Evaluation on resistance of Botrytis cinerea to four fungicides in Mile county

Abstract:【Objective】Grape grey mould, caused by Botrytis cinerea, brings about damage to grapes (Vitis vinifera) in vineyards throughout the world. The first signs of disease on grapes often appear dur- ing the bloom period, when the fungus attacks the flower. The infected berries may become covered with greyish-tan conidia of the fungus. Berry stalks and cluster stems may be invaded, causing them to shrivel, and berries that have split or have been punctured are often easily attacked by other organisms. Gray mold is one of the most serious fungal diseases in grape. Due to the lack of registered broad-spec- trum compounds, effective control of grape grey mould is usually based on repeated fungicide applica- tions during the growing season, especially in mild and humid climates. But the fungus is known to rap- idly adapt to its environment and to develop fungicide resistance in the field. Because of these problems, the pesticide resistance management strategies have now been widely adopted, which restrict the usage of the same fungicide. However, increasing occurrence of isolates with low-level cross-resistance to various fungicides has been observed in Chinese vineyards. In Mile grape production, the following fungicide groups are in current use against B. cinerea: procymidone, pyrimethanil, fludioxonil and fluo- pyram. However, little is known about the pesticide resistance in B. cinerea populations from the grape in Mile county, which is the primary region for grape production in Yunnan province. The objectives of   this study were to: (1) assess the resistance to procymidone and pyrimethanil and establish the resis- tance distribution of B. cinerea to procymidone and pyrimethanil in Mile county; (2) assess the stability of resistance to fludioxonil; (3) estimate the stability of resistance to fluopyram; (4) evaluate the effica- cy of the four fungicides for controlling grape gray mold.【Methods】The diseased samples were col- lected from Mile county in Yunan province. One hundred and ninety-seven strains of B. cinerea were isolated and purified. All isolates were then single-spore cultured and stored as mycelium plugs either in 15% glycerol at −80 °C for long-term storage or in sterile water at 4 °C for short-term storage. All iso- lates were screened for resistance to fungicides. Technical-grade fungicide was dissolved in dimethyl sulfoxide (DMSO) to make a 10 mg · mL- 1 stock solution. To detect fungicide resistance, conidia from sporulating colonies growing on PDA were harvested in 5 mL of sterile ddH2O. Final conidial concentra- tion was adjusted at 2 × 105 conidia · mL- 1 using a hemocytometer. For determining pathogen sensitivity to the pyrimethanil, a minimal medium instead of PDA was used containing 10 g glucose, 1.5 g K2HPO4, 2 g KH2PO4, 1 g (NH4)2SO4, 0.5 g MgSO4 · 7H2O, 2 g yeast extract, and 12.5 g agar per liter. Colony diameters were measured after incubation for 4 days at 20 °C in the dark and the inhibition of mycelial growth was calculated. The sensitivity assays were performed at the discriminatory concentra- tions of fungicides in the medium. Mycelial growth at a given discriminatory concentrations implies a fungicide-resistant phenotype. Resistance standards employed for procymidone, pyrimethanil, fludioxo- nil and fluopyram were 0.31, 0.091 1, 0.013 5 and 0.13 mg·L-1, respectively. Three replicate plates were used for each treatment and the experiments were performed three times for all tested fungicides.【Results】The study showed the existence of procymidone- and pyrimethanil-resistant strains was at frequencies of 71.1% and 100.0%, respectively. High resistance frequencies of 13.7% and 23.8% were observed for procymidone and pyrimethanil, respectively. In procymidone-resistant strains, forty-four were low-resistant to procymidone (frequency: 23.4%), sixty-seven were medium-resistant to procymi- done (frequency: 34.1%), and twenty-seven were high-resistant to procymidone (frequency: 13.7%). In pyrimethanil-resistant strains, thirty-six were low-resistant to pyrimethanil (frequency: 18.3%), one hun- dred and fourteen were medium-resistant to pyrimethanil (frequency: 57.9%), forty-seven were high-re- sistant to pyrimethanil (frequency: 23.8%). Frequencies of 71.1% were resistant to both procymidone and pyrimethanil. In the forty-seven pyrimethanil-high-resistance strains, thirty-eight were resistant to procymidone. Of thirty-eight strains, two were high-resistant to procymidone, sixteen were medium-resistant to procymidone, and twenty were low-resistant to procymidone. In this study, two isolates were found to be resistant to fludioxonil (frequency: 1.0%), while no strain resistant to fluopyram was detect- ed.【Conclusion】Our research has raised a serious concern in grape production in Mile county regard- ing the resistance of B. cinerea to the four fungicides most widely used to control gray mold. The pres- ent monitoring revealed the fungicide resistance phenotypes in B. cinerea, showing different levels of resistance to single fungicides and/or multiple resistance to different combinations of fungicides. The high levels of resistance observed with procymidone- and pyrimethanil-resistant strains and the high number of isolates with double-resistance to the two fungicides are alarming data. It’s worth noting that the strains isolated were high-sensitive to fludioxonil and fluopyram, though two strains were resistant to fludioxonil. Thus, we suggest that procymidone and pyrimethanil should be restricted to use for con- trolling grape gray mold. We recommend that fludioxonil and fluopyram may be adopted for control and fungicide-resistance-management in B. cinerea.