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 10-30 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 (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 0Á004 to 1904Á8 pg lL À1 . 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.

Effect of white mustard cover crop residue, soil chemical fumigation andTrichodermaspp. root treatment on black‐foot disease control in grapevine

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Dysfunction in the arbuscular mycorrhizal symbiosis has consistent but small effects on the establishment of the fungal microbiota in Lotus japonicus

“…After harvesting from plates, fungal isolates were grown on MYP medium and pure genomic DNA was extracted as described (Cenis, 1992). Dactylonectria torresensis 107 DNA was quantified with primers YT2F/Cyl_R and cycling conditions (95°C for 10 min, 60 cycles: 95°C for 10 s, 60°C for 10 s, 72°C for 30 s) as described (Agust ı-Brisach et al, 2014). DNA of T. olida isolates 57 and 102 was quantified with primer pair YT2F/ 102_Cyl_R using the same cycling conditions.…”

Metabarcoding and development of new real‐time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain

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