Bacillus subtilis strain and application thereof
A Bacillus subtilis, strain technology, applied in the application, bacteria, fungicides and other directions, can solve problems such as narrow control spectrum, and achieve the effect of strong inhibitory activity
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Embodiment 1
[0019] Isolation and purification of strains
[0020] From the soil of the lily field in Fanjiaping, Lanzhou City, Gansu Province, select an appropriate location, take a soil sample at a distance of 5-20 mm, and isolate bacteria from it by the dilution plate method:
[0021] (1) Preparation of soil dilution: weigh 10 g of soil sample, put it into 100 ml of sterile water, and oscillate on a shaker for 30 min. Set aside to clarify. Aseptically draw 100 μl of the supernatant and add it to 900 μl sterile water, dilute to 10 with sterile water -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 A total of eight concentration gradients.
[0022] (2) Cultivation: add sterilized NA (beef extract 3g, peptone 5g, glucose 10g, yeast extract 1g, agar 15g, distilled water 1000ml, pH 7.0) agar medium cooled to 55~60℃ for each dilution concentration, Made into a flat plate. Incubate at 30°C in an inverted incubator.
[0023] (3) Picking a single colony: After 3 to 4 days, after t...
Embodiment 2
[0028] Identification characteristics of strains
[0029] 1. Bacterial morphology observation and staining reaction
[0030] M22BH3 was inoculated in NA liquid medium and cultured with shaking at constant temperature for a suitable period of time, and then stained for microscopic examination. Including Gram staining (crystal shoxenamide staining method), flagella staining (silver nitrate staining method), capsule staining (wet ink method), spore staining (modified Schaeffer-Fulton's staining method).
[0031] Staining results: M22BH3 is rod-shaped bacteria, G + Sex; flagella grow laterally, with 8 to 20 flagella growing on each cell, the length of which is about 2 to 3 times that of the cell; capsulated: spore-forming, mesozoic, and spore cysts swell.
[0032] 2. Effects of environmental factors on strain M22BH3
[0033] 2.1 O 2 Effect on strain M22BH3
[0034] Determination of O by the deep agar method 2 For the effect on the growth of M22BH3, insert M22BH3 into a test ...
Embodiment 3
[0080] Preparation of cell-free culture medium of M22BH3 strain
[0081] ① Inoculate M22BH3 in 25ml small amount of NA liquid medium (pH7.0) at a ratio of 1:100, 120rpm / min, 30°C, shake culture for 24h;
[0082] ② Then inoculate the above-mentioned culture solution into 1000ml large amount of NA liquid medium (pH7.0) at a ratio of 1:20, culture at 120rpm / min, 30°C, and shake for 72h;
[0083] ③ Centrifuge the fermented liquid at 8000-12000r / min to get the supernatant, and the collected supernatant is filtered through a bacterial filter (0.22μm) to obtain a concentrated solution, which is spread on a flat plate, and no viable bacteria are detected. It is the M22BH3 strain preparation.
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