A kind of Trichoderma viridans for preventing and treating pepper phytophthora, application and pepper cultivation method
A technology of pepper phytophthora and cultivation methods, applied in the field of biological control of plant diseases, can solve problems such as difficult to effectively control, ecological and environmental problems, achieve high-efficiency inhibition, reduce the incidence and degree of incidence, and have good biological control applications foreground effect
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Embodiment 1
[0026] Isolation, screening and identification of strains.
[0027] 1. Isolation of Trichoderma
[0028] 40 soil samples were collected from 8 field plots in Hangzhou City, Zhejiang Province, which were seriously infested by the pathogenic fungus of Fusarium wilt of pepper. After retrieval, the samples were stored in a refrigerator at 4°C. Take 1g of soil sample and dissolve it in 9ml of sterile water to make a series of soil suspensions. Take 1ml of soil suspension and evenly spread it on Trichoderma screening medium (TSM: 0.2g MgSO 4 7H 2 O; 0.9 g K 2 HPO 4 ; 3.0g glucose; 0.25g chloramphenicol; 0.3g sodium p-dimethylaminobenzenediazepine sulfonate; 0.2g pentachloronitrobenzene; 0.15g Bengal red; 20g agar; add water to 1L), at 27 After culturing at ±1°C for 4 days, the colonies grown on the medium were transferred to potato dextrose medium (PDA) for isolation of single spores. For short-term storage of strains, put them in a 4°C refrigerator. For long-term storage, p...
Embodiment 2
[0037] Detection of antibacterial activity of metabolites.
[0038] The 15 strains that were screened and purified were taken with a hole punch to take a 5mm diameter bacterial cake into a conical flask containing 100ml of PDB medium, and cultured in a ZWY-211B shaker at 27±1°C and 150rpm for 4 days. First filter and centrifuge the mycelium with sterilized gauze to obtain the supernatant containing metabolites. Dilute the resulting supernatant to 50% and 20% concentrations, filter with a bacterial filter with a pore size of 0.22 μm, pour 1 ml of 50% and 25% metabolite solutions together with 10 ml of melted PDA medium into a 9 cm petri dish, and then Insert the Phytophthora capsici cake with a diameter of 5mm and cultivate it at 25°C. The metabolite-free treatment was used as a control, and measurements were taken when control-treated Phytophthora hyphae grew close to the edge of the dish.
[0039] The inhibitory effect test of 15 strains of Trichoderma metabolites on the gr...
Embodiment 3
[0044] Purification and identification of active metabolites.
[0045] 1. Purification and activity testing of active metabolites
[0046] Based on the results of the inhibitory activity, possible active compounds produced by Trichoderma viridans HZA14 were isolated, purified and identified. The screened Trichoderma viridans HZA14 was inoculated into conical flasks containing PDB, and cultured in a shaker for 14 days, using the above method. 2 L of culture fluid was obtained and metabolites were extracted using ethyl acetate. Metabolite residues were obtained by evaporation under reduced pressure. The residue was purified by silica gel column chromatography (particle size: 200-300 mesh), and purified by preparative silica gel TLC (GF254) to obtain four fractions A, B, C and D. A small amount of components A, B, C, and D were dissolved in DMSO (dimethyl sulfoxide) for biological activity determination. 1 ml of each fraction (100 μg / ml) solution was added to a 10 ml Petri di...
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