Method for separating active components of cotton endophytic fungi CEF-082 for inhibiting cotton verticillium wilt bacteria
A technology of CEF-082 and cotton Verticillium dahliae, applied in the field of agricultural biology, can solve the problems of unidentified active components, etc., and achieve the effects of crop safety, enhanced defense response, and growth promotion
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Embodiment 1
[0029] The inhibitory action of embodiment 1 crude extract to Verticillium dahliae Vd080 spore
[0030] Inoculate the CEF-082 bacterial mass grown on PDA into 1000mL Erlenmeyer flasks containing 400mL PDB, a total of 10 flasks, at 25°C, 150rpm, culture for 15d, filter mycelium with a single layer of gauze, and extract with ethyl acetate equal volume for 2 The second time, 37 ° C rotary evaporation to dryness, to obtain the crude extract of the fermentation broth, and then dissolved in methanol, 0.22 μm microporous membrane filter to sterilize.
[0031] Inoculate Verticillium dahliae Vd080 bacterial block into Czapek medium, culture at 25° C. and 150 rpm for 7 days, filter out hyphae with sterile gauze, and obtain spore suspension. Spread 200 μL of Vd080 spore suspension on a PDA plate, and then place two sterile Oxford cups symmetrically, add 200 μL of CEF-082 crude extract to the Oxford cups of the treatment group, and add 200 μL of sterile methanol to the control group. Inc...
Embodiment 2
[0033] The effect of embodiment 2 crude extract on Verticillium dahliae spore cell wall
[0034] The cultured Verticillium dahliae Vd080 spore suspension was centrifuged to collect Vd080 spores with a volume of rice grain size, and 200 μL of CEF-082 crude extract (5 mg / mL) was added, and the control was sterile water. Incubate at 28°C for 24 hours, and then wash 4 times with phosphate buffered saline (PBS, 0.1M). The spores were fixed with 3% glutaraldehyde at 4°C for 2h, washed with PBS 4 times, 10min each time, then soaked in 1% starvation acid solution, placed at 4°C for 1h, washed 4 times with PBS, 10min each time, and then washed with 50%, 70%, 90% ethanol gradient dehydration for 15 minutes, acetone series dehydration, 100% acetone dehydration for 3 times, 10 minutes each time, and finally embedding with Epon812 epoxy resin as embedding agent, after embedding, carry out polymerization at constant temperature, and then Ultrathin sections were made and stained with uranyl...
Embodiment 3
[0036] The antibacterial concentration of embodiment 3CEF-082 metabolism crude extract
[0037] The CEF-082 crude extract was diluted to 5, 4, 3, and 2 mg / mL, and sterilized by filtration with a 0.22 μm Millipore membrane filter. Drill 5 wells evenly in the PDA plate, first add 20 μL of CEF-082 crude extract, the control group is sterile methanol, after air-drying, add 20 μLVd080 spore suspension to the wells respectively, after air-drying, culture at 28 °C, repeat 3 times, after 4 days, the colony expansion diameter was measured and photographed.
[0038] In order to study the antibacterial concentration of the crude extract of CEF-082, 5 wells were evenly punched in the PDA plate, and 20 μL of the crude extract of CEF-082 was added to the wells, and then 20 μL of Vd080 spore suspension was added after air-drying, and the colony expansion diameter was measured after 4 days . In the control group, the expansion diameter of Verticillium dahliae Vd080 is 0.8cm, and the expansi...
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