Establishment Of GFP-labeled Transformation Of Trichoderma Hamatumand Its Growth Promoting And Biological Control Against Phytophthora Capsici For Pepper Plants

Trichodermaspecies is one kind of important resources of biocontrol fungi,which have significant prospect and value for biological control of crop diseases.The reserches of interactions between Trichoderma species and host plants,its growth-promoting and biocontrol for plant diseases are the theoretical basis for developing stable and highly effective Trichoderma biocontrolagents.The Agrobacterium tumefaciens-mediated transformation(ATMT)of Green Fluorescence Protein(GFP)gene is a strong tool for the research of interaction between microorganisms and host plants.T.hamatum ACCC31649 is an excellent biocontrol strain which have significant growth-promoting effects on cucumber plants performed by previous research.In this study,GFP-labeled transformation of T.hamatumwas established by ATMT method,and the GFP-labeled T.hamatum-GF21 strain which inherits stably was selected.The observations of pepper tissue sections and rhizosphere soils of pepper plants by fluorescence microscopy were performed for the colonizing patterns of the GFP-labeled strain.In addition,the growth-promoting effects on pepper plants caused by T.hamatum and the primary inhibition of T.hamatum-GF21 against Phytophthora capsici were further proved.The main results are as follows:1.Establishment of transformation of GFP gene to T.hamatum strain via ATMT method and screening of the stable GFP-labeled transformants of T.hamatum.In the present study,the dyes of DAPI(4’,6-diamidine-2-phenylindole dihydrochloride)and Calcoflour White were combinedly used for dying the nucleuses of T.hamatum strain ACCC31649,from which showed that hyphae and spores of this strain are mononuclear.The protocol of GFP-labeled transformation of T.hamatum through ATMT method were optimized,including the concentrations of acetosyringone solution and conidium solution,the time induction culture and co-cultivation.Ten stable GFP-labeled transformants of T.hamatum with strong fluorescence were selected by Hygromycin B resistance and PCR of Hyg B gene of continuous subcultures from 100 transformants.Furthermore,the consistency of the morphology,growth rate,sporulation yield and biomass between GFP-labeled strain ACCC31649 and the wild strain were determined and compared respectively.2.The colonizing characteristics of T.hamatum in pepper tissues and its growth-promoting effects on pepper seedlings.The four-leaf pepper seedlings were inoculated with conidium solution of T.hamatum by pouring root treatment,the T.hamatum-GF21 was detected in rhizospheresoil of pepper and the population increased gradually and then declind.Fluorescence microscopy observation confirmed that the T.hamatum-GF21 colonized in the roots,stems and leaves of the pepper.After 30 days,the trement of T.hamatum-GF21 increased the pepper height by 13.52%,root length by 16.19%,fresh weight by 43.75% and dry weight by 45.28% respectively,compared with the water control.There is no significant difference between the T.hamatum-GF21 and the wide strain in the effect on the plant growth promoting.3.Theprimary research of the biocontrol effect of T.hamatuma against Phytophthora capsici.The dual-culture assay confirmed that T.hamatum inhibited P.capsici by 40.87% of growth inhibition rate.The fermentation liquid of T.hamatum strongly inhibited hypha growth of P.capsici,and its biocontrol mechanisms against P.capsici were mycoparasitic and secretion of cell lysis by primary assays.The inoculation trials of pepper fruits of T.hamatumagainst P.capsici showed that T.hamatum strain had a biocontrol effect on the Phytophthorablight of pepper fruits by 19.01% of inhibitory rate.