Stimulatory Effects Of Sublethal Doses Of Carbendazim On Virulence Of Botrytis Cinerea

The plant pathogen Botrytis cinerea is a necrotrophic fungus belonging to the genus Botrytis of the phylum Ascomycota.In this dissertation,hormetic effects of carbendazim on the virulence of two sensitive isolates?HBtom459 and HBtom536?and two carbendazim-resistant isolates?HBtom451 and HBstr491?of B.cinerea were studied.Sublethal doses of carbendazim significantly?P<0.05?increased the virulence of B.cinerea to cucumber plants.The maximum percent stimulations of virulence for the resistant isolates HBtom451 and HBstr491 were 16.7%and 13.5%,occurring at carbendazim doses of 10?g/mL and 50?g/mL,respectively.The maximum percent stimulations for the sensitive isolates HBtom536 and HBtom459 were 18.8%and 12.5%,occurring at carbendazim doses of 0.02?g/mL and 0.005?g/mL,respectively.After the mycelia were grown for 72 h on PDA media amended with carbendazim and inoculated on cucumber leaves,the maximum percent stimulations of virulence for the resistant isolates HBtom451 and HBstr491 were 7.9%and 9.5%,respectively,occurring at a carbendazim dose of 50?g/mL.The maximum percent stimulations for the sensitive isolates HBtom536 and HBtom459 were 12.9%and 9.1%,occurring at carbendazim doses of 0.02?g/mL and 0.005?g/mL,respectively.Mycelia of the resistant isolates HBtom451 and HBstr491 were grown for 48 h on PDA amended with carbendazim at 200?g/mL as the preconditioning treatment.After the preconditioned mycelia were inoculated on cucumber leaves sprayed with sublethal doses of carbendazim,the maximum percent stimulations for the isolates HBtom451 and HBstr491 were 18.0%and27.4%,occurring at spraying concentrations of 200?g/mL and 400?g/mL,respectively.As the preconditioning treatment,mycelia of the sensitive isolates HBtom536 and HBtom459 were grown for 48 h on PDA amended with carbendazim at 0.05?g/mL and0.02?g/mL,respectively.The maximum percent stimulation of virulence for the preconditioned isolates HBtom536 and HBtom459 were 22.2%and 17.2%,occurring at spraying concentrations of 0.05?g/mL and 0.02?g/mL,respectively.Sublethal doses of carbendazim significantly?P<0.05?increased the sclerotia number and weight of B.cinerea.For the resistant isolate HBtom451,the maximum percent stimulations on sclerotia number and weight were 19.8%and 10.3%,respectively,occurring at carbendazim dose of 50?g/mL.For the resistant isolate HBstr491,the maximum percent stimulations on sclerotia number,weight,and conidial germination were 26.6%,12.6%,and 4.4%,respectively,occurring at carbendazim dose of 50?g/mL.For the sensitive isolate HBtom536,the maximum percent stimulations on sclerotia number and weight were 22.6%and 20.8%,respectively,occurring at carbendazim dose of 0.02?g/mL.For the sensitive isolate HBtom459,the maximum percent stimulations on sclerotia number,weight,and conidial germination were 19.7%,14.8%,and 4.0%,respectively,occurring at carbendazim dose of 0.005?g/mL.Studies on the time-course of virulence stimulations with the isolates HBstr491showed that the earliest symptom of infection could be detected at 24 hour post inoculation?HPI?on cucumber leaves spayed with carbendazim at 50?g/mL,whereas no necrotic symptom could be discerned by the naked eye for the nontreated control.Scanning electron microscopy observations demonstrated that infection cushions were visible at 16 HPI for cucumber leaves treated with carbendazim,whereas no infection cushions could be detected for the nontreated control.These results indicate that stimulatory effect of sublethal doses of carbendazim on the virulence of B.cinerea manifests itself in the early infection stage and thus is a direct stimulation rather than an overcompensation for a disruption of homeostasis.Studies on the biochemical mechanisms of hormesis demonstrated that stimulatory effects of carbendazim on virulence were not caused by oxalic acid production,laccase activity,or tolerance to hydrogen dioxide?H₂O₂?.Molecular studies showed that sublethal doses of carbendazim did not increase the expression of virulence factor pectin methylesterase gene Bcpme1,endopolygalacturonase gene Bcpg2,cutinase gene CutA,xylanase gene Xyn11A,and NADPH oxidase gene BcnoxA.Concurrent spraying carbendazim and iprodione at the following concentrations 3?carbendazim?+0.0001?iprodione??g/mL,10+0.0005?g/mL,50+0.0025?g/mL,and200+0.0125?g/mL on leaves of cucumber plants exhibited hormetic effects on the virulence of the resistant isolates of B.cinerea.The maximum percent stimulations for the isolates HBtom451 and HBstr491 were 18.4%and 22.8%,respectively.Compared with spraying carbendazim or iprodione alone,the maximum percent stimulation did not increase significantly for concurrent spraying the two fungicides.None of the differences in tolerance to H₂O₂,cell membrane permeability,or exopolysaccharide content was statistically significant?P<0.05?between spraying a single fungicide and concurrent spraying the two fungicides.