Development and use of a PCR assay to detect Rhizoctonia cerealis, the cause of sharp eyespot in wheat

Random amplified polymorphic DNA assays were carried out on a range of isolates of Rhizoctonia cerealis to identify markers common to all isolates. Two fragments were isolated, cloned and used to probe Southern blots of DNA from R. cerealis and isolates from a range of anastomosis groups of Rhizoctonia solani. The two fragments hybridized specifically to DNA of R. cerealis and not to DNA of any of the isolates of R. solani. Both fragments were partially sequenced and two pairs of primers were generated for use in the polymerase chain reaction (PCR). Amplification of a fragment of the anticipated size occurred following PCR of all isolates of R. cerealis and not from any of a range of fungal species associated with disease of the stem base of cereals. The primer pairs for R. cerealis were also used, along with those for Microdochium nivale and W and R-type of Pseudocercosporell herpotrichoides , to deled these pathogens in extracts from field-grown wheat plants exhibiting symptoms of sharp eyespot, eyespot, foot rot or a combination of the diseases. No relationship was found between visual disease assessment of sharp eyespot at growth stage 37 and the results of PCR However, the results of PCR and visual disease assessment at growth stage 75 were similar, indicating that visual disease assessment may not be reliable until later growth stages. This system offers the potential to detect the presence of R. cerealis in cereals and avoid problems commonly associated with conventional diagnosis of this disease and isolation of the pathogen.     

Relationship between brown foot rot and DNA of Microdochium nivale, determined by quantitative PCR, in stem bases of winter wheat

Relationships between the incidence and severity of brown foot rot and of pathogenic fungi, determined by diagnostic and quantitative PCR, were investigated during the growth of nine winter wheat crops in three cropping seasons. Microdochium nivale vars nivale and majus were the only brown foot rot pathogens present in significant amounts. Relationships between disease symptoms and amounts of pathogen DNA were often weak in early spring (when shoot-base symptoms are usually most difficult to ascribe to particular pathogens by visual examination) because of indistinct symptoms and small amounts of pathogen. Relationships were strongest during stem elongation. The amount of M. nivale in the tissues tended to decline in the summer as the plants matured, apparently disappearing partially from necrotic lesions to which it contributed, resulting in a weakened relationship between symptoms and pathogen DNA. Regression analyses of brown foot rot on amounts of M. nivale DNA for different wheat cultivars generally produced lines with similar slopes but were often most significant for the cultivar with most eyespot resistance (i.e. with least confounding eyespot) or most apparently genuine brown foot rot. DNA of Fusarium spp. was rarely present in amounts sufficient to quantify.     

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.     

Development of Stem-base Pathogens on Different Cultivars of Winter Wheat Determined by Quantitative PCR

The progress of development of stem-base pathogens in crops of second winter wheat was plotted in nine experiments in three years. The amount of each pathogen present was determined by quantitative PCR. Where Tapesia yallundae was present in quantifiable amounts, it usually developed earlier than the other eyespot pathogen, T. acuformis. Both species were usually present in greater amounts on cultivars which are more susceptible to eyespot. The sharp eyespot pathogen, Rhizoctonia cerealis, developed more erratically than either of the Tapesia spp. and there were no consistent effects on different cultivars. Fusarium spp., the cause of brown foot rot, were rarely present in quantifiable amounts, but Microdochium nivale was usually present as one or both of the varieties nivale and majus. Late-season (after anthesis) decreases in M. nivale suggest that any brown foot rot symptoms attributable to this fungus would have fully developed earlier. Cultivar differences in amounts of M. nivale were most clear in stems during internode extension and when relatively large amounts of DNA were present. Such differences approximately reflected eyespot susceptibility, cv. Soissons containing most and cv. Lynx containing least DNA. The results emphasise the difficulty in relating diagnoses, by quantitative PCR or other means, at early growth stages when decisions to apply fungicides against stem-base disease are made, to later disease severity.     

Evaluation of diagnostic and quantitative PCR for the identification and severity assessment of eyespot and sharp eyespot in winter wheat

Diagnostic and quantitative polymerase chain reaction (PCR) provided clarification of the causes of symptoms and the extent of infection by eyespot ( Tapesia spp.) and sharp eyespot ( Rhizoctonia cerealis ) on winter wheat at early growth stages. Disease assessments made before stem extension, when decisions to apply fungicides are usually made, often did not agree with the pathogen diagnoses using PCR, suggesting that such early visual diagnoses may be unreliable. Visual and PCR diagnoses made on stems in summer generally supported each other, but there were often discrepancies in relating disease severity to amounts of pathogen present when determined by regression analyses of incidence or severity of symptoms on amount of pathogen DNA. Mixed symptoms caused by different pathogens may sometimes have been confounded. Relationships between symptoms and DNA of eyespot pathogens were less clear on some cultivars, often those with least disease. Sharp eyespot symptoms had a stronger relationship to DNA of its pathogen. Significant regressions often accounted for a small percentage of the variance, suggesting either that pathogens not assayed were contributing to symptoms or that lesions were in some cases persisting longer into the season than pathogen DNA. The frequency of pathogen detection before stem extension was a poor predictor of the amounts of pathogen DNA measured later in the season.     

小麦纹枯病菌的分子检测

小麦茎基部可寄居多种真菌,并可导致发褐、坏死等症状,与小麦纹枯病相混淆.为准确诊断小麦纹枯病,根据江苏省小麦茎基部常见寄居菌禾谷丝核菌Rhizoctonia cerealis、立枯丝核菌Rhizoctonia solani、长蠕孢菌Helminthosporium spp.、交链孢菌Alternaria spp.、小麦全蚀病菌Gaeumannomyces graminis var.tritici和禾谷镰刀菌Fsarium graminearum rDNA的ITS区段序列差别,设计合成了2对特异性扩增引物(DNF77 DNR554,DNF81 DNR564)用于小麦纹枯病菌检测.2对引物均能从小麦纹枯病菌株扩增到特异性的分子片段,说明设计的特异性引物可以用来检测小麦纹枯病菌.采集田间具有小麦纹枯病症状、茎基部变褐及健康麦苗,进行病原菌分离,同时提取这些麦苗茎基部DNA,利用上述2对引物进行扩增.结果表明,仅从能分离到禾谷丝核菌的麦组织中扩增到约480bp的特异性条带.说明设计的特异性引物可以对田间小麦纹枯病进行早期、快速分子诊断.     

Evidence for differential host preference in Microdochium nivale var. majus and Microdochiumnivale var. nivale

The pathogenicity of Microdochium nivale var. majus and var. nivale was tested on wheat, rye and oat seedlings using both visual disease scoring and quantitative PCR measurements. In an individual inoculation trial at 10°C var. majus and var. nivale were strongly pathogenic towards wheat and rye, with var. nivale causing slightly greater disease in rye. At this temperature only var. nivale caused significant disease of oats. In a further experiment M. nivale was inoculated as a series of mixtures of the two varieties and incubated at 15°C. The ratio of the varieties present in the inoculum and present at harvest was analysed by quantitative PCR and this enabled a coefficient of selection to be calculated for the varieties on each host. M. nivale var. majus showed a weak selective advantage over var. nivale on wheat (0.33 ± 0.08) and oat seedlings (0.35 ± 0.016) and M. nivale var. nivale showed a strong selective advantage over var. majus on rye seedlings (0.92 ± 0.26). The isolates were also compared for sensitivity to benzoxazolinone (BOA), a hydroxamic acid compound derived from rye leaves. M. nivale var majus was found to be significantly more sensitive to BOA than M. nivale var. nivale , indicating a possible mechanism for the selective advantage of var. nivale growing on rye. This is the first substantiated indication of a significant difference in host preference between Microdochium nivale var. majus and var. nivale .     

The use of species-specific PCR-based assays to analyse Fusarium ear blight of wheat

Polymerase chain reaction (PCR) assays for the detection of various Fusarium species and Microdochium nivale subspecies were compared with conventional visual disease assessment using a field plot of wheat in which the central subplot was inoculated with F. culmorum . Visual disease assessment was performed on a range of samples taken from each of 15 subplots at growth stage 80. At harvest, each sample was divided into its component parts, i.e. grain, glume and rachis, and species-specific PCR analysis was used to detect the presence of F. culmorum , F. poae , F. avenaceum , F. graminearum , M. nivale var. majus and M. nivale var. nivale . Within the inoculated subplot there was good correlation between visual disease assessment and PCR analysis, both techniques indicating a high incidence of F. culmorum in this region. According to the visual disease assessment results, there was also a relatively high incidence of F. culmorum in most other regions of the field plot. However, according to PCR analysis the incidence of F. culmorum in many of the other subplots was relatively low and F. poae , M. nivale var. majus and var. nivale , and F. avenaceum were detected within the grain, glume and rachis tissues of many of the ear samples from these subplots. F. poae predominated in the glume component of ears and M. nivale var. majus and var. nivale in the rachis component. M. nivale PCR results revealed that 64% of infected samples involved var. majus , and 36% var. nivale . PCR analysis has highlighted some difficulties that may arise when using visual assessment for studying disease complexes.     

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.     

Damping-off of sugar beet caused by Rhizoctonia cerealis

Rhizoctonia cerealis was isolated from diseased sugar beet seedlings collected from crops in Ireland. In pathogenicity tests, isolates of R. cerealis from beet seedlings, and from sharp eyespot lesions on wheat crops, caused severe damping-off of sugar beet. Isolates from beet seedlings also caused symptoms of sharp eyespot on wheat.     

中国小麦纹枯病化学防治研究进展

随着秸秆还田等耕作栽培措施的推广,中国小麦纹枯病发生日趋严重,对小麦的高产、稳产造成了很大威胁。由于缺乏免疫及高抗病性小麦品种,生产中对纹枯病一直采用播期拌种及春季喷雾相结合的化学防治方法。文章总结了当前中国小麦纹枯病的发生现状及主要病原;评述了三唑类药剂对纹枯病菌的毒力及对纹枯病的防治效果,介绍了生产中小麦纹枯病菌对三唑类药剂的抗药性现状及机理,分析了三唑类药剂对小麦的安全性;同时阐述了井冈霉素、甲基立枯磷及其他种类药剂在小麦纹枯病综合防治中的应用;指出小麦纹枯病化学防治的发展方向应是将生防菌剂同化学药剂相结合,实现生物防治与化学防治的协同应用。     

Biological control of cereal seed-borne diseases by seed bacterization with greenhouse-selected bacteria

About 400 bacterial strains, isolated from roots of wild and cultivated plants, were screened for effects against diseases caused by Drechslera teres and/or Microdochium nivale in greenhouse tests and against common bunt caused by Tilletia caries in field tests. Four of the strains showed good biocontrol activity <70% disease reduction) against D. teres and T. caries both in screenings and field tests. One Pseudomonas isolate, MA 342, strongly and reliably suppressed both D. teres and T. caries in the field, while effects against M. nivale were weaker. The effects could not be enhanced by varying pre-application or seed application procedures. This isolate could be stored as a suspension in a refrigerator, frozen or applied to seeds for at least one month without loosing its disease controlling ability.     

利用荧光定量PCR检测禾谷丝核菌的相对生物量

小麦纹枯病是以禾谷丝核菌(Rhizoctonia cerealis)侵染为主的小麦土传病害。为建立检测禾谷丝核菌在寄主小麦(Triticum aestivum)中的相对生物量的可靠方法,促进小麦抗纹枯病机制的研究,本研究克隆了禾谷丝核菌肌动蛋白基因RcActin的部分(3′端)cDNA,并设计了RcActin的特异引物。该引物不仅能区分禾谷丝核菌与寄主小麦,还能区分全蚀病菌(Gaeumannomyces graminis)、根腐病菌(Bipolaris sorokiniana)和立枯丝核菌(Rhizoctonia solani)等常见小麦土传病害的病原菌,表明该引物能用于小麦纹枯病的分子检测,也能用于相对表达量的测定。利用相对定量法,以RcActin相对于寄主管家基因的相对表达量作为禾谷丝核菌相对生物量的指标,结果表明,此方法能准确反映禾谷丝核菌在寄主中的相对生物量和对小麦纹枯病抗性程度进行快速鉴定。     

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.     

Stem-base disease and fungal colonisation of winter wheat grown in compost inoculated with Fusarium culmorum, F. graminearum and Microdochium nivale

Fungal colonisation of winter wheat cv. Cadenza by Fusarium culmorum, F. graminearum and Microdochium nivale was studied under conditions designed to avoid the splash dispersal of conidia from infested compost, to evaluate the possibility that systemic growth may transfer infection from the stem-base to the head. At decimal growth stages 33, 59, 77–87 and 95 the extent of fungal growth was assessed using a sample of 72 plants, by the recovery of fungal species from the stem-base, from each node and from the ear. Each of the fungi was recovered from stem tissues above soil level in some, apparently symptomless, plants. Symptoms of Fusarium foot rot were seen in an increasing proportion of plants during grain-fill and desiccation. There was an inverse relationship between recovery and the height above stem-base from which the stem tissue was excised. F. culmorum was the most frequently isolated fungus and it was also recovered from the highest position in plants. Only 3% of plants were colonised above the second node and none of the fungal species were recovered from either the fifth node or the ear. This suggests that colonisation and systemic growth from Fusarium infested compost is unlikely to contribute to the development of ear blight symptoms in winter wheat.     

Infection of potato and wheat by isolates of Rhizoctonia solani and R. cerealis

Isolates of Rhizoctonia were obtained from potato crops with stem canker or black scurf and from cereal crops with sharp eyespot. Those with many nuclei per cell, wide cells, darkening colonies and fast growth were assigned to R. solani ; those with two nuclei per cell, narrow cells, pale colonies and slow growth were assigned to R. cerealis . Only R. solani was obtained from potatoes and only R. cerealis from cereals. On young plants in the glasshouse, the isolates of R. solani infected potato substantially but not wheat; R. cerealis infected wheat substantially and potato slightly. This host preference was shown at temperatures between 10 and 25°C in growth rooms. R. solani on potato caused more disease with increasing temperature; no trend with temperature was observed for R. cerealis on wheat.     

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.     

The escalating threat of Rhizoctonia cerealis, the causal agent of sharp eyespot in wheat

Rhizoctonia cerealis, the causal agent of sharp eyespot on wheat, was not considered to be an important pathogen for many years. Recently, the disease has become endemic in many countries except for South America. The disease has created a new threat to world wheat production because the damage of wheat sharp eyespot has become increasingly severe. In this paper, previous studies on this pathogen, including the disease geographical distribution, pathogen identification, life cycle, symptoms, favourable environmental conditions, effects on wheat yield and control strategy, are reviewed. Such information will be helpful in management of sharp eyespot.     

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.     

Screening winter rye cultivars for snow mould (Microdochium nivale) resistance

The snow mould ( Microdochium nivale ) resistance of 13 winter rye cultivars was studied in field trials and in three different laboratory tests: snow mould chamber tests, enzymatic assay tests and leaf segment tests. On the basis of the results, it is suggested that both the field trials and the snow mould chamber tests describe more the general winterhardiness of plants involved in the survival of the crown tissue of plants during prolonged incubation under the snow cover, than the snow mould resistance. The results from the enzymatic assay and the leaf segment tests indicate that there are other, more specialized snow mould resistance mechanisms in the plant that act also at the single leaf level. At least some of these resistance reactions seem to be induced by the lytic enzymes secreted by M. nivale.