Studies On Optimization Of Fermentation Conditions And Antimicrobial Activive Compositions And Nematicidal Activity Of The Fermentation Liquid Of T. Longibrachiatum T6

Trichoderma longibrachiatum is one species of Trichoderma genus that has biocontrol potential. There are some reports on application to control plant diseases, but the biocontrol mechanism is still not known. Trichoderm T6 strain was isolated from the local forest soil in Gansu. The strain has great inhibition on pathogene, and has a strong lethal function on cysts eggs and second stage juveniles of Heterodera avenae. In order to determine the mechanism of Trichoderma bio-control, the current research studies the metabolites from Trichoderma longibrachiatum T6. The main results of this research are as follows:1. The antimicrobial activity effect of T. longibrachiatum T6 strain was studied through the single factor. O n the basis of the previous results, according to the central composite experimental design principles, the response surface method with 3 factors and 3 levels was adopted. The results showed that the optimal carbon source were the mannitol, quality scores for 4.0 percent. The optimal nitrogen source was peptone, quality scores for 0.6 percent. The optimum conditions were that the inoculated with 1.1%, linoculums in 150 m L with 74.5 m L fermented liquid for 4 days. Under these conditions, the predicted value of the antifungal activity could be up to96.3%, in basic agreement with the actual value of 96.88%. The absolute error is 0.58%.2. On the basis of optimized cultural conditions, using 11 plant pathogens as inhibitory spectrum tested the antifungal activity of fermentation. The results of bioassay showed that the fermentation had best inhibition against Valsa mail、Alternaria mali、Rhizoctonia solani(the inhibition rates were 95.97%、80.33% and 81.97% respectively). The weakest inhibition was against Trichothecium roseum and Rhizopus nigricans(the inhibition rates were 56.56% and 52.61% respectively). Growth rate method was used to determine stability of the fermentation products on Valsa mail. Stability tests showed that: fermentation broth could endure the treatme nt of a high temperature of 121℃, acid, alkali, light and ultraviolet; it could be stored at 4℃ and remain active for at least 60 days unchanged; long time microwave treatmentand Zn2+、Fe3+ 、Fe2+have large effect on its stability; oxidizer and reducing agent have insignificant effect within the scope of the concentration.3. Using the separation and analysis methods of extraction, TLC and column chromotography, the antifungal secondary metabolites from the fermentation liquid of T. longibrachiatum T6. The antifungal part of secondary metabolites was determined with the method of biological activity tracing. The results indicated that the extracts obtained with direct extraction method and ethyl acetate extract had the optimum inhibiting effect on mycelial growth of Valsa mail and Botrytis cinerea, with the inhibition rates of 87.96% and 70.04%, respectively. Ethyl acetate: petroleum ether(10:l) was the optimum developer for TLC. Seven point of samples were observed under UV light and with I2. Nine components were obtained by silica column chromatography, different ratio of ethyl acetate and petroleum ether as eluent, the antifungal activity was tested after condensing and drying solvent. We selected the flows which had high-activity matter. According to the results of high flow of CZ-4 antibacterial component were analyzed with GC-MS, Main antibacterial compone nts are Di- isobutyl phthalate, O leic acid, 2-Ethylhexyl hydrogen phthalate and Erucylamide.4. Through the antifungal activity tests of DIBP, O leic acid, MEHP and Erucylamide on Valsa mail, Alternaria mali, Fusarium oxysporum, Rhizoctonia solani and Trichothecium roseum. The results showed that DIBP exhibited strong antibacterial activity against Rhizoctonia solani and Trichothec Ium roseum with EC50 values of 64.7 μl/m L, 1.8 μl/m L. MEHP showed strong antibacterial activity against Valsa mail with EC50 value of 40.7 μ g/m L. O leic acid and Erucylamide were hardly any inhibition to the test pathogenic fungis.5. Study of lethal effects of culture filtrate of T. longibrachiatum T6 at different concentrations on hatching of individual eggs and secondary juvenile of Heterodera avenae, and observe the morphological characteristics of Heterodera avenae eggs and secondary juvenile infected. The results showed that the fermentation of T. longibrachiatum T6 has a strong lethal function on eggs and second stage juveniles ofHeterodera avenae. The two times concentrated of T.longibrachiatum T6 fermentation on Heterodera avenae eggs, second stage juveniles, The hatching of eggs were suppressed by 46.47%; And the corrected mortality of second stage juveniles were 44.45%, respectively. The microscopic observation showed that the coelom inclusions of died nematode was dissolved.