Biocontrol strain hw17 for controlling cucumber green mottle mosaic virus and its application
A technology for green mottled flowers and biocontrol strains, applied in the field of agricultural microorganisms, can solve the problems of difficult prevention and control, complex genetic mechanism and background, and difficulty in cultivating varieties with stable resistance, etc., and achieves the effect of reducing residues and saving expenses.
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Embodiment 1
[0020] Screening method: Bacillus subtilis HW17 (CGMCC NO.11025) was isolated from healthy watermelon rhizosphere in Dingji Town, Huaian City, Jiangsu Province. Cut the root tissue of watermelon into small pieces of 1 cm, soak it in 1% sodium hypochlorite for 5 minutes, soak it in 70% alcohol for 1-2 minutes, and wash it with sterile water for 3 times (take 100 μl of the last cleaning solution and apply it on R 2 A solid culture medium, if sterile growth after 48h, it is considered that the surface is clean and sterile). Take 3g of the sterilized sample and place it in a sterile mortar, add 27ml of sterile 0.85% NaCl, macerate the tissue, grind it, and dilute with sterile 0.85% NaCl gradient. take 10 -1 、10 -2 、10 -3 100 μl each of the three gradient dilutions were applied to R 2 On A, culture at 28°C for 48h. Pick the largest single colony to inoculate into fresh R 2 A solid medium, through repeated transfer to obtain purified strains. The purified strains were stored ...
Embodiment 2
[0046] The preparation of embodiment 2 HW17 biocontrol agent
[0047] Inoculate the Bacillus subtilis HW17 (CGMCC NO.11025) strain into the LB culture medium for 16 hours at 28°C with shaking at 200rpm, then take a sample every 2 hours to measure its OD value at 600nm at 600nm, when the OD value is 0.8 When the culture is over, this bacterial solution is used as the seed bacterial solution. Inoculate the seed bacteria solution into LB fermentation medium at a volume ratio of 1:500 for fermentation, culture at 28°C and 200 rpm for 48 hours, then centrifuge at 6000 rpm for 10 minutes, and dilute the precipitate to obtain the biocontrol agent HW17. The total concentration of viable bacteria in the resulting biocontrol agent was 1×10 9 ~1×10 10 CFU / mL.
Embodiment 3
[0048] Example 3 Greenhouse Test Determination of the Control Effect of HW17 on Cucumber Green Mottled Mosaic Virus
[0049] The cucumbers are transplanted at the 2-3 leaf stage, and the roots are irrigated and sprayed at the same time. Example 2 The preparation of HW17 (CGMCCNO.11025) bacterial agent irrigation root concentration is 1 × 10 8 CFU / mL, the dosage for each seedling is 30ml. The spray concentration is 1×10 7 CFU / mL, the spray standard is that there is a mist-like droplet distribution on each leaf, subject to not falling, spray evenly. After 7 days of biocontrol agent treatment, inoculate cucumber green mottle mosaic virus (the pathogenic bacteria used is the virus CGMMV, donated by Zhejiang Academy of Agricultural Sciences, it is an infectious cloned Agrobacterium, the strain name is called EHA105 in the experiment, and the transformed plasmid is pCB-CGMMV Prepare EHA105 in LB culture solution containing rifampicin and kanamycin (the final concentration is 50mg...
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