Detection of Pseudocercosporella herpotrichoides (Fron) Deighton in Wheat by Indirect ELISA

An indirect ELISA for quantitative detection of P. herpotrichoides in fections in wheat is presented. All tested isolates of the virulent varieties P. herpotrichoides var. herpotrichoides, P. h. var. acuformis or the W- and R-type react on a high level in the test, while the less virulent P. anguioides is assessed only with 40% and the avirulent P. aestiva with 20% of the homologous reaction. No crossreactions occur with extracts of 11 other species of in, vitro cultivated fungi nor with plant material infected with other pathogens. The infection profile throughout the leaf-sheaths was clearly reflected by ELISA. The examination of 24 stembase samples from the field showed that the values assessed by ELISA correlate well also with the disease indices of naturally infected plant material.     

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.     

Negative cross-resistance between benomyl and MDPC in British isolates of Botrytis cinerea, Pseudocercosporella herpotrichoides and Verticillium fungicola var. fungicola

Fifteen isolates of Pseudocercosporella herpotrichoides, four isolates of Botrytis cinerea and four isolates of Verticillium fungicola var. fungicola were examined on potato dextrose agar amended with benomyl or methyl N-(3, 5-dichlorophenyl)-carbamate (MDPC). Negatively correlated cross-resistance was clearly demonstrated with the isolates of P. herpotrichoides and B. cinerea. There were indications that the same phenomenon might also operate with the isolates of V. fungicola var. fungicola.     

The Development of Eyespot on Seedling Leaf Sheaths in Winter Wheat and Winter Barley Crops Inoculated with W-type or R-type Isolates of Pseudocercosporella herpotrichoides

In 1986–88 the development of eyespot lesions in winter wheat or winter barley differed plots inoculated with W-type isolates of Pseudocercosporella herpotrichoides and plots inoculated with R-type isolates. In the spring of 1986, after a cold winter, the incidence (%shoots infected) and severity (number of leaf sheaths penetrated) of eyespot lesions in wheat before GS 30/31 were greater in plots inoculated with R-type isolates than in those inoculated with W-type isolates. In 1987, after amild winter, eyespot incidence and severity in both wheat and barley were initially greater in W-type plots than in R-type plots. However, by GS 30/31 or 1987. In 1988, when the crop was October-sown, eyespot incidence and severity were greater in W-type than in R-type plots at GS 30/31. Differences in eyespot incidence and severity between W-type and R-type plots were smaller in barley than in wheat. Both the incidence and severity of eyespot were greater in early-sown than in late-sown plots. Seed rate, had little effect on the rate of lesion development in 1987, but in 1988 the rate of penetration was less at the low seed rate for both wheat and barley.     

Effects of water potential and temperature on the development of eyespot lesions in wheat

Growth of Pseudocercosporella herpotrichoides on potato dextrose agar at water potentials from -0.5 to -6.9 MPa was optimal at 20°C. At 12 and 20°C, six isolates of P. herpotrichoides grew more rapidly at -0.5 to -2 MPa than at -6.9 MPa. Wheat plants inoculated with P. herpotrichoides and grown in columns of soil at either 15 or 20°C developed more severe eyespot lesions under a heavy watering regime than under medium or light watering regimes. P. herpotrichoides penetrated leaf sheaths of inoculated plants grown in compost more rapidly at 10°C night/15°C day temperatures than at 5/10°C; death of leaf sheaths was also more rapid and consequently there were fewer living infected leaf sheaths at 10/15°C than at 5/10°C. Irrigating for 5 wk before harvest increased the severity of eyespot lesions in a 1983 wheat crop.     

Strategies for control of eyespot (Pseudocercosporella herpotrichoides) in UK winter wheat and winter barley

This review considers strategies for control of eyespot (Pseudocercosporella herpotrichoides) in winter wheat and winter barley in the UK. Varietal resistance to eyespot may take the form of direct resistance to the growth of the pathogen in the stem base or of indirect tolerance to eyespot through resistance to lodging. The French variety Cappelle-Desprez has been a source of resistance for most UK wheat varieties and a new source of resistance in wild goat grass has been discovered recently. Use of fungicides for control of eyespot increased rapidly after the introduction of the highly effective MBC fungicides in the 1970s, but has decreased recently because UK populations of P. herpotrichoides are now predominantly resistant to MBC fungicides and alternative fungicides are more expensive. Historically, cultural methods, especially crop rotation, have been important in the control of eyespot. The importance of cultural control of eyespot is now increasing because more non-cereal break crops are being grown and fungicide use is declining.     

Tapesia yallundae collected from wheat stubble in South Africa

Tapesia yallundae var.yallundae is newly recorded from wheat stubble collected near Moorresburg in the western Cape Province of South Africa. Apothecia were observed on wheat stubble incubated for 8 months at 10°C under near-ultraviolet light. Single ascospore isolates produced colonies typical of the Wheat (W)-type in culture. Apothecia were also induced after single-conidial isolates were mated on wheat stubble and incubated at 10–15°C for 6 months. All single-conidial isolates were of the W-pathotype (Ramulispora herpotrichoides var.herpotrichoides).     

Prediction of Eyespot Severity on Winter Wheat or Winter Barley Inoculated with W-type or R-type Isolates of Pseudocercosporella herpotrichoides

In crops of winter wheat (1986—88) or winter barley (1987—88) inoculated with W-type or R-type isolates of Pseudocercosporella herpotrichoides and sown on different dates (1986) or at different seed rates (1987,1988) eyespot epidemics developed in different ways. Methods of measuring eyespot incidence/severity during crop growth were compared for their ability to predict eyespot severity at grain filling. Regressions were calculated for eyespot severity score at GS 71 on earlier measurements, either at GS 30/31 (11 methods) or from GS 22 to GS 65 (3 methods). Based on measurements at GS 30/31, all the methods predicted eyespot severity at GS 71 well in plots of winter barley inoculated with W-type isolates (r, 0.83—0.97) but the accuracy of predictions in plots inoculated with R-type isolates was very variable (r, 0.09—0.71). Predictions for 1987 and 1988 were less accurate in wheat than in W-type plots of barley, but did not differ between W-type and R-type plots (r, 0.70—0.89). When the wheat data for 1986 were also included predictions were less accurate, especially in R-type plots (r, 0—0.59). Generally, it was easier to predict eyespot severity at GS 71 in W-type than in R-type plots, especially in barley and in wheat before GS 37/39. Predictions of eyespot severity at GS 71 based on measurements before GS 25 were inaccurate for both wheat and barley. After GS 25 the accuracy of the prediction was generally good in W-type plots and did not improve greatly except in wheat after GS 59. However, there was a steady improvement in the accuracy of the prediction in R-type plots of barley from GS 24 to GS 53. Assessments of eyespot incidence on stems predicted eyespot severity at GS 71 more accurately than assessments on leaf sheaths on wheat after GS 37/39, but were not as good on barley until GS 53.     

Réactions histologiques de blés, résistants ou non àľinfection par Pseudocercosporella herpotrichoïdes

Histological reactions of wheat resistant or sensitive against infection of Pseudocercosporella herpotrichoides The microscopic observations reveal important histological differences between the sensitive or resistant varieties. The disease rapidly affect the whole of the first three leafsheath tissues in the “Étoile de Choisy” variety; in the “Roazon” variety, it only affects the first two sheaths. The inter- or intracellular mycelia can be observed both under the form of filaments more or less dilated or under the form of dense segments in the sensitive variety. However in the resistant variety, only short segments can be observed. The tissues of those two varieties are gradually destroyed when they are in contact with P. herpotrichoides hyphae, whereas the hyphae remain undamaged. In “Etoile de Choisy” the hyphae develop in great quantity whereas in “Roazon”, the development remains limited. In the variety Ae. ventricosa no. 11, which is very resistant, the parasite rarely penetrates into the more external cell layers of the first leaf sheath,‘those invaded host cells are destroyed by the mycelium but, at the same time, the mycelian hyphae degenerate and dilate. Thus, the Ae. ventricosa no. 11 tissues seem to be the seat of a strong fungic toxic reaction.     

Growth and isoflavonoid accumulation of Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

We established cell suspension cultures derived from leaf, stem, and root calli of Pueraria candollei var. candollei and P. candollei var. mirifica using liquid Murashige and Skoog (MS) medium supplemented with 0.56 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Growth of the cell suspension cultures progressed to the stationary phase within 15–24 days. Methanolic extracts of cell suspension cultures of both varieties of P. candollei were analyzed using a validated HPLC protocol. All cell lines derived from leaf, stem, and root explants produced four major isoflavonoids: daidzein, daidzin, genistein, and genistin; these isoflavonoids were detected only in the roots of intact plants. Furthermore, the isoflavonoid contents of the cell suspension cultures were higher than those of intact plants. Thus, cell suspension culture of both varieties of P. candollei may be an effective tool for isoflavonoid production.     

Surveys of stem base diseases and fusarium ear diseases in winter wheat in England, Wales and Scotland, 1989–1990

An investigation into the incidence of stem base diseases and the pathogens associated with them was undertaken on winter wheat samples collected at growth stages 31 and 73–75 in 1989 and 1990. Symptoms at growth stage 31 were classified into 10 different types based on visual characteristics. Although Pseudocercosporella herpotrichoides was associated mainly with eyespot lesions and Rhizoctonia spp. with sharp eyespot lesions, a significant number of isolates of each pathogen were taken from lesions classified as fusarium. Different types of fusarium lesion on stem bases at both growth stages were not associated with any one particular Fusarium or Microdochium species. 98% of P. herpotrichoides isolates obtained from eyespot lesions at growth stage 31 in 1989, and 87% of those obtained in 1990 were identified as the sub-species P. herpotrichoides var. acuformis, Microdochium nivale was more common than any of the Fusarium species at both growth stages, and was particularly prevalent in samples from Scotland at the earlier growth stage in 1989. Isolations from the top internodes at growth stage 73–75 indicated that systemic infection by Fusarium species, although present in some stems, was of little importance. Fusarium ear blight affected 0.4% of ears in 1989 and 0.5% in 1990. Glume spot lesions on the ears in 1990 were predominantly associated with Fusarium poae.     

Isolate Virulence and Cultivar Response in the Winter Wheat: Pyrenophora tritici-repentis (Tan Spot) Pathosystem in Oklahoma

Prevalence of tan spot of wheat caused by the fungus Pyrenophora tritici-repentis has become more prevalent in Oklahoma as no-till cultivation in wheat has increased. Hence, developing wheat varieties resistant to tan spot has been emphasized, and selecting pathogen isolates to screen for resistance to this disease is critical. Twelve isolates of P. tritici-repentis were used to inoculate 11 wheat cultivars in a greenhouse study in split-plot experiments. Virulence of isolates and cultivar resistance were measured in percent leaf area infection for all possible isolate x cultivar interactions. Isolates differed significantly (P < 0.01) in virulence on wheat cultivars, and cultivars differed significantly in disease reaction to isolates. Increased virulence of isolates detected increased variability in cultivar response (percent leaf area infection) (r = 0.56, P < 0.05) while increased susceptibility in cultivars detected increased variance in virulence of the isolates (r = 0.76, P < 0.01). A significant isolate × cultivar interaction indicated specificity between isolates and cultivars, however, cluster analysis indicated low to moderate physiological specialization. Similarity in wheat cultivars in response to pathogen isolates also was determined by cluster analysis. The use of diverse isolates of the fungus would facilitate evaluation of resistance in wheat cultivars to tan spot.     

Epidemiology of eyespot (Pseudocercosporella herpotrichoides) on winter wheat, with particular reference to the period of infection

The period of infection by Pseudocercosporella herpotrichoides was investigated by transplanting winter wheat from uninfested to infested sites at Cambridge. Maximum infection was found on plants moved between November and March; most conidia were trapped during winter but infection was not closely related to the numbers of conidia. Plants exposed at intervals on infested land and incubated in a standard environment became infected throughout the period from October to July; infection was not closely related to the numbers of conidia trapped or plant age, but was positively correlated with the number of wet days/wk. Thus, while enough P. herpotrichoides conidia were dispersed throughout the life of winter wheat crops to cause substantial infection, environmental factors limited the development of new infections from April onwards. Practical implications of these results for the choice of sowing date, the development of resistant varieties, and fungicide use, are discussed.     

The effect of temperature on eyespot (Cercosporella herpotrichoides) in wheat seedlings

When seedlings of the winter wheat cultivars Cappelle-Desprez, Champlein and Hybrid 46 were artificially infected with mycelial inoculum of Cercosporella herpotrichoides Fron, the number of leaf sheaths penetrated by the pathogen increased with temperature over the range 6-18^oC. Rate of growth of the pathogen on agar also increased with temperature over this range. Temperature had little effect on differences between cultivars in number of leaf sheaths penetrated.     

Sensitivity to prochloraz in populations of the eyespot fungus, Pseudocercosporella herpotrichoides, in relation to fungicide treatments and their efficacy in continuous winter wheat

Eyespot was assessed and grain yields determined in the eighth and ninth years (1992 and 1993) of a field experiment in which the fungicides carbendazim and prochloraz were applied, separately or in mixtures, to plots of successive crops of winter wheat. Populations of the eyespot fungus, Pseudocercosporella herpotrichoides, were characterised by the proportions of cultures grown on agar from infected stems that were W-type or R-type, or were carbendazim-resistant or carbendazim-sensitive. Sensitivity to prochloraz in agar was determined for isolates from populations sampled in 1992 using restricted maximum likelihood analysis of EC50s (concentrations needed to decrease colony growth by 50%), to deal with the unbalanced data, and comparisons were made by Wald statistics. Control by prochloraz was maintained but in 1992, as in some previous years, its application with carbendazim was more effective than its application alone. Selection by prochloraz for strains sensitive to carbendazim continued to occur and may have contributed to its sometimes relatively poorer performance in the absence of carbendazim. R-type isolates taken from prochloraz-treated plots, with or without carbendazim, in 1992 were less sensitive to prochloraz than were isolates from plots untreated with prochloraz. There was evidence of a greater range of sensitivities to prochloraz in R-type than in W-type isolates (although some uncertainty results from the small numbers of W-type isolates obtained from some treatments), which would explain the selection of the R-type by prochloraz. The significance of these findings to modern wheat growing practices is considered.     

Analysis of Microdochium nivale isolates from wheat in the UK during 1993

A total of 144 isolates of Microdochium nivale from stem bases of winter wheat were taken from 30 sites and 91 isolates from grain were taken from seven sites throughout the UK. Identification by polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region followed by restriction enzyme digestion of the PCR product revealed that 70% of stem base isolates were M. nivale var. majus and 30% var. nivale while 93% grain isolates were var. majus and 7% var. nivale. Almost all isolates were resistant to the benzimidazole fungicide benomyl. Perithecial production in vitro was more common in var. majus isolates and occurred in almost all grain isolates, but was less common in stem base isolates. The implications of the findings in terms of epidemiology and chemical control of this important cereal pathogen are discussed.     

Dispersal of Pseudocercosporella herpotrichoides Spores from Infected Wheat Straw

Evidence from spore samples collected amongst infected straw spread on fallow ground supported the conclusion that spores of Pseudocercosporella herpotrichoides are dispersed mostly by rainsplash. Most spores travelled a short distance in the larger ballistic splash droplets, although some may have travelled further in smaller airborne droplets. Weekly spore counts from microscope slides under rainshields, a funnel and an impinger, evaluated as samplers for spores of P. herpotrichoides, showed a similar seasonal pattern. The funnel, as the largest sampler, generally collected most spores, but the impinger collected more spores per unit area of sampling surface. Slides sometimes collected spores when none was recovered from other samplers. Young wheat plants, exposed with the samplers and changed weekly, subsequently developed eyespot symptoms for most of the season.     

Molecular and physiological diversity among Verticillium fungicola var. fungicola

The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible for Agaricus bisporus dry bubble disease in North America is well documented but little is known about the var. fungicola affecting European crops. Variability was assessed within this variety and compared with that reported for the var. aleophilum. Eighteen isolates of V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymorphism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homogeneous group containing the other var. fungicola isolates, but no correlation could be drawn between DNA polymorphism and the various traits studied. The var. fungicola isolates were more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only mycelial growth rate at 23 °C could explain the variability in aggressiveness among the European isolates. The putative effect of the post-incubation temperature on contamination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices.     

Recovery of Pseudocercosporella herpotrichoides Spores from Rain-Splash Samples

Methods for recovery of P. herpotrichoides spores from rain-splash were tested, initially with pure spore suspensions and then with field samples. Provided surfactant was added and samples were processed immediately, centrifugation gave good recovery or spores from field samples which contained few soil particles. Foam-flotation, glycerol and sucrose methods for separation of spores from soil particles were not satisfactory for P. herpotrichoides spores. A paraffin oil method proved to be the most reliable because it gave good recovery of P. herpotrichoides spores from field samples containing many soil particles.