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MOLECULAR DETECTION AND QUANTIFICATION OF NECTRIA GALLIGENA FROM APPLE WOOD
MOLECULAR DETECTION AND QUANTIFICATION OF NECTRIA GALLIGENA FROM APPLE WOOD SRH LANGRELLl, DJ BARBARA2 and TR SWINBURNEl l Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK; 2 Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK. Background and Objectives Nectria canker is prevalent in apple and pear orchards in all temperate growing areas of the world. The disease, incited by Nectria gailigena (anamorph Cylindrocarpon heteronema), causes direct loss of yield by damage to trees, and is a major cause of rotting in stored fruit. Current control measures are designed to protect infection sites, notably leaf scars from external inoculum. However, recent research has indicated that young trees can be infected symptomlessly during propagation [1]. In the absence of reliable selective media for conventional diagnosis, the objective of the present work, funded as part of a MAFF-APRC-DANI project, was to develop a robust, species specific PCR based detection assay for such symptomless infection Results and Conclusions A pair of species specific primers to N. galligena were designed from a detailed sequence characterisation survey of the internal transcribed spacer (ITS) regions (1 and 2) of 30 isolates of diverse global and host origin through direct comparison with ITS sequence data generated from closely described Nectria species (N. ditissima, N. coccinea, N. punicea, N. hederae and N. fuckeliana), including N. radicicola. Under stringent PCR conditions, primers Ch 1 (5'-AAC CCC TGT GAA CAT ACC CAT C-3') and Ch 2 (5'-GTG GCC GCG CTG CTC TTC CG-3') amplify a 412 bp fragment specific to N. galligena DNA but not from other Nectria, associated fungal apple endophyte species or rosaceous host tissues. Assay development has followed two strategies to facilitate direct research applications; to assess infection levels, and the reliable routine screening of asymptomatic tissues, repectively. A 500 bp heterologous internal standard containing identical 5' and 3' recognition termini to the specific primer pair was generated initially as an internal positive control for individual PCR test reactions and later developed as a quantitative assay based on competitive PCR [2]. This approach enabled the determination of infection levels in different apple cultivars and may lead to a more definitive assay in measuring host responses in resistance studies. To eliminate the inhibitory effect of certain PCR interfering compounds inherent in DNA extracts from lignified tissue samples, an 81 bp single stranded biolinylated probe was developed as part of a magnetic capture-hybidisation (MCH-PCR) assay to increase detection sensitivity for routine screening of suspect asymptomatic tissue. The hybridisation probe, designed complementary to the region between Ch 1 and the conserved 5.8S gene in the ITS 1 region, allows the exclusive removal of target template from inhibitors through specific hybrid formation and magnetic separation of bead-probe-template conjugate, resulting in increased levels of sensitivity with PCR. Both variations of the detection assay are currently being employed as research tools in appraising the level of contribution of cryptically infected stock to the spread and epidemiology of this enigmatic fungal disease. References 1.Swinburne TR, Langrell SRH, Coventry J, Li R, 1998. Plant Pathology (accepted). 2.Forster E, 1994. Biotechniques 16, 1006-1008.