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

Abstract

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 Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10-30cm, and 10-20cm from the base of the plant. Three replicate subsamples (10g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species-specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma 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 Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0004 to 19048pgL(-1). In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black-foot pathogens.