diversity. There was wide variation among isolates for the phenotypic characteristics measured, but there was no evidence that a genotypically unique, highly aggressive, and fungicide resistant isolate had been introduced or evolved. The predominant genotype, TX1, which contained 154 isolates, was found in every county and field population.     

Control of lettuce drop disease, caused by Sclerotinia sclerotiorum, with metham-sodium soil treatment and foliar application of benomyl

The effects of metham-sodium (MES) soil treatment, a varying number of benomyl foliar sprays (two, three and six) and combined treatments on control of lettuce drop disease were studied at two sites in Israel naturally infested with the pathogen Sclerotinia sclerotiorum. MES killed 85% of the S. sclerotiorum sclerotia in the top 10 cm of soil where initial populations were 0.6 and 1.6 viable sclerotia per kg soil at the two sites. Of the remaining viable sclerotia. only 30% produced apothecia. MES treatment alone reduced numbers of apothecia to 5% of those counted in the unsprayed control treatment. Numbers of ascospores deposited on the crop in the MES-treated plots were 7-20% of those deposited on control plots. Lettuce drop decreased the total yield by 30% in unsprayed control plots; MES application reduced the loss to 4% and also increased the number of marketable plants. Benomyl sprays alone also significantly reduced numbers of apothecia and disease development and consequently increased yield, although not to the extent observed with the MES treatment alone. Combined treatments produced the best disease control, but this improvement was not accompanied by any major increase in yield compared to that with MES treatment alone.     

Characterization of monilia disease caused by Monilinia linhartiana on quince in southern Spain

This study elucidates the aetiology and epidemiology of monilia disease of quince caused by the fungus Monilinia linhartiana in Spain. Disease incidence and the dynamics of apothecial development and ascospore discharge were quantified and the pathogen was characterized using morphological and molecular methods. The pathogen did not produce conidia or apothecia on agar media but produced conidia on leaves showing symptoms and apothecia on mummified young quince fruit. Monilinia linhartiana was not pathogenic on ripe quince fruit but was readily isolated from developing, mummified fruit (pseudosclerotia). Phylogenetic analysis based on 5·8S‐ITS region sequences placed M. linhartiana in the Disjuntoriae section of Monilinia species infecting rosaceous hosts. Studies during 2004–2008 in four commercial orchards in southern Spain determined two major infection periods for the disease. The first coincided with the unfolding of the first leaves and resulted in leaf blotch and shoot blight. The second coincided with flowering and led to mummification of developing young fruit. Foliar infection was apparently initiated by airborne ascospores produced on pseudosclerotia that overwintered on the soil surface, while flower infection was probably initiated by conidia produced on leaf lesions. Incidence of diseased shoots ranged from 1 to 91% and was correlated with calculated inoculum potential, based on the density and maturity of apothecia formed on pseudosclerotia. This epidemiological study has made it possible to characterize the life cycle of monilia disease on quince in southern Spain, which will help the development of new control strategies.     

Factors affecting the production of apothecia and longevity of sclerotia of Sclerotinia sclerotiorum

Sclerotia of Sclerotinia sclerotiorum produced naturally on winter oilseed rape and by an isolate from this host on sterile wheat grain were placed in two different soil types in pots, either on the soil surface or 1. 2, 3, 4 or 6 cm deep. Nitrogen (as TN 34, Thames Nitrogen Co.) was applied to one set of pots in autumn 1982 and spring 1983. Production of apothecia was recorded in this set in 1983 only. In pots without added N apothecia were counted in 1983 and 1984 and sclerotia were recovered from these pots at the end of the experiment.
Overall, production of apothecia was not affected by soil type. More apothecia formed from cultured sclerotia and they appeared earlier than from natural sclerotia. More apothecia were produced from sclerotia placed at 0–2 cm than from those buried deeper. Fewer apothecia were produced in pots with added N and their production was delayed. Fewer sclerotia were recovered from the upper layers of soil with a higher clay content, and when sclerotia were obtained from oilseed rape rather than from cultures.     

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.