Methylotrophic Bacillus ar3, Bacillus subtilis ar4, Bacillus amyloliquefaciens ar10 and their applications
A technology of Bacillus subtilis and methylotrophic type is applied in the application field of three biocontrol Bacillus strains and bacterial wilt fungicide, which can solve the problems of large pollution, low efficiency of chemical fungicides, weak pertinence, etc., and achieve the effect. Good, rich results, highly targeted effects
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Embodiment 1
[0025] Embodiment 1 prepares three kinds of bacillus AR3, AR4, AR10
[0026] 1. Screening of Bacteria
[0027] Soil samples were taken from healthy tobacco fields in Xuan'en County and brought back to the laboratory for analysis. Weigh 5 grams of soil sample and place it in a sterilized 150mL Erlenmeyer flask containing 15-20 small glass beads, add 45mL of sterile water, shake the Erlenmeyer flask for 20min, and let stand for 30min to obtain the supernatant which is 10 -1 Diluted gradient soil drench. Then use a 1mL sterile pipette to draw 10 -1 Transfer 1mL of the diluent into a test tube filled with 9mL of sterile water, blow and inhale 3 times, let the bacteria solution mix evenly, and serve as 10 -2 Diluent, take 100μl concentration as 10 -2 ~10 -4 The soil immersion solution was evenly spread on the plates with a coating stick, and after standing for 20 minutes, the culture plates were turned over and incubated at 37°C for 24 hours. Select the plate that is most sui...
Embodiment 2 3
[0032] Embodiment 2 Three strains of biological control bacteria activity verification
[0033] Pick a single colony from the three preserved strains into 5mL LB medium, culture at 37°C, 180rpm / min shaker for 72h, take 1mL of fermentation broth in a 1.5mL centrifuge tube, centrifuge at 12000r / min for 5min, and collect the supernatant solution, and the supernatant was aspirated with a syringe and sterilized by filtration with a filter. Prepare a PDA plate, take 100 μl of R. solanacearum cultured for 24 hours and evenly spread it on the PDA plate with a spreader, and then punch holes (5 mm in diameter) on the plate with a sterilized hole puncher. Inject 50 μl of the fermented liquid after filter sterilization into the round hole, and use an equal amount of sterile water as a control, and finally cover the round hole with a sterilized filter paper. Place the plate in a 37°C incubator and incubate for 48 hours, observe and record the antibacterial results, such as figure 2 (1: ...
Embodiment 3
[0034] The identification of embodiment 3 biocontrol bacteria
[0035] Pick a single colony from the three preserved strains into 5mL LB medium, culture at 37°C, 180rpm / min on a shaker for 5-6h, take out 1mL of the cultured bacterial solution into a 1.5mL centrifuge tube, and centrifuge at 12000r / min for 5min. Discard the supernatant to collect the bacteria, add 400 μl of deionized water to the centrifuge tube, cook in boiling water for 10 minutes, centrifuge at 12000 r / min for 5 minutes, and take the supernatant as a template.
[0036] The total volume of the PCR system is 50 μl: template DNA 2 μl, forward and reverse primers 2 μl each, 2×Easy Taq PCR super Mix 25 μl, ddH 2 O 19 μl.
[0037] Use the prepared supernatant as a template for PCR amplification. The reaction was carried out on a Thermo-Hybaid PCR instrument, and the program was 30 cycles of denaturation at 94°C for 3 min, denaturation at 94°C for 45 s, renaturation at 59°C for 45 s, extension at 72°C for 45 s, an...
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