First Report of Fenhexamid Resistant Isolates of Botrytis cinerea on Grapevine in Chile

Abstract

Botrytis cinerea Pers. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is a haploid, filamentous ascomycete that causes gray mold on many economically important crops in temperate regions, especially grapevine. The management of gray mold on table grape in Chile involves cultural and chemical methods. Currently, protection programs are based on several fungicide families (dicarboximides, anilinopyrimidines, mixture of anilinopyrimidines and phenylpyrroles, and hydroxyanilides [fenhexamid]). During the last 25 years, B. cinerea developed resistance to virtually all specific fungicides used to control gray mold. Field resistance to benzimidazoles, phenylcarbamates, and dicarboximides was detected soon after their introduction. Recent studies using PCR-duplex and specific primers for the detection of transposable elements on Chilean B. cinerea isolates recovered from different table grape cultivars corroborated the presence of two sibling cryptic populations, transposa and vacuma (3). Some vacuma isolates have shown natural resistance to fenhexamide (HydR1) and it has been separated into two groups on a molecular basis using a marker gene (Bc-hch): Group I, fenhexamid-resistant vacuma isolates; Group II, vacuma and transposa isolates sensitive to this fungicide (HydS) (2). Group I and II isolates can not interbred (1,2). Other B. cinerea resistant phenotypes, HydR2 and HydR3, have been reported as belonging to Group II (1,4). Single-spore isolates of B. cinerea (472) were collected from different table grape cultivars from 13 locations in the Chilean Central Valley. The isolation was done during harvest time from rotting berries. Fenhexamid (Teldor; Bayer CropScience, Monheim, Germany) was diluted to 10 μg a.i./ml and added to the solid medium (10 g of glucose, 1.5 g of K2HPO4, 2 g of KH2PO4, 1 g of (NH4)2SO4, 0.5 g of MgSO4·H2O, 2 g of yeast extract, and 12.5 g of agar in 1 liter) to reach concentrations of 0, 0.025, 0.05, and 0.1 μg a.i./ml. A 5-mm mycelial plug from each isolate of B. cinerea was cut from the edge of 4-day-old colonies placed in the center of petri dishes with the described fungicide-amended medium and incubated at 20°C for 5 days. Two measurements, octogonal diameters, were taken from each of three replicates per treatment. Means were calculated and the diameter of the inoculated plug was subtracted from each mean. For each isolate, a linear regression of the percent inhibition of mycelial growth versus the Log10 transformation for each of the four concentrations of fenhexamid was obtained. The 50% effective concentration of fenhexamid (EC50) was calculated with the regression equation for each isolate. So, 95.3% of B. cinerea isolates were sensitive (EC50 under 0.083 μg/ml), 1.9% were less sensitive (EC50 between 0.084 and 0.1 μg/ml), and 2.8% (13 isolates) were resistant EC50 values ranging from 0.1 to 8.4 μg/ml. Through PCR-restriction fragment length polymorphism, according to the Bc-hch gene restriction pattern, all resistant isolates analyzed belong to Group II of B. cinerea (Bc-hch2) (2). To our knowledge, this is the first report of fenhexamid resistant isolates of B. cinerea on grapevine in Chile and South America. It would be necessary to study the population dynamics of these isolates, although failure of botrytis control in the field with this compound has not been reported. References: (1) C. Albertini et al. Mycol. Res. 106:1171, 2002. (2) E. Fournier et al. Mycologia 97:1251, 2005. (3) T. Giraud et al. Mol. Biol. Evol. 14:1177, 1997. (4) P. Leroux et al. Phytoma 599:31, 2006.