Paenibacillus with bacteriostatic activity, and extraction method and application thereof
A technology for bacillus and bacteriostatic activity, which is applied in the field of Paenibacillus and its extraction, and can solve problems such as failure to large-scale application and no registration of biocontrol agents for root cancer.
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Embodiment 1
[0028] Embodiment 1: the separation and screening of soil antagonistic bacteria
[0029] Select 10g of soil near the root system of a plant with a low incidence of cherry root cancer, pour 10ml of sterile distilled water into it, vortex and mix well, let stand for 10 minutes, wait for the soil to settle and take the supernatant. After the soil is left standing, record the supernatant as the stock solution, carry out gradient dilution with sterile water in the ultra-clean bench, and mark it as 10 1 -10 3 . Then pipette 50 μl of the diluted solution and apply it to blank YEB solid medium for overnight culture at 28°C. On the second day, pick the uncontaminated single clone and culture it in a small amount in YEB liquid medium, and culture it overnight at 28°C and 200rpm on a shaker. On the third day, each bacterial solution was concentrated, and the inhibition zone test plate was prepared (the first layer was a blank YEB medium, the second layer was a YEB solid medium added w...
Embodiment 2
[0030] Embodiment 2: Identification of antagonistic bacteria
[0031]The identification of antagonistic bacteria was analyzed from three aspects: morphology, physiology and biochemistry, and molecular biology. Morphology: Streak the activated bacteria on a YEB solid plate, culture at 28°C for about 16 hours, select freshly grown single clones, treat them with negative staining, observe under a transmission electron microscope, and select Whole cells were photographed. For the identification of physiological and biochemical indicators, refer to the "Common Bacteria System Identification Manual", and carry out related inventions for the identification of Bacillus races. Determination inventions include: catalase, oxidase, anaerobic growth, nitrate reduction, utilization of citrate, fermentation of mannitol, maltose, xylose, and hydrolysis of DNA. The reagents used were purchased from Guangdong Huankai Microbial Reagent Co., Ltd. Molecular biology identification The bacterial u...
Embodiment 3
[0032] Embodiment 3: Antagonism analysis of antagonistic bacteria
[0033] Antagonism analysis of antagonistic bacteria includes two parts in vitro and in vivo. The in vitro analysis is colony counting after co-cultivation. The specific method is as follows: mix the separately activated and resuspended Agrobacterium and antagonistic bacteria in equal volumes, and place them at 28°C for 1-2 days with shaking at 60 rpm. At 0, 6, 12, 24 and 48 hours, the mixed bacterial solution was drawn and serially diluted. Choose a dilution of 10 4 、10 5 Multiple dilutions were used to count the number of bacteria. A total of three experiments were carried out, with 3 repetitions each time. The experimental results were statistically analyzed by t test. The in vivo analysis method is as follows: the pathogenic Agrobacterium tumefaciens ACCC19197 and the antagonistic bacteria purchased from the China Agricultural Microorganism Culture Collection were selected and cultured separately until t...
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