Sensitivity of Plasmopara viticola Italian populations to QoI fungicides

Sensitivity of Plasmopara viticola Italian populations to QoI fungicides Marina Collina, Lucia Landi, Maria Barbara Branzanti, Agostino Brunelli First and fourth authors: Dipartimento di Protezione e Valorizzazione Agroalimentare, Università di Bologna, Viale Fanin 46, 40127 Bologna, Italy, mcollina@agrsci.unibo.it; second and third authors: Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy ___________________________________________________________________________ Strobilurin and some non-strobilurin fungicides belong to a new cross-resistance group that has been designated as QoIs (Quinol outside inhibitors). They specifically inhibit fungal respiration by binding to the subunit protein of the cytochrome bc1 complex of the electron chain (Sauter et al., 1995). It is well known that site-specific fungicides generally possess a high risk of resistance development if resistant isolates of the pathogen are not impaired in their ability to survive and multiply in the agricultural environment. A single point mutation (GGT to GCT) in the cytochrome b gene, that results in substitution of glycine by alanine at position 143 in the gene product, has been found in the most part of resistant isolates to QoI. To date, this particular mutation has been identified and characterized in sub-populations of pathogenic fungi resistant to QoIs under practical conditions of disease control such as Venturia inaequalis, (Zheng et al., 2000), Mycosphaerella fijiensis (Sierotski et al., 2000), Blumeria graminis (Fraaije et al., 2000), Pseudoperonospora cubensis and Podosphaera fusca (Ishii et al., 2001), Plasmopara viticola (Heaney et al., 2000). The sensitivity of pathogens to fungicides has been generally tested using biological assays, but this classical methodology can be now combined with a new molecular analysis relating to the specific G143A mutation from the DNA to screen for fungicide resistance. This can be sensitive and cost-effective, especially for obligate pathogens that are unable to grow on artificial media. Thus, to determine the frequency of the mutant allele, quantitative real-time PCR was reported the first time in P. viticola by Sirven et al. (2002). A failure in grapevine downy mildew control with azoxystrobin occurred in 2000 in north-eastern Italy in some farms located in Emilia-Romagna Region (Ravenna area, where the fungicide had been largely and successfully applied through 1998 and 1999). In the experimental farm of Bologna University, where azoxystrobin was showing excellent results in field experiments since 1994, azoxystrobin failed to control downy mildew in an experiment carried out in 2000, with applications every 10 days. In the same trial, the activity of a famoxadone plus cymoxanil ready to use mixture was also unsuccessful. Laboratory and greenhouse sensitivity tests, carried out from 2000 with P. viticola populations collected from both commercial vineyards and trial plots, have clearly demonstrated the first occurrence of field resistance in Italy (Brunelli et al., 2001; 2002). We report the results of our studies based on a wider monitoring of P. viticola sensitivity to QoIs in north-eastern Italy, through the use of biological assays and real-time PCR analysis to quantify the G143A mutation in each sampled population. Material and methods More than 200 P. viticola populations were collected in early growing seasons and summers from 2000 to 2005, in vineyards located in Emilia-Romagna, Friuli-Venezia Giulia and Lombardia Regions. Twenty to fifty infected vine leaves were randomly collected from field or trial plots and used as representative samples of each location. Sporangia obtained by washing the leaves with distilled water were used both in bioassays for the identification of the resistant/sensitive phenotype and in quantitative PCR analysis. Three doses (30, 60 and 250 mg/l) of azoxystr...