SENSITIVITY TO FUNGICIDES AND GENETIC STRUCTURE OF BOTRYTIS CINEREA POPULATIONS ISOLATED IN LOMBARDY.

The repeated use of fungicides can select resistant strains in Botrytis cinerea population, the causal agent of grapevine grey mould. A better understanding of the population biology and genetics of B. cinerea can be useful in designing more sustainable disease management strategies. To date the effect of fungicide treatments, the presence and distribution of transposons and the genetic structure in B. cinerea populations isolated in Italian vineyards are scarcely investigated. The aim of the present research was the characterization of Italian B. cinerea populations taking into account fungicide sensitivity and population biology. Seven hundred and twenty B. cinerea monoconidial strains were isolated in three years of study (320 in 2011, 240 in 2012 and 160 in 2013) from infected grapes collected at harvest from vineyards located in the provinces of Brescia, Sondrio, Pavia and Mantova. The vineyards of Sirmione in the province of Brescia, Chiuro in the province of Sondrio and TorrazzaCoste in Pavia, have been sampled in the three years of study in order to have a comparison between years. All the strains were tested for their sensitivity to the following classes of fungicides: anilinopyrimidines (cyprodinil), hydroxyanilides (fenhexamid), phenylpyrroles (fludioxonil) and pyridine carboxamides (boscalid). For each strains the EC50 (the effective concentration of fungicide that reduces mycelial growth by 50%), the IC and the Rf (Resistant factor) were assessed. The DNA was extracted from all the strains to analyze the distribution of the mating type and the presence/absence of transposable elements Boty and Flipper. The presence of the main mutations in the SdhB subunit (BH272R, BH272Y) associated with boscalid resistance was assessed in the strains with Rf greater than 10. 317 B. cinerea strains, arbitrarily chosen, were completely genotyped for six microsatellites loci and genetic differentiation and the mode of recombination were investigated among B. cinerea populations. Several strains with a reduced sensitivity to boscalid, cyprodinil and fludioxonil were found in B. cinerea populations. However, the status of the B. cinerea populations in Lombardy assessed in the present study denotes a good sensitivity to the four active substances tested. Particular attention should be taken when using anilinopyrimidines, limiting their application to a single treatment per season. In the present study, the mutation H272Y, found at high frequency, was associated to high resistance levels but also to sensitivity, so it is possible to conclude that molecular methods able to discriminate strains resistant to boscalid can not be based simply on the detection of H272Y point mutation. Genotypic differentiation was detected among the haplotypes and the presence/absence of transposable elements: the Flipper molecular type seems to be completely different from all the others. Indeed, very weak differentiation was found between geographic origin and years of sampling, suggesting that B. cinerea population in Lombardy are similar. Application of the index of association, the chi-square test, and the phi test consistently indicated that the population of northern Italian isolates of B.cinerea undergoes sexual reproduction and therefore a mixture of a sexual and asexual reproduction can be assumed. This finding is in accord with the successful adaptability of B. cinerea.