A strain of Bacillus subtilis with antagonistic action to degrade cellulose at low temperature and its application
A technology of Bacillus subtilis and cellulose degradation, which is applied in the fields of application, chemicals and bacteria for biological control, and can solve the problems of broad-spectrum antagonism and different antagonism capabilities
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Embodiment 1
[0019] The screening and identification process of Bacillus subtilis involved in the present invention are as follows:
[0020] 1. Screening of strains
[0021] Take the straw from the stacked straw pile, rinse the surface with sterile water, spread the rinsed liquid on the beef extract peptone medium, and cultivate it at 15°C.
[0022] 2. Preliminary screening of cellulose degrading strains
[0023] (1) Congo red staining: Inoculate the strains on the beef extract peptone medium onto the sodium carboxymethylcellulose plate by spot inoculation method, inoculate 3 spots on each plate, culture at 15°C for 3 days, and then inoculate with 1 mg / mL Congo red staining solution was used for staining for 30 minutes, the dyeing solution was discarded, and 1mol / L NaCl solution was added to decolorize for 20 minutes. Observe whether there is a hydrolysis circle near the bacterial colony, if there is, it means that the strain has the ability to degrade cellulose, and record the size of t...
Embodiment 2
[0035] In this embodiment, cellulase activity was measured on GN19, and the process was as follows:
[0036] Inoculate the primary screened strains into the seed culture medium, and cultivate them at 15°C and 150r / min for 12h to obtain the seed liquid. The seed solution was inoculated into 100mL liquid fermentation medium at 2%, and cultivated under the same conditions for 72h. The obtained fermentation liquid was centrifuged at 4°C and 10,000 r / min at high speed for 15 min, and the supernatant was collected to be the crude enzyme liquid.
[0037] (1) Endoglucanase activity: get 4 test tubes, 1 as a control and 3 as a test tube, add 1.5 mL of 1% CMC-Na solution respectively, add 0.5 mL of crude enzyme solution in the test tube, wherein Add 2mL DNS to the control to inactivate the enzyme activity, and react at 50°C for 30min. After the reaction, add 2mL DNS to the 3 test tubes to terminate the reaction, shake well and then boil in water bath for 10min, distill the volume to 2...
Embodiment 3
[0045] In this embodiment, antagonism test is carried out on GN19 bacterial strain, and the process is as follows:
[0046] Inoculate the plant pathogenic bacteria cake in the center of the PDA solid medium, use a toothpick to pick up Bacillus subtilis GN19 at a distance of 2cm from the pathogenic bacteria cake, directly inoculate the pathogenic bacteria cake as a control, and culture at a constant temperature of 28°C for 3 to 7 days, measure the radius of the colony, and calculate the antibacterial effect rate, the results are shown in Table 3, with another Bacillus subtilis DN5 as a comparison.
[0047] Bacterial inhibition rate = [(control colony radius - treatment colony radius) / control colony radius] × 100%
[0048] Table 3 Inhibitory rate of Bacillus subtilis GN19
[0049]
[0050]
[0051] It can be seen from the above table that the GN19 strain has an antagonistic effect on a variety of pathogenic bacteria, especially the bacteriostatic rate of Sclerotinia scler...
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