Detection and quantification of llyonectria spp. associated with black-foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

摘要

Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central-eastern Spain) were surveyed in July 2011 to detect the presence and to quantify llyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10-30 cm, and 10-20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species-specific primer pairs (Macl/MaPa2, Lirl/Lir2 and Paul/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, llyonectria spp. were detected in 172 of them. llyonectria rnacrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open-root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma-complex DNA in the soil samples. Detection of llyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of llyonectria spp. ranged from 0-004 to 1904-8 pg μL~(-1). In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify llyonectria spp. genomic DNA in soil samples from nursery fields and root-stock mother fields confirms soils from both field types as important inoculum sources for black-foot pathogens.