| Clonostachys rosea（syn.Gliocladium roseum）is a promising mycoparasite against various plant pathogenic fungi,such as Sclerotiniasclerotiorum,Botrytis cinerea,Fusarium spp.and Rhizoctonia solani,and it has been used in the management of plant fungal diseases.However,unpredictable efficacy in fields has hindered the application of the biocontrol agents.Understanding the modes of action of these biocontrol fungi will facilitate their commercialization and large scale application.In order to clarify the modes of action C.rosea,we selected a highly efficient isolate 67-1 originally marked with Green Fluorescent Protein（GFP）.The growth,sporulation ability,antagonistic ability,the mycoparasitic ability of the transformants,and their control efficacy to Sclerotinia stem rot were tested,from which the mutants had no biological difference with the wild type strain determined.This strong fluorescent emitting transformant（G67-1）was used against Botrytis cinerea and Sclerotinia sclerotium for monitoring the mycoparasitic and antagonistic partiality.Clonostachys developed different special structures like papillae and infection pegs,by which the biocontrol fungus penetrates the host and absorbs nutrients,and secrets different secondary tabolites for controlling the pathogen.The exploration of parasitism-related genes provides better knowledge about the mechanisms of parasitizing to the pathogens.Transcriptome data from the whole genome sequence and quantitative real-time PCR techniques were used to investigate the differentially expressed genes in 67-1 associating with sclerotia parasitizing at 8 h,24 h,and 48 h.Nine genes with remarkably up-regulated expression levels（protease encoding gene crprs5A and crprs,sodium P-type ATPase encoding gene cratp,heterokaryon incompatibility protein-encoding gene crhep,ethanolamine utilization protein-encoding gene cruet and cretu,short-chain dehydrogenase protein-encoding gene crscd,triose-phosphate isomerase encoding gene crtpi,and Hypothetical protein-encoding gene crhyp）.Furthermore,the function of protease encoding gene crprs5A was testified during the mycoparasitic process.The full DNA sequence of crprs5A was obtained from the whole genome of C.rosea 67-1.The coding region of crprs5A was 1419 bp,which was remarkably up-regulated expression at 8 h,which encodes a protein,consists of 852 amino acids.Phylogeny analysis showed that crprs5A had 73.8%similarity with26S proteasome regulatory subunit S5A,a multiubiquitin chain binding protein from Trichoderma.Phenotypes analysis showed that the growth rate and sporulation ofΔcrprs5A were significantly lower than that of wild type and the complemented strain.The parasitic ability ofΔcrprs5A to sclerotia was reduced from 4th grade to 1st grade compared with the WT and complemented mutant.Its control efficiency of soybean Sclerotinia stem rot in the greenhouse decreased half significantly（p<0.05）compared to the wild type.This study will significantly improve the current understanding of the mode of action of C.rosea and provide a basis for further exploring the genes related to the mechanisms of action against pathogens,which has essential controlling significance to improve the biocontrol effect of C.rosea67-1. |
