Studies On Trichoderma Harzianum As A Biocontrol Agent On Plant Disease

A biocontrol strain of strong inhibition to Fusarium spp. had been isolated from 13 soil samples in different counties in Taiwan and in Fujian, which had been identified to be Trichoderma harzianum, named Trichoderma harzianum FJAT-9040. In the assay on biology, the effects of the different source of carbon, nitrogen, carbon nitrogen ratio, inorganic salt and different culture conditions such as cultural time, inoculation quantity, initial pH value, temperature on the hypha growth and spore germination of T. harzianum FJAT-9040 had been studied. The relationship among the hypha growth, the spore germination, the sorts and the contents of fatty acid of T. harzianum FJAT-9040 had been discussed. The gfp gene genetic transformation system had also been setup to obtain a srain of T. harzianum with gfp gene marked, named FJAT-9295. The colonization character of FJAT-9295 in defferent types of soil and its effects on soil microbial community structure had observed. The results were as follows:The best carbon source for T. harzianum FJAT-9040 was glucose, the optimum nitrogen source for T. harzianum FJAT-9040 was beef extract, and the best carbon nitrogen ratio was 3:1, and the best mineral composition in the orthogonal test for spores germination was the magnesium sulfate. By orthogonal test optimization, the optimal solid medium contmpositon for T. harzianum FJAT-9040 was: glucose 18 g, NaNO₃ 16.6 g, MnSO₄ 1 g, KH₂PO₄ 2 g, water 1000 mL. The better cultural times for T. harzianum FJAT-9040 growing were 3 days, in 4 days was the best time for spores germination. The initial pH values between 4 to 6 were good for the hypha growth, pH 6 to 8 good for spore growing. 25℃was the optimal temperature for the hypha growth, the best temperature for spores germination was 30℃.The sorts and contents of fatty acids biomarkers (PLFAs) could be changed in the different nutritional conditions, such that the contents of four main fatty acids biomarkers were shown as C18:2ω6,9c/18:0 ANTE > C16:0 > C18:1ω9c > C18:0. By clustering analysis the different nutrient conditions of T. harzianem, the five groups had been classified according to the fatty acids biomarkers , which demonstrated that the hypha growth and spores germination T. harzianum, had been correlative to the average of fatty acids sorts and content. The content of C18:2ω6,9c/18:0 ANTE had been highly correlated with the hypha growth and spores germination.The inhibition rate of the strain T. harzianum FJAT-9040 against the Fusarium strain FJAT-141, FJAT-136, FJAT-282, FJAT-370 ranged from 80% to 90%. When the fusarium spore concentration at 107 magnitude, the diameters of inhibition zone were 16.00 mm, 16.78 mm, 27.9 mm, 14.54 mm for the strains, respectively. The metabolic liquid of T. harzianum FJAT-9040 caused a series damage such as, the hypha distortions, protoplasm inflating, protoplasm concentrating, a large vacuoles occuring within hypha. T. harzianum FJAT-9040's bacteriostatic action had achieved mainly through the strain competition and inhibition.The GFP transformation system of T. harzianum was established mediated by PEG-CaC12, throuh which we integrated the plasmid pCT74 with amphotericin B resistance genes and GFP marker genes into the chromosome of T.harzianum successfully and acquired a transform strain FJAT-9295 with stability genetic and high fluorescence intensity. Compared to the wide type, the biomass for the hypha growth and the spores germination were a little lower, the inhibition to fusaruim also declined dslightly, but its entifungal efficiency was still significantly strong.The colonization characteristics of transformants in different soils were studied, the results showed that colonization situation was different according to different types of soil, the colonization order were: sandy soil>nutritional soil>eggplants soil >loess. In 15 days after colonization, the number of FJAT-9295 in plant-exsiting treatment was significantly higher than the no plant-exsiting treatment, but later this advantage had gradually reduced, the quantity of FJAT-9295 in the soil became stable about 105 quantity until 32 days. The PLFAs analysis showed that FJAT-9295 could obviously restrain bacteria, fungi, actinomyces in soil and changed the microbial community structure of the soil.