Development of a dot blot technique for rapid identification of Botrytis cinerea, the causal organism of grey mould in greenhouse tomatoes

摘要

A dot blot assay, based on the internal transcribed spacer of the ribosomal region, was developed for rapid diagnosis and identification of Botrytis cinerea, the causal agent of grey mould of greenhouse tomatoes (cv. Rapsodie). In this assay, DNA from fungal cultures and diseased plant tissue were amplified by PCR using primers specific to septate fungi, fixed to nylon membranes, and hybridized with digoxigenin-d-UTP labelled oligonucleotides specific to B. cinerea. The hybridized probes were detected by chemiluminescence. Six isolates of B. cinerea, four isolates of Botrytis species, one isolate each of B. fabae, B. squamosa, B. streptothrix, B. allii, B. porri [Botryotinia porri] and B. tulipae, and some common greenhouse pathogens were tested using this assay. Of four probes screened, probe Bot1 gave a positive reaction with all isolates of B. cinerea and with fresh or frozen plant tissue affected with grey mould from research centres and commercial greenhouses. All other greenhouse fungi tested negative with probe Bot1. This dot blot diagnoses infection by B. cinerea in large numbers of samples rapidly and reliably and distinguishes isolates of B. cinerea from other epiphyte fungi found on greenhouse crops.