Studies On The Screening And Properties Of Trichoderma Spp.Producing Chitinase

118 strains of Trichoderma spp. were isolated from 49 soil samples collected from 4 cities (Yuxi, Wenshan, Dali and Lijiang) of Yunnan province.The optimized medium for isolation contained yeast extract 5g/L, KH2PO4 10g/L, MgSO4?7H2O 0.5g/L, NaNO3 3g/L, KCl 0.5g/L, FeSO4?7H2O 0.02g/L, Agar 18g/L, and sucrose 30g/L. There was much KH2PO4 in this improved medium, which made the medium acidulated, so bacteria and a few fungi could not grow with the medium. As the color of the improved medium was darker, which made the character of Trichoderma spp. colony and spore's color very visible, it was very easy to discriminate Trichoderma spp. colony.Direct dilution method and trapping method were optimal for soil samples disposal. Compared with plating dilution method, direct dilution method and trapping method were more fit to process large number of soil samples. The trapping method could help us gain objective strains.In this experiment, we found a new method that can screen visually and accuratly Trichoderma spp. producing chitinase. 118 isolates of Trichoderma spp. were cultured in liquid medium with chitin or colloidal chitin as the sole carbon source for 4 days. The supernatant of the fermented broth was mixed with colloidal chitin and heated in water-bath at 37℃ for 30 minute, then 3, 5-dinitrosalicylic acid reagent (DNS) was added to the mixture, and let them react for 10 min in water-bath. According to the different colours of the mixture from dark brownish red to light brownish red, the isolates of Trichoderma spp.which can produce chitinase could be screened. The result showed that compared with the traditional plating diaphanmetry method, thenew method was desirable for screening Trichoderma spp. producing chitinase.The result showed that all the isolated Trichoderma spp. could use chitin or colloidal chitin as the sole carbon source for growth. All strains of Trichoderma spp. could produce chitinase. Both chitin and colloidal chitin had inductive effect for Trichoderma spp. to produce chitinase. But the inductive effects of them for Trichoderma spp. to produce chitinase were different. When chitin was used as the sole carbon source, 15 strains of Trichoderma spp. (TYM-1, TYM-2, TYM-8, TYM-10, TJQ-1, TJQ-2, THS-1, TCS-1, TWD-1, TYB-GL-1, TYZH-1, TCZ-DL-1, TCZ-DL-2, TXM-DL-5, TXM-DL-6) were higher chitinase producer, 11 strains of Trichoderma spp.(TWD-2, TSQA-1, TSQC-3, TSQC-5, TBC-LJ-1, TSYL-LJ-1, TSYL-LJ-2, TSYL-LJ-4, TSYL-LJ-7, TSS-ZY-3, TBC-ZY-2) were lower chitinase producer. When colloidal chitin was used as the sole carbon source, 7 strains of Trichoderma spp. (TYM-4, TJQ-1, TYT-1, TYT-2, TYZH-1, TCZ-DL-3, TXM-DL-2) were higher chitinase producer, 10 strains of Trichoderma spp. (TWD-4, TSQC-3, TXM-LJ-1, TXM-LJ-3, TYC-LJ-4, TSYL-LJ-1, TSYL-LJ-2, TSS-ZY-1, TSS-ZY-2, TBC-ZY-1) were lower chitinase producer.With method of measuring reducing sugar, Quantitative inoculating spore and inoculating colony had the same result of measuring Trichoderma spp.'s character of producing chitinase. With method of measuring reducing sugar, quantitative inoculating spore fermentation test showed that TYB-GL-1 strain isolated by trapping method was the highest chitinase producer.4 Trichoderma spp. strains (TYB-GL-1, YWD-1, TXM-DL-6, TYM-2)with higher chitinase activity and 2 Trichoderma spp. strains (TBC-LJ-1, TSYL-LJ-7) with lower chitinase activity isolated with method of measuring reducing sugar, all showed strong inhibitory effect on the growth of 6 phytopathogen Colletotrichum nicotianae Averna (1923), Alternaria alternata (freis) kissler, Thielaviopsis basicola (Berk.and Br.) Ferraris, Phytophthora nicotianae var. nicotianae, Pythium aphanidermatum. The chitinase activity didn't appeare to be directly related to the antifungal ability.