Pseudocercosporella Herpotrichoides Research Articles

Screening for potential antagonists of Pseudocercosporella herpotrichoides, the causal agent of eyespot disease of cereals

The eyespot fungus Pseudocercosporella herpotrichoides survives on straw colonized during infection of a previous cereal crop, which then serves as an inoculum source for subsequent infection cycles. Tests were devised to identify fungal antagonists capable of competing with the pathogen on straw, and suppressing inoculum production and host infection. The 228 fungal isolates from wheat seedlings, plants or straw were screened for activity, along with 13 fungi and a commercial strain of Streptomyces griseoviridis which had proven biocontrol activity against other fungal pathogens. Potential antagonists were selected on the basis of inhibition or overgrowth of P. herpotrichoides on several contrasting nutrient media. Co‐inoculation of straw with the pathogen and test antagonists reduced disease severity in pot trials using this straw as an inoculum source. When straw pre‐inoculated with the pathogen was used, fewer antagonists proved effective in suppressing eyespot disease. However, one isolate, a Trichoderma sp., gave positive results both in vitro and in infection trials using different inoculation procedures, and may therefore be an effective antagonist of P. herpotrichoides during the saprophytic survival phase of the pathogen life cycle.

Crop rotation with pure stands and mixtures of barley and wheat to control stem and root rot diseases

Crop rotations are known to be a natural barrier against a wide range of fungal soil-borne pathogens. Results shown in this paper are based on observations that were made in the third growing period of a 3-year cereal crop rotation (1989–1991). Mixtures as pre-crops and main crops were compared with pure stands of barley and wheat also grown as pre- and main crops. After this 3-year period, field experiments showed that interspecific mixtures of winter barley and winter wheat reduced stem rot caused by Pseudocercosporella herpotrichoides, Fusarium spp., Gaeumannomyces graminis and Rhizoctonia cerealis. Growing barley and wheat after a cereal mixture resulted in an increased number of healthy plant stems. The pre-crop mixture significantly reduced the disease incidence of Fusarium spp. on barley (> 50%) during stem elongation and ripening. Disease incidence of G. graminis on barley during ripening decreased from 44% after barley to 9% when grown after the mixture. Differences in take-all incidence on wheat due to different pre-crops were not significant. The percentage of P. herpotrichoides lesions on wheat grown in a mixture increased after pre-crops containing barley. Furthermore, interactions occurred between pathogens, indicating differences in competition. Additional effects on plant health were observed where barley-wheat mixtures were grown for 2 years. In general, the incidence of stem base diseases was lowest in mixtures that were grown after a mixture. Disease reductions on wheat grown in a mixture after a mixed pre-crop were comparable to those disease reductions brought about by chemical fungicides applied in pure stands of wheat. Pathogen frequency on wheat hypocotyl and root tissue was estimated using a selective medium: the frequency of G. graminis on wheat tissue decreased from 14% after the pre-crop barley and 7% after the pre-crop wheat, to 1% after the pre-crop mixture. The frequency of Fusarium spp. was generally not affected, but the spectrum of species changed quantitatively. A greater number of weak or non-pathogenic fungi were isolated from wheat segments when the plants were grown after a mixed pre-crop.

Parasexual recombination between W and R pathotypes of Pseudocercosporella herpotrichoides through protoplast fusion

Vegetative incompatibility between W and R pathotype strains of Pseudocercosporella herpotrichoides was overcome by fusion of protoplasts of two auxotrophically marked strains, yielding viable fusion products. Fusion frequency was low ( P. herpotrichoides usually contain only a single nucleus. Consequently fusion products were therefore considered to represent diploid colonies. Stable progeny were recovered from three separate fusion products after exposure to carbendazim, the N-phenylcarbamate fungicide MDPC, or both compounds together. Spontaneous breakdown of the fusion products was also shown. Recombination between the pathotypes was proven by the recovery of progeny combining auxotrophic markers of the two parental strains. Analysis of isozyme banding phenotypes for five enzyme systems (malate dehydrogenase glutamate dehydrogenase, glucose phosphate isomerase, mannose phosphate isomerase, α-esterase) confirmed the recombinant status of progeny.

Development of disease symptoms and fungal pathogens on shoot bases in continuous winter wheat, and effects of fungicides

Field plots in three consecutive crops of winter wheat were sampled at approximately 2‐week intervals from April to July in 1989, 1990 and 1991. Culm and stem bases were examined for symptoms of eyespot, sharp eyespot and brown foot rot. The W‐type and R‐type of Pseudocercosporella herpotrichoides, P. anguioides, Fusarium culmorum, F. avenaceum and Microdochium nivale grown from this plant material on agar were identified. Eyespot was most severe in 1991, when plant development was least rapid following cool weather in late winter and the summer was relatively cool and wet. Sharp eyespot was most severe in 1990, which had a warm summer with moderate rainfall. The other warm summer, 1989, was drier and these conditions favoured late development of brown foot rot, associated mainly with F. culmorum which was scarce at other times. Sharp eyespot sometimes increased where prochloraz, which decreased eyespot, was applied. Distinct symptoms of more than one disease occurred less frequently on the same stem than expected from the individual total occurrences, but co‐occurrences of different fungi were often more frequent than expected. In July 1990, Fusarium spp. co‐occurred with R‐type, but not W‐type, P. herpotrichoides more frequently than expected, and in July 1990 and 1991 M. nivale and both W‐type and R‐type co‐occurred more frequently than expected. Fusarium spp. and M. nivale were more frequent, especially in the earlier samples, on nodes than on internodes, whilst P. herpotrichoides normally infected at the internodes. The results suggest that stems weakened or altered by a primary colonizer are often a suitable substrate for a secondary colonizer, often a Fusarium sp., which may begin infection at a distance from the original lesion and often not cause distinct symptoms itself.

Mechanism of control of eyespot disease in winter wheat by the fungicide prochloraz

Winter wheat plants grown in troughs and infected with the eyespot pathogen ( Pseudocercosporella herpotrichoides) were sprayed with prochloraz at growth stage (GS) 31 and 39 using a hydraulic nozzle sprayer and with a drop-leg spray system at GS 39. Some plants received artificial rain (4 mm) 1 or 4 days after spraying and were compared with those not receiving rain, with respect to foliar and stem-base fungicide deposits, as well as control of eyespot disease. Single pot-grown plants were treated topically with prochloraz inside the leaf axils and examined for redistribution of the fungicide by rain as well as the effect on eyespot disease. Rain redistributed the fungicide deposited by the hydraulic nozzle spray system from the initial site of deposition to the base of the crop at GS 31 and 39 and significantly suppressed eyespot disease development. No similar effects were found on the crop sprayed by the drop-leg system. The effect of rain on plants treated by topical placement of the fungicide in leaf axils was to redistribute prochloraz downwards between the outer leaf sheath and the stem, thus suppressing eyespot disease.

Development of a monoclonal antibody immunoassay for the eyespot pathogen Pseudocercosporella herpotrichoides

A double‐antibody‐sandwich ELISA test has been developed for the detection of Pseudocercosporella herpotrichoides using a highly specific monoclonal antibody PH‐10 as the capture antibody and genus‐specific rabbit polyclonal antiserum as the detector antibody. The assay recognizes extracts from plants both artificially and naturally infected with P. herpotrichoides giving at least three‐fold higher absorbance values with extracts from Pseudocercosporella‐infected tissue than with extracts from healthy tissues or from tissues naturally infected with Microdochium nivale, Rhizoctonia cerealis or material artificially inoculated with P. anguioides. The assay tested positively against all isolates of P. herpotrichoides, including both W‐type and R‐type isolates. In this assay system, extraction of the antigen from the stem bases of infected plants is a one‐step process not requiring any dilution procedures. The assay can be used to detect the pathogen in presymptomatic infected seedlings. The immunogen used to generate the specific monoclonal antibody and the rabbit antiserum was a mycelial extract from which the high‐molecular‐weight proteins and glycoproteins had been removed by ammonium sulphate precipitation. The high‐molecular‐weight fraction was shown to contain cross‐reactive antigens; it induced antiserum in mice that cross‐reacted with the other stem‐base fungi even at high dilutions. The monoclonal antibody PH‐10 is an IgM antibody. Heat and periodate treatment of the antigen indicate that it is a glycoprotein and that the epitope recognized by the antibody is a protein.

Classification of a world‐wide collection of isolates of Pseudocercosporella herpotrichoides by RFLP analysis of mitochondrial and ribosomal DNA and host range

Two profiles of mitochondrial DNA (mtDNA) were detected among 59 isolates of Pseudocercosporella herpotrichoides from diverse geographical sources. One profile (type II) was present only among isolates pathogenic on rye (R‐type), and a second profile (type I) was present in W, C and S‐pathotypes of the fungus. Mitochondrial DNA from P. herpotrichoides hybridized strongly to DNA of P. anguioides, but very weakly to DNA from P. aestiva, suggesting that the latter is not closely related to P. herpotrichoides. Fifteen profiles of ribosomal DNA (rDNA) were observed among isolates with type I mtDNA, and five of these were only found among seven isolates from Ireland. The other profiles present among type‐I isolates appeared to be largely independent of geographic origin. Three rDNA profiles were observed among R‐type (type II mtDNA) isolates.

Effects of rotation, stubble retention and cultivation on take-all and eyespot of wheat in northeastern Victoria, Australia

The effects of different rotations and methods of seed-bed preparation on take-all ( Gaeumannomyces graminis var. tritici) ( Ggt) and eyespot ( Tapesia yallundae; anomorph Pseudocercosporella herpotrichoides) of wheat ( Triticum aestivum) were investigated in 1984–1986 in a long-term field experiment (established in 1981), on a fine sandy-clay loam in northeastern Victoria. One treatment, representing the conventional practice, involved burning stubble from the preceding crop followed by four passes with a scarifier in the autumn before sowing wheat each year. Other treatments were direct drilled each year (sown without prior cultivation) and included two stubble treatments (retained or burnt) in either continuously cropped wheat or in lupin ( Lupinus angustifolius)-wheat rotations. High levels of take-all and eyespot developed in the continuously cropped wheat (up to 79% and 66% plants affected, respectively). On the other hand, the incidence of plants with either disease was very low or negligible in wheat that was preceded by lupins. In continuous wheat the incidence of plants with Ggt lesions were up to two times higher in the conventionally cultivated treatment (14% 74% and 79% plants affected in 1984, 1985 and 1986, respectively) than in direct drilled treatment (5%, 36% and 39% plants affected, respectively). Generally, the incidence of plants with Ggt lesions and the severity of root symptoms in direct drilled wheat did not differ significantly between the two stubble treatments. The incidence of eyespot in direct drilled wheat was exacerbated by the retention of wheat stubble on the uncultivated plots (3%, 46% and 66% plants affected in 1984, 1985, and 1986, respectively) but was reduced by burning the stubble (4%, 19% and 33% plants affected in 1984, 1985, 1986, respectively). However, cultivating soil after burning stubble resulted in more plants with eyespot (14%, 39% and 46% plants affected in 1984, 1985 and 1986, respectively) than did burning stubble alone, and in one year the incidence of affected plants in the cultivated treatment was as high as in wheat direct drilled into stubble. The incidence of eyespot was positively correlated ( r=0.85, 0.84 and 0.61 in 1984, 1985 and 1986, respectively) with the incidence of lodged tillers. The incidence of lodging was, generally, highest each year in wheat sown directly into wheat stubble and was reduced by burning wheat stubble or by cropping wheat after lupins. Wojnowicia graminis was frequently isolated, as was Tapesia yallundae, from eyespot lesions suggesting that perhaps not all symptoms of lodging in Victoria can be attributed to Tapesia yallundae.

Screening Selected Fungi for Antagonism towardsPseudocercosporella herpotrichoides(Fron) Deighton, the Cause of Eyespot Disease of Cereals

In the laboratory, a dual culture primary screen using wheat straw agar (WSA) showed that 64 out of 152 selected fungi inhibited the in vitro growth of Pseudocercosporella herpotrichoides by 50% or more. An ‘asymmetric’ species of Penicillium isolated from wheat stubble was the most effective, inhibiting growth of the pathogen by 70%. The results were similar using both W‐type and R‐type isolates of P. herpotrichoides. Other fungi showing in vitro antagonism included: Trichoderma viride, T. koningii, T. harzianum, Chaetomium globosum, Acremonium persicum, Botryotrichum piluliferum, Sordaria alcina, Microdochium bolleyi, Rhizoctonia cerealis and Laetisaria arvalis. In a glasshouse secondary screen, the 64 fungi showing antagonism in vitro were assessed further for their ability to reduce eyespot disease symptoms of wheat seedlings using a straw collar co‐inoculation technique. Of them, 13 fungi reduced disease symptoms significantly: a Chaetomium sp. from stubble, two Fusarium spp., a F. culmorum isolate T. viride, T. koningii, T. harzianum, B. piluliferum, M. bolleyi, L. arvalis, the ‘asymmetric’ Penicillium sp. and two unidentified fungi from stubble. A F. culmorum isolate was the most effective antagonist on average, giving disease reductions of 54 and 58% for the W‐type and R‐type of P. herpotrichoides respectively. M. bolleyi, T. harzianum and the F. culmorum isolate were the most effective antagonists of the W‐type, all three reducing disease by 54%. T. viride was most effective against the R‐type, reducing disease by 64%.

Pathogenicity to Wheat of Isolates of Pseudocercosporella herpotrichoides (Fron) Deighton from Five Graminaceous Hosts

Eleven isolates of Pseudocercosporella herpotrichoides were obtained from field material of the Gramineae Agropyron repens, Alopecurus myosuroides, Apera spica-venti, Hordeum vulgare and Triticum aestivum. Four of these isolates belonged to the variety acuformis and 7 isolates to the variety herpotrichoides of the fungus. In a greenhouse experiment, winter wheat was inoculated with these isolates. All isolates were pathogenic on wheat. They differed in virulence, but these differences could not be related to the nature of their original hosts nor to their classification as variety acuformis or herpotrichoides.

Identification of rye- and wheat-types of Pseudocercosporella herpotrichoides with DNA probes

A partial genomic library of the plant-pathogenic fungus Pseudocercosporella herpotrichoides was prepared in a bacterial plasmid vector. Plasmids from 20 random recombinant clones were radioactively labelled and used as probes in Southern blot experiments with genomic DNA isolated from seven isolates belonging to the rye-type and four isolates of the wheat-type of P. herpotrichoides. Most of the probes tested revealed polymorphisms between the two types. In addition, some of them distinguished different isolates within each type. One probe that gave strong hybridization signals may correspond with repetitive DNA sequences. It was shown by dot-blot hybridization to be specific to all the wheat-type isolates tested.

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.

Eyespot resistance gene Pch-1 in H-93 wheat lines. Evidence of linkage to markers of chromosome group 7 and resolution from the endopeptidase locus Ep-D1b

Gene Pch1, which confers resistance to eyespot disease (Pseudocercosporella herpotrichoides Fron), has been located on chromosome 7D in the H-93 wheat-Aegilops ventricosa transfer lines using isozyme markers and DNA probes corresponding to group 7 chromosomes. Previous experiments had failed to ascertain this location. The lack of segregation of the resistance trait in progeny from reciprocal crosses between lines H-93-70 and VPM1 indicates that their respective resistance factors are allelic. Line H-93-51 carries the endopeptidase allele Ep-D1b but is susceptible to eyespot, which indicates that resistance to eyespot is not a product of the Ep-D locus, as had been proposed in a previous hypohesis.

Measuring the Severity of Eyespot Disease Induced by Pseudocercosporella herpotrichoides (Fron) Deighton in Wheat Cultivars at Different Growth Stages

AbstractFrom the total soluble protein of Pseudocercosporella herpotrichoides (Fron) Deighton, specific proteins were isolated and used as antigens. One antiserum proved to be highly sensitive and was used for quantitative determination of eyespot severity in 16 wheat cultivars. The detection limit in ELISA was calculated as 2.2 μg total fungus protein per ml plant sap. From anthesis, genotypes showed the most characteristic and reliable differentiation for a longer period of time, viz. at growth stages 60 and 75. These quantitative differences could be more successfully demonstrated with ELISA than with eye‐spot scoring. There was, however, a close correlation between both traits. The most resistant genotypes carried the gene Pch‐1 with the resistance originating from ‘Capelle Desprez’. Some genotypes originating from this cultivar showed rather low levels of susceptibility, which might be explained by additional effects of the genotypic background.

Pseudocercosporella herpotrichoides, Fusarium spp. and Rhizoctonia cerealis stem rot in pure stands and interspecific mixtures of cereals

Cereal species mixtures were used to study the effect of increased genetic diversity in a crop stand on major fungal pathogens causing stem-rot diseases. Mixtures of spring-barley/oats, winter barley/winter wheat and winter barley/wheat/oats were tested in field experiments between 1986 and 1990. Disease incidence of Pseudocercosporella herpotrichoides, Fusarium spp. and Rhizoctonia cerealis was greatest on barley and wheat. Lesions caused by Fusarium spp. were prevalent on oats. Stem-rot symptoms were reduced by 50% in barley/oats and barley/wheat mixtures. The reduction was comparable to the effects obtained with fungicide applications. The effects of mixed cropping of barley, wheat and oats varied greatly and the results were dependent on mixture composition. Disease reductions in barley and oats grown in mixtures were greater than those in wheat. In general, higher proportions of oats in a mixture favoured plant health by decreasing the incidence of diseases caused by P. herpotrichoides and Fusarium spp. A change of pathogen dominance often, occurred after the application of fungicides in pure stands. In mixtures, however, total disease pressure was the result of an interacting complex of different pathogens that was less influenced by fungicides. The field experiments demonstrated that the level of disease reductions varied between pathogens, year and mixture composition.

Effects of fungicides on eyespot in five successive crops of winter wheat in plots with different initial or fungicide-selected populations of Pseudocercosporella herpotrichoides

The effects on eyespot of carbendazim, prochloraz or a mixture of carbendazim and prochloraz, applied twice yearly and to the same plots in each year, were studied in five successive crops of winter wheat. Some plots were uninoculated (with a background population of the eyespot fungus, Pseudocercosporella herpotrichoides, which was initially mainly MBC-sensitive W-type), whereas others were inoculated artificially in the first autumn of the experiment (1984) with mainly MBC-sensitive W-type, MBC-resistant W-type, MBC-sensitive R-type or MBC-resistant R-type fungus. Samples were taken in April (1985–1987) and July (1985–1989). When all inoculation treatments are considered together, carbendazim was ineffective at all sampling times after April 1985 because of its rapid selection for MBC-resistance, and increased eyespot incidence and severity in some samples. Prochloraz applied alone decreased eyespot incidence and severity at all sample times except July 1988. Where applied with carbendazim, prochloraz always decreased eyespot incidence and severity, sometimes more so than when applied alone. The control of eyespot by the fungicides was influenced by the initial populations in the first two seasons only. Carbendazim decreased eyespot incidence in 1985 only where no artificial inoculum was added (both samples) and where mainly MBC-sensitive R-type inoculum was added (April 1985). Prochloraz applied alone was ineffective in July 1985 where mainly MBC-sensitive R-type fungus was added, and, applied with carbendazim, was ineffective in July 1986 where mainly MBC-resistant fungus was added. Subsequently, the populations were unrelated to the initial populations and were mainly a result of selection by fungicides where these were applied. Although selecting for the R-type fungus and, where no carbendazim was applied, for MBC-sensitivity, prochloraz remained generally effective after repeated applications over five seasons.

A comparison of cultural, morphological and DNA markers for the classification of Pseudocercosporella herpotrichoides

Isolates of Pseudocercosporella herpotrichoides were classified by a variety of established techniques including colony morphology, colour, pathogenicity on seedlings and spore morphology. In all cases, certain isolates could not be clearly classified and correlation between the systems was incomplete. In particular, the typing system of Nirenberg in which two varieties of P. herpotrichoides (var. herpotrichoides and var. acuformis) are recognized did not correlate with the widely used UK system based on colony morphology on PDA: fast, even (F E) and slow, feathery (S/F). Restriction fragment length polymorphisms in total DNA and rDNA indicated groupings related to morphology (and pathogenicity) with considerable polymorphisms in rDNA of F/E isolates. Generally, good correlation was found between RFLP markers and other tests.

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).

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.

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.