First Report of High Boscalid Resistance in Botrytis cinerea Associated with the H272L Mutation in Grapevine in Chile.

Gray mold (Botrytis cinerea Pers.) is a major disease of grapevine (Vitis vinifera L.) worldwide. Integrated control strategies, including canopy management and fungicide treatments, are needed to control gray mold. Chemical control relies on the use of single mode of action fungicides. The aim of this research was to study the sensitivity of B. cinerea to boscalid, which is a single mode of action fungicide of the succinate dehydrogenase inhibitor (SDHI) fungicide group. Fifty isolates were obtained in 2012 to 2013 from commercial vineyards in central Chile. Vineyards had received two boscalid applications at least for four consecutive years. Briefly, the percent mycelial growth inhibition (MGI) was determined on minimal medium (MM) (2) plus 50 μg m of boscalid (Cantus 50 WP, BASF Chile). Each isolate was tested in triplicate, obtaining 2% highly resistant (HR, MGI ≤25%), 32% moderately resistant (MR, MGI 26 to 50%), 64% low resistant (LR, MGI 51 to 80%), and 2% sensitive (S, MGI ≥81%) phenotypes. Nine isolates were arbitrary selected and compared for MGI on MM plus 50 μg ml of boscalid (1) and conidial germination inhibition (CGI) on yeast extract-bacto peptone-Na acetate (YBA) plus 5 μg ml of boscalid (2,3). Isolates previously determined to be S and HR had the same phenotype for both MGI and CGI. However, all of the MR and LR isolates, determined based on the MGI tests, were identified as S isolates in the CGI tests. Using primer-introduced restriction analysis (PIRA)-PCR (4), the SdhB mutations were detected only in the HR isolate. The amplifications were performed with H272L-fw/H272-rev and were digested by the enzyme BglII, yielding 35- and 85-bp fragments and confirming a mutation at codon 272 (H272L) in the HR phenotype. The efficacy of the label-rate (0.4 g liter) boscalid in controlling gray mold was determined on 'Granny Smith' apples. The apples were surface-disinfested (75% ethanol, 30 s), wounded with a sterile syringe, and inoculated with a mycelium plug (5 mm in diameter) or 20 μl of a conidial suspension (10 conidia/ml) of one HR, MR, and S isolate. The inoculum was placed on the wounded sites after boscalid application. Apples were incubated for 7 days at 21°C. Each test had four replicates and the experiment was conducted three times. Boscalid slightly controlled (<6.7% efficacy) gray mold on the apples that were inoculated with mycelium or conidia of the HR phenotype isolate, while the sensitive isolate was highly controlled (>95% efficacy), and the MR isolate was moderately controlled (27 to 34% efficacy). These results demonstrate that mycelium or conidia assays using MM + 50 μg ml boscalid or YBA+5 μg ml boscalid consistently detected HR isolates. The S isolates detected using MGI were also S according with the CGI tests. The presence of the boscalid HR strains of B. cinerea associated with the H272L mutation in grapevine in Chile is reported for the first time in this study. This finding suggests that resistance to boscalid needs to be considered in the design of gray mold control strategies in commercial grapevine orchards. References: (1) D. Fernandez-Ortuño et al Plant Dis. 96:1198, 2012. (2) M.-J. Hu et al. J. Phytopathol. 159:616, 2011. (3) Y. K. Kim and C. L. Xiao. Plant Dis. 94:604, 2010. (4) T. Veloukas et al. Plant Dis. 95:1302, 2011.