1Horticulture Research International, East Malling, West Malling, Kent ME19 6JB, UK; 2John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Background and objectives
Facultative pathogens are involved in a number of diseases of cereals and may often be found together forming a disease complex. Two such complexes are of economic importance in the UK. The stem-base disease complex involves several Fusarium spp., two varieties of Microdochium nivale (all of which cause foot rot ), two Tapesia spp. (which cause eyespot) and Rhizoctonia cerealis (which causes sharp eyespot). The Fusarium spp. and M. nivale varieties are also involved in seedling and ear blight, the latter of which is of additional concern because of the potential of many of these species to produce mycotoxins which are harmful to human and animal health. The inability to identify and quantify the individual fungal species present has hindered epidemiological studies and efforts to investigate host resistance and fungicide efficacy. Molecular techniques offer the opportunity to dissect disease complexes and evaluate the role and interaction of the individual pathogen components. The present work aimed to exploit these opportunities and develop assays for the detection and quantification of the major fungi involved in these two disease complexes of cereals.

Materials and methods
Culture collections of facultative pathogens were screened by Random Amplified Polymorphic DNA (RAPD) assay to confirm the identity of isolates and to determine the degree of variability within species. The RAPD assay was used to identify fragments common to all members of a species and specific to that species. Selected fragments were used as probes to identify those fragments hybridising only to the species of interest. Species specific fragments were cloned, sequenced and used to design primer pairs for use in conventional polymerase chain reaction (PCR) assays. Primers were designed to each of the major pathogens involved in the stem-base and ear blight complexes. Primers were chosen to enable multiplexing to detect several fungi simultaneously. Further to this, competitor fragments were generated for each of the pathogens to permit the development of quantitative PCR assays.

Results and conclusions
RAPD analysis highlighted both the distinction between the two varieties of M. nivale and similarities between F. culmorum and F. graminearum. Occasionally, during the development of primers, unsuspected relationships between species were revealed, such as that between F. avenaceum and F. tricinctum. Diagnostic assays were developed for each of the major stem-base and ear blight pathogens [ 1 1. The assays were highly specific and sensitive, being capable of detecting approximately 30 genome-equivalents of the selected species. Further to this, competitive PCR assays have been established to permit the quantification of each pathogen in extracts from plant tissue [2]. Diagnostic PCR has revealed that, in a UK survey of stem-base pathogens in seedlings of winter wheat, eyespot was almost exclusively due to T. acuformis whereas foot rot was predominantly caused by M. nivale var. nivale. Quantitative PCR has been used to study fungicide efficacy against Fusarium ear blight caused by F. culmorum and F. poae; this work highlighted some shortcomings of visual disease assessment. Similar assays to study host resistance toT. acuformis and T. yallundae have suggested that the genetic basis of resistance to the two species may not be identical. In addition, PCR assays are being used to study the effect of selected fungicides against stem-base disease in an on-going programme.

This work demonstrates the use of DNA fingerprinting assays, such as RAPDS, as a generic approach to the isolation of species-specific primers. Using this system, both diagnostic and quantitative PCR assays have been developed against a range of facultative pathogens of cereals and used in studies involving a variety of host tissues from artificially and naturally inoculated plants. Examples of the current use of these assays in studies of host resistance, fungicide efficacy, pathogenicity, and epidemiology of stem base and ear diseases of cereals will be given, along with a discussion of challenges and possible future developments.

1. Parry DW, Nicholson P, 1996. Plant Pathology 45, 383-91.
2. Nicholson P, Rezanoor HN, Simpson DR, Joyce D, 1997. Plant Pathology 46, 482-56.