Bacillus pumilus NMCC 46 and application thereof
A technology of Bacillus pumilus, nmcc46cgmccno.3378, applied in application, bacteria, fungicides, etc., can solve problems such as environmental pollution and soil fertility decline
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Embodiment 1
[0019] Using the five-point sampling method, use a small shovel to remove the topsoil, take dozens of grams of soil samples at a distance of 5-25 mm, put them into a sampling bag, and mark the sampling time, location, and vegetation. Weigh 5g of soil sample and 45ml of normal saline, shake it on a shaker for 30 minutes, and let it stand for clarification. Dilute the supernatant with sterile water for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Five concentration gradients, 80°C water bath for 10min. Aspirate 200μl coated LB plates and culture overnight at 37°C. After colonies grew on the plate, single colonies were picked, streaked and purified, and numbered and stored, and screened to obtain Bacillus pumilus NMCC46C GMCC No.3378.
Embodiment 2
[0021] The Bacillus pumilus (Bacillus pumilus) NMCC46CGMCC No.3378 of embodiment 1 is carried out morphological characteristic observation and physiological and biochemical characteristic identification, the result shows, this bacterium is Gram-positive, rod-shaped, produces spore (as figure 1 shown). Not only can grow at 10°C, but also can grow at 50°C, can tolerate 7% NaCl culture, but can not tolerate pH 5.7 culture, contact enzyme test, citrate utilization test, V-P test, glucose fermentation and casein The tests were all positive, and the starch hydrolysis test, nitrate reduction test, anaerobic growth test and propionate utilization test were all negative.
Embodiment 3
[0023] The Bacillus pumilus (Bacillus pumilus) NMCC46CGMCC No.3378 strain of Example 1 was inoculated in liquid LB medium, 180 rpm, 37 ° C shaking culture for 10 hours, centrifuged to collect the bacteria, after suspension, lysozyme and SDS were added to break the wall, and the phenol - Chloroform method to extract genomic DNA. Using the extracted total DNA as a template, the 16S rDNA gene of the strain was amplified by PCR with forward primer PRB1601 (5'-GGATCCTAATACATGCAAGTCGAGCGG-3') and reverse primer PRB1602 (5'-GGATCCACGTATTACCGCGGCTGCTGGC-3'). The PCR conditions are: 95°C, 4min; 94°C, 1min; 50°C, 1min; 72°C, 2min; 34cycle; 72°C, 10min, stored at 4°C. After the PCR product was recovered, it was sequenced, and its length was confirmed to be 482bp. The sequencing result was shown in the sequence table, and the similarity with Bacillus pumilusEF157741.1 in GenBank was 94%.
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