Sensitivity to carboxylic acid amide fungicides of Plasmopara viticola populations in northern Italy

Dimethomorph was the first carboxylic acid amide (CAA) fungicide used to control downy mildews (Albert et al., 1988). In Italy it has been authorized on grape since 1994 while the other compounds belonging to CAA class have been in use since the early 2000s: iprovalicarb (2002), benthiavalicarb (2007), valifenalate and mandipropamid (2009). Cytological studies have demonstrated that CAA inhibit processes involved in cell wall formation (Kuhn et al., 1991; Jende et al., 2002) but only recently was the mode of action of CAA elucidated as corresponding to the inhibition of cellulose biosynthesis (Blum et al., 2010a). The recessive nature of CAA resistance led the FRAC group to classify the resistance risk as moderate, and cross resistance among its members was clearly demonstrated (Gisi et al., 2007). At the end of the 2000s the single point mutation leading to an amino acid change from glycine to serine at codon 1105 (G1105S) was discovered in the CesA3 protein, conferring CAA resistance in P. viticola (Blum et al., 2010b). The occurrence of CAA fungicide-resistant populations of Plasmopara viticola was described for the first time in France in 1994 (Chabane et al., 1996) for dimethomorph, but it did not create problems in the field. During the following years, resistance increased gradually in Europe although field performance has remained good. In order to evaluate the sensitivity of P. viticola to CAA, thirty-fifty downy-mildew-infected leaves were collected from 12 vineyards located in Northern Italy (principally in Friuli Venezia Giulia) during the seasons 2010-2011. Biossays on foliar discs and PCR analysis were carried out on populations obtained after 1-2 sporangia inoculations on fresh plant material (cv Sangiovese). Nine concentrations (from 0.1 to 300 mg/L) of mandipropamid (Pergado SC, Syngenta) and dimethomorph (Forum 50 WP, Basf) were applied in bioassays. For each concentration (including an untreated one), a total of 15 discs (22 mm ) of leaves were soaked in formulated products. After 30-45 min, the leaf material was removed and dried on a grate at room temperature, then transferred to survival agar medium (1.5%) complemented with 2 mg/L of kinetin, in petri dishes in triplicates The inoculation was done by depositing two 20 ml droplets of sporangial suspension (50,000 spores/ml) onto the adaxial face of each leaf disc. The petri dishes were incubated under controlled conditions (20°C) with a 12-h photoperiod (Wong & Wilcox, 2000). The sporulation was assessed 8-10 days after the treatment, evaluating the sporulated leaf surface matching with the droplets, and the phenotypes were defined as resistant when sporangia were observed up to 300 mg/L or sensitive when sporulation was observed not beyond 3 mg/L. To confirm the presence of CAA fungicide resistance phenotype observed in the biological assays, a molecular method for rapid detection of the mutation in the PvCesA3 gene was developed. Samples were subjected to PCR analyses, using two PCR mixes, with primers amplifying either the sensitive or the resistant genotype (Blum et al., 2010b). Most of the samples were amplified by the two different PCR mixes, confirming the presence of a heterozygote genotype. Selected amplicons were subjected to direct sequencing and the sequences obtained confirmed the presence of a substitution at the expected position. To improve the molecular method, a new reverse primer, common for both genotypes, was designed, and PCR-RFLP analyses were performed. Amplicons obtained with the new primer pair were digested with selected restriction enzymes, leading to the visualization of different restriction profiles. Seven populations were considered resistant and five appeared sensitive in bioassays, while all populations revealed the presence of the mutated allele by PCR-RFLP analysis (except one totally resistant in both tests). On the basis of these first results, disc leaf assay appears able to better discriminate the sensitivity of P. viticola populations compared to PCR-RFLP analysis. As in many other cases, the development of a quantitative molecular method to quantify mutated alleles could represent the best approach to evaluate, together with traditional bioassays, the P. viticola sensitivity to CAA to predict the performance of these fungicides in the field.