Screening And Identification Of Antagonistic Bacteria Against Sclerotinia Sclerotiorum And Their Application Bases

Sclerotinia sclerotiorum(Ss)is a necrotrophic fungal phytopathogen with extremly broad host plant range.The white mould or stem rot disease caused by this fungus is the most important disease in oilseed rape in China.Given lacking of highly resistance resources,labour-and time-consuming and low efficiency of agricultural control due to sclerotium production by this pathogen,and easily causing chemical resistance and environmental pullution by chemical control,biological control of Ss is thought to have great potential to be exploited.In this study,antagonistic microbes against Ss were screened,mechanisms underlying the antagonistic function were revealed and effects on rape growth and resistance were analyzed in order to provide potential beneficial microbe resource for exploiting novel biocontrol agents.The main results are as follow.(1)A batch of antagonistic microbes against Ss were screened out.Five hundred and ninety bacterial and 75 fungal strains were isolated from rape root rhizosphere and Ss sclerotia.From which 45 antagonistic microbes including 35 bacterial and 10 fungal strains were screened out using plate co-culture approach.Among them,24 strains exhibited stable genetic inheritance of the antagonistic trait for over 10 generations.Six of them,named S4,S5,S6,S7,S10 and S14,showed consistantly high inhibitory efficiency in the detached leaf and pot growing whole plant tests.Strains S4,S5 and S6 are the best with consistant inhibitory efficiency of over 81%with the maximum of 97%.(2)Seven antagonistic strains were identified.The antagonistic strains S4,S5 and S6 were identified as Bacillus cereus through morphological observation,16S rDNA sequence analysis,fatty acid identification and biological mass spectrum identification.Additionally,strains 83 and 84,whose culture caused decay in sprayed rape plants,were identified as Pectobacterium carotovorum according to 16S rDNA sequencing result.Strains 111 and 250 produced nondiffusible blue pigment and diffusible yellowish green fluorescence,and were identified as Pseudomonas fluorescens according to results of 16S rDNA sequencing and hypercensitive response inducing test in tobacco.(3)The mechanisms underlying the antagonistic function and the potential in biocontrol of strains S4,S5 and S6 were elucidated.The three strains owned antagonistic ability against a broad spectrum of fungal phytopathogens.Both metabolic volatiles produced by the strains and the secreted nonvolatile compounds had the inhibitory role to Ss.They could secret chitinases,proteases and cellulases.The ethyl acetate extracts displayed inhibitory activity to Ss.Spraying with the bactrial culture significantly induced rape plant resistance to Ss with disease lesion inhibitory efficiency of 66%-83%,while root drenching,seed immersing and leaf brusing with bacterial culture all could not induce systemic resistance to Ss in the up untreated parts of plants.However,co-culture of the bacteria with rape was able to induce systemic resistance to Ss with disease lesion inhibitory efficiency of 43%-66%.The antagonistic bacteria were enriched to the rape roots to repress taproot growth while promote lateral root formation and growth.Proteomics analysis for rape response to strain S6 was conducted.Two hundred and seventy and 259 proteins differentially for over two folds accumulated in between S6-co-cultured and non-cultured rape roots and leaves,respectively,including dozens of proteins involved in resistance as well as reactive oxygen species(ROS)accumulation and redox regulation.ROS accumulation and redox status in roots and leaves differed significantly.The anatagonistic bacterium S6 could suppress ROS accumulation in roots to benefit its growth and colonization.