Development of a qPCR detection procedure for fruit tree canker caused by Neonectria ditissima

Fruit tree canker, caused by the fungus Neonectria ditissima, is considered to be a serious economic problem in apple orchards, especially in north-western Europe. This fungus produces conidia and ascospores. They are dispersed and cause infection and eventually life-threatening cankers during prolonged periods of rainy weather. Moreover, spores produced on the infected wood can act as an infection source in the orchards and spread easily during the year. Preventing the spread of the fungus is important, and therefore a fast, sensitive and reliable method to detect N. ditissima in infected apple trees would be desirable. A quantitative PCR (qPCR) method was developed for the detection and quantification of N. ditissima in infected apple cultivars. Primers, specific for N. ditissima, were designed and used for qPCR analysis on genomic DNA (gDNA) extracted from infected trees. N. ditissima gDNA was detected at variable amounts in the samples from the infection sites of different cultivars. This quantification method proved to be very accurate, since the relative quantities of gDNA were correlated to the lesion size measured after artificial inoculation of these apple cultivars. The results demonstrate that the qPCR assay is a sensitive method for detection of N. ditissima, and that it could be very useful for screening levels of resistance in germplasm collections.