qPCR and RT-qPCR assays for the quantification of Trichoderma asperellum icc012 and Trichoderma gamsii icc080 used as biocontrol agents against Phaeomoniella chlamydospora in grapevine nursery.

Trichoderma asperellum strain icc012 (TA) and Trichoderma gamsii strain icc080 (TG) (Remedier, Isagro S.p.A., Italy) are biological control agents (BCAs) proposed against esca disease on grapevine. This research was aimed at evaluating their effectiveness against esca in the nursery with particular regard to Phaeomoniella chlamydospora population and at developing a fast and reliable method to quantify the two BCAs. The effectiveness of TA and TG was evaluated on 1103 P. rootstocks, artificially inoculated at the hydration stage with conidia of the benomyl-resistant mutant C1.A43.1 of P. chlamydospora and successively treated or not with Remedier at different time combinations (end of hydration, at planting, and three times during plant growth). Eight different combination programs were compared. At uprooting of rootstocks, wood fragments were sampled at 1 cm and 2–15 cm over the crown from at least 90 rootstocks per program. The incidence of fungi, including the C1.A43.1 mutant and the BCAs, was evaluated on malt extract agar (MEA) and MEA amended with 10 mg L-1 of benomyl. Natural infections of P. chlamydospora were rarely recovered while the population of the artificially inoculated mutant C1.A43.1 was always reduced by BCAs (P≤0.05, ranging from 18% with only one application at hydration to 36% with four applications). Fomitopsis sp., Acremonium sp., and Fusarium sp. were also reduced by BCAs. qPCR and RT-qPCR detection methods based on specific primers and TaqMan probes were set up and applied to quantify singularly and in duplex the populations of TA and TG. The primers/probe sets were designed on the polymorphic region of the rpb2 gene and the method allows to specifically detect up to 10 pg of DNA and 150 pg of RNA. Further studies are in progress to evaluate the effectiveness of the BCAs and define the most appropriate usage in grapevine nursery for reducing infection of esca-associated fungi.