Functional Analyses Of Botrytis Cinerea Alcohol Dehydrogenases Mediating The Fungal Development, Environmental Adaptation And Pathogenicity

Botrytis cinerea causes gray mold disease on various important vegetables,fruits,and crops.The disease annually causes huge economic losses worldwide.This problem can be generally solved upon usage of chemical fungicides.However,most of them are expensive,non-environmentally friendly,and ineffective for long term use.This is due to the ability of B.cinerea to develop resistance to those chemicals.Alternatively,determination of the pathogenesis of B.cinerea on the molecular levels emerged as effective and environmental benign approach for gray mold diseases.Recently,the sequencing of the whole genome and analysis of two model strains of the pathogen led to a practical model outlining the molecular strategies used by B.cinerea in the infection process.More extensive studies are discussed the status of the functional analysis of genes which are involved in pathogenesis.Our global transcriptome analysis of tomato-B.cinerea interaction at the early stage of infection found that the transcriptional levels of three genes encoding alcohol dehydrogenase?ADH?BcADH1,BcADH2 and BcADH3 in B.cinerea strain B0510 were highly up-regulated.Using gene replacement approach via homologous recombination,we generated the target gene knockout mutants and their functional complementary strains.The variability,growth phenotypes and pathogenicity of the generated B.cinerea strains were investigated and compared to their wild type?WT?control.In addition,their responses to oxidative stress,alcoholic fermentation,hypoxic conditions and cell wall integrity were also investigated.The results clearly demonstrate that the mutant strains of ?bcadhl and ?bcadh2 revealed a strong difference in morphological observations of conidiospore in the shape and size.The ?bcadhl and ?bcadh2 mutants have semi-spherical shape and smaller,comparison with the WT strain.While the ?bcadh3 mutant is approximately close to the WT and complementary strains.Correspondingly,the characteristic analysis of the strains during conidial germination was determined by calculating the percentage of germination rate and branches formed after 6 and 12 hours of incubation,respectively.Notably,in the germination rate,there is slightly difference among the examined strains with more than 90%germinating conidia except for?bcadh2mutant with only 60%?P<0.01,n=200?.As well,after 12 hours post incubation,the ?bcadhl and Abcadh2 mutants are noticed significantly different in the number of branches compared with the WT.On the other hand,there are significantly different between the Abcadhl and ?bcadh2 mutants and other related strains in the sporulation and sclerotia formations.The production of conidia was dramatically decreased,while the sclerotia were increased on the plates of Abcadhl and Abcadh2 mutants.Pathogenicity test was analyzed based on measurements of the average size of the lesions on tomato leaves.The lesion sizes caused by the mutants were recorded a significantly smaller than their counterpart WT strains.As a result,the stress responses were investigated by measuring the radial diameter of strains growing on the Potato Dextrose Agar?PDA?medium supplemented with ethanol,SDS?a cell wall damage agent?,H₂O₂?an oxidative stress agent?and COCl2?a chemical mimicking hypoxic condition?.The results indicate that disruption of ADHs in B.cinerea impaired the Abcadh mutants abilities to fermentation alcoholic when compared to the WT and complemented strains.Moreover they were more sensitive to the reactive oxygenated species?ROS?,oxygen limitation?hypoxia?and SDS??bcadhl and ?bcadh2?.Taken together,all these results constitute strong evidence demonstrating that both of ADHI and ADH2 genes in B.cinerea are required for the fungal development,environmental adaptation and full pathogenicity.