Suppression Mechanism Of Wuyiencin On Grape Gray Mold And Its Role In Deferral Of Fungicide Resistance To Botrytis Cinerea

In order to further expand the resources of biocontrol agents on grape gray mold,the biological control effect of wuyiencin on grape gray mold was determined using the in vitro inoculation method,and the mechanism of wuyiencin against Botiytis cinerea was clarified by fluorescence staining,electron microscopy observation,and real-time fluorescence relative quantitative detection.On this basis,the Horsfall and CTC co-virulence coefficient methods were applied for the combination of wuyiencin and chemical fungicides and the deferral of fungicide resistance in B.cinerea through combination of wuyiencin and chemical fungicides,to further explore the mechanism of wuyiencin deferring fungicide resistance in B.cinerea.The main results are as follows:(1)Wuyiencin had good effect on the control of grape gray mold.When the concentration of wuyiencin reached 120 μg/mL,its control effect on the leaves and fruits could reach 88.1%and 96.6%,by spraying wuyiencin,and the disease symptoms of leaves and fruits were significantly reduced or no symptoms appeared.Wuyiencin could effectively reduce the pathogenicity of gray mold,and the inhibition rate of mycelium and spores reached 94.3%and 97.8%when it reached 120μg/mL.Wuyiencin could destroy the growth and development of mycelium and spores in B.cinerea,reduce the number of infestation mats and deform their morphological structure,decrease the oxalic acid production capacity and the expression level of disease-causing genes,induce the rapid production and increase the accumulation of callus and reactive oxygen in grapes,and effectively improve the defense ability of grapes.(2)The combination of wuyiencin with three chemical fungicides(pyrimethanil,iprodion and flumioxazin)was found to have synergistic effects.The highest co-virulence factor of 391.4 and the smallest EC50 were found for the combination of wuyiencin:pyrimethanil with a volume ratio of 7:3,which was the best synergistic combination.The effect of the combination was evaluated and it was found that the effect of the combination on the control of gray mold on detached grape leaves and fruits could reach 51.1%and 66.1%,respectively,which was significantly better than the control effect of wuyiencin or pyrimethanil alone.(3)The combination of wuyiencin and the chemical fungicide pyrimethanil could effectively defer the resistance of B.cinerea.Through the induction of highly resistant strains in vitro in cultivation of B.cinerea for 10 generations on media with pyrimethanil,the resistance multiplier of induced strains was 9.18 times,while the resistance multiplier of the compound induced strains grown on media with wuyiencin and pyrimethanil was only 1.27 times,proving that the addition of wuyiencin could effectively defer the development of fungicide resistance.On this basis,the preliminary investigation of the mechanism of wuyiencin to defer the resistance of B.cinerea was conducted,and it was found that wuyiencin had a significant inhibitory effect on the methionine synthesis gene,and the gene expression of CGS and CBL was down-regulated by 4.38 and 2.85 times,respectively,when the concentration of wuyiencin reached 60 μg/mL.In addition,wuyiencin could significantly reduce the activity level of cell wall degrading enzymes.Except for β-glucosidase activity,the activities of pectinase and cellulase showed significant decreases,from about 38 U/g and 150 U/g to about 20 U/g and 50 U/g,respectively.In conclusion,it was demonstrated that wuyiencin could effectively defer the development of resistance to fungicides in B.cinerea.