Screening of Trichoderma strains tolerant to benzimidazole

The screening of isolates and the assay of biocontrol mechanisms of Trichoderma were studied systematically in laboratory and greenhouse in vivo. The proteins tolerant to benzimidazole in Trichoderma strains were purified, and their physical and chemical properties were detected. Compared their biological activities in vitro and vivo in greenhouse, nine biocontrol strains (including Ty-10-2,LTR-2,Tj-5-1,Tj-5-4,Ty-11-1,Tj-11-3,Ty-11-3,Tj-3-3-2,Tj-3-3-4) were screened. These biocontrol strains had faster rates of growth and higher inhibition to gray mould (Bortrytis cinerea), and the inhibition was stable. The effects of controlling gray mould in greenhouse with the screened Trichoderma strains were 70% and 50% in vivo. Induced by the substrates including benzimidazole, UV rays and nitrosoguanidine, four strains were screened. They were Ty-10-2,LTR-2,Tj-5-4,Ty-11-3. After being cultured for 24 h, conidiospores of tolerant strains germinated but the sensitive ones didn’t. Compared with the hyphae of the tolerant strains, the sensitive hyphae, front became thinner and shorter. Experiments of biocontrol mechanisms showed as followed: some genes had mutated, transcripted at different levels and translated into some tolerant proteins, while some genes had opposite changes. In the whole, the soluble proteins in cells had increased in quantity. The esterase isoenzymes in the tolerant strains decreased at different levels. After being induced, some genes mutated and some genes, transcription decreased and even diminished. None were screened from UV rays and nitrosoguanidine.

Effect of Trichoderma viride on activities of polygalacturonase of Rhizoctonia

The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and causes the maceration of tissue and the death of protoplast. Polygalacturonase (PG) can decompose the galacturonic acid of disease tissue. The research defined the PG activities of extracellular metabolite of the different virulence Rhizoctonia isolates, and testifid the effect of Trichoderma viride to PG activities, and clarified the mechanisms of biocontrol by Trichoderma. The test methods as following: Firstly, to select the isolates of different virulence: WK-47, WK-141 and WK-160 strain of Rhizoctonia AG-D and YW-2 strain of Rhizoctonia AG1-IA and TCS-1 strain of Trichoderma viride. Secondly, to culture TCS-1 on PD, and draw a group of fermented liquid in every 24 hours, and draw 7 times. Thirdly, to culture quietly Rhizoctonia isolates with Czapek-Dox at 25℃ for 15 days, filter and centrifuge (2350 g×30 min), fetch the clear liquid, put it into the ammonium sulfate according to 60% saturation degree, put it quietly for 30 min at 4℃, centrifuge (21000 g×30 min) at 4℃, remove the clear liquid, dissolve the deposit with sodium acetate buffer (25 mmol/L, pH5.5), dialysis for 48 h in the same buffer,and change the buffer every 12 h, Fourthly, to put TCS-1 fermented broth of different times in the tubes, one mL per a tube, add 0.5 mL PG to every tube, react for 4 h in 30 ℃ water, the same time fetch the test tube filled with the same treated liquid that was not dealed in 30℃ water.Finally,to testify PG activities with DNS’s test. In all, PG of Rhizoctonia had high activities and virulence. The conrtrol efficacy of T.viride to PG activities of WK-47, WK-141, WK-160 and YW-2 were 95%,94%,95%,92% separately, fermented time had a great influence to control efficacy, the third fermented broth did the best. Through effect to PG activities T. viride can reduce the virulence of Rhizoctonia, and protect the hosts. The specific mechanism, qualitative and quantitative research of antagonistic substance in the fermented broth will be further carried out.

Study on the character of chitinase produced by Trichoderma spp. with measuring reducing sugar

A trusty and intuitionistic method for screening chitinase produced by Trichoderma spp. was developed. 38 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 min, 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 colour of the mixture, the isolates of Trichoderma spp. which can produce chitinase could be screened.

Studies on the control of Fusarium oxysporum f.sp. cubense with Trichoderma

One hundred and fifty one isolates of Trichoderma were collected mainly from more than 40 soil samples and other materials in Guangdong Province (including Chigang, Zhanjiang, Wuchuan, Panyu, Zhaoqing, Dongguan, Humen, Qingyuan, Guanzhou) and the soil samples were also from different plant rhizosphere (including rice, different fruits and different vegetables). It was shown that 39 isolates of Trichoderma grew faster than other isolates using growth velocity experiments. The 39 isolates could effectively inhibit Fusarium oxysporum f.sp. cubense (E.F.Sm) Sny.&Hans. by dual cultural experiments. The inhibited activity included the antifungal activities of its metabolite, mycoparasitic activities and the lytic enzymes by dual culture, electronic microcopy and enzyme assay. At present, studies on the taxonomy of the 151 isolates of Trichoderma are carried out in our experiments, some Trichoderma species aggregates will be identified according to the taxonomy system revised by Rifai and Bissett.