Isolation And Purification Of Gliotoxin From TRICHODERMA VIRENS Strain TY009 And Its Gfp-Labeling

In our previous studies,it was found that one fraction of the metabolites isolatedfrom Trichoderma virens strain TY009 inhibited strongly the mycelial growth ofRhizoctonia solani,the causal agent of rice sheath blight.The aim of this study was toevaluate the potential of Trichoderma virens strain TY009 to control the rice disease.In the present work we purified and identified the active component of the metabolitesfrom the strain TY009,the inhibition in vitro of the mycelial growth and the sclerotialgermination of Rhizoctonia solani by the purified gliotoxin was observed.Also thevector pSilent-EGFP containing GFP gene for its expression in Trichoderma virensstrain was constructed.The strain of Trichoderma virens TY009 was labeled with GFPgene through genetic transformation.The colonization on sclerotia of Rhizoctoniasolani was investigated by using of the GFP-labeling transformant of Trichodermavirens strain TY009.The main research results are as follows:1 Extraction,purification and identification of gliotoxin from the metabolites of T.virens andthe inhibition of gliotoxin to Rhizoctonia solaniThis active fraction of the metabolites of T virens strain TY009 was extractedwith ethyl acetate,purified with chromatographic column and thin layerchromatography(TLC).Through mass spectrum analysis,the active component wasidentified as gliotoxin.Its molecular weight is 326.The purified gliotoxin inhibited invitro the mycelial growth of Rhizoctonia solani.That inhibition was promoted by lowpH in the medium.The mycelial growth of Rhizoctonia solani was totally suppressed at the gliotoxinconcentration of 80μg/ml.The purified gliotoxin inhibited in vitro also thegermination of sclerotia of the pathogen,the germination rate was 0.00 % and 71.67 %at the concentration of 160μg/ml and 40μg/ml,respectively;While that was 100 % atthe concentration of 5μg/ml and 10μg/ml,as same as the control.2 Labeling of Trichoderma virens GFP geneThe vector pSilent-EGFP containing GFP gene for its expression in Trichodermastrain was constructed and transformed into Trichoderma virens strain TY009 usingPEG-CaCl₂ mediated transformation method with an efficiency of 3-4transformants/μg plasmid DNA.The transformants with gfp gene were detected withPCR analysis and the fluorescence expression.The expression of fluorescence in young hyphae was stronger than that in the oldhyphae and the fluorescence of immature chlamdospores was stronger than that ofmature chlamdospores.Biological characteristic detection of transformants manifestedthat there was no significant difference in spore production but significant difference in the mycelial growth between the wild strain and the three transformants.There wasalso significant difference in the mycelial growth among the three transformants.Thin-layer chromatography analyses manifested that the metabolites produced by thethree transformants were same with that by the wild strain.3 The colonization of GFP-labeling Triehoderma virens on sclerotia of Rhizoctonia solaniIt was found that the hyphae of Trichoderma virens transformant wraped thesclerotia of Rhizoctonia solani 2 days after innoculation and penetrated the sclerotiaafter 5 days,then they extended deeply in sclerotia after 20 days and finally reachedcore of sclerotia appearing soft after 40 days.Trichoderma virens formedchlamydospores in sclerotia.The fluorescence of chlamydospores in sclerotia wasweek,but strong fluorescence was expressed when the chlamydospores germinated.