Bacillus subtillis HMB28948 and application thereof

[0050] Example 1 Preparation of HMB28948 microbial agent

[0051] Follow the steps below:

[0052] (1) Strain activation: Bacillus subtilis strain HMB28948 stored at -80°C (this strain has been deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on October 28, 2016, and the deposit number is CGMCCNo.13212) Activated (30℃) on LB plate medium (its composition and weight ratio are: tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL), pick a single colony on LB Inclined medium (its composition and weight ratio are: tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL) cultured at 30°C for 12 hours to obtain activated strains;

[0053] (2) Preparation of seed solution: Prepare LB liquid medium according to conventional methods (its composition and weight ratio are: tryptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL), and fill it in a 250mL Erlenmeyer flask Add 100 mL of LB broth, sterilize with high pressure, humid heat, and after the temperature drops to room temperature, connect each bottle to an inoculation loop of the above-mentioned activated strains in step (1), and shake at 30°C and shaker rotation speed 180 rpm. Cultivate for 12 hours to obtain seed liquid;

[0054] (3) Preparation of corn flour and soy flour medium: According to weight percentage, corn flour 1.5%, soy flour 2.0%, NaCl 0.5%, MnSO 4 ·H 2 Add 0.6% of O to water, stir and mix evenly to obtain corn flour and soy flour medium; divide it into 500 mL Erlenmeyer flasks, 200 mL each; sterilize corn flour and soy flour medium at 121°C for 30 minutes, and then cool to 30℃ spare;

[0055] (4) Fermentation culture: inoculate 2 mL of the seed liquid obtained in step (2) into each bottle of corn flour and soybean meal medium obtained in step (3) 200 mL; carry out fermentation and culture for 36 hours at 30°C and shaker rotation speed of 180 rpm. Take samples from the Erlenmeyer flask for microscopic examination every 30 minutes, count the number of spores and total bacteria in the field of view, and calculate the spore rate (spore rate (%) = number of mature spores/(number of mature spores + number of bacteria)× 100); When the spore rate reaches 90%, stop the fermentation and culture; the total fermentation culture is 48 hours to obtain the liquid preparation of Bacillus subtilis HMB28948.

[0056] Example 2 The antagonistic effect test of the HMB28948 strain of the present invention on Rhizoctonia solani

[0057] (1) The source of the tested cotton Rhizoctonia solani: The cotton Rhizoctonia solani RH-2 strain was collected from the cotton wilt disease strain in Qiuxian County, Handan City, Hebei Province, and was isolated and purified by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences. Plants of Hebei Agricultural University The Department of Plant Pathology of Conservation College identified Rhizoctonia solani as strong pathogenicity.

[0058] (2) Test method

[0059] Plate determination test: First, R. solanacearum RH-2 was activated and cultured on a PDA plate for 3 days, and then a hole punch with a diameter of 6 mm was used to make a plate at the edge of the colony, and then the R. solanacearum plate was made. Transfer to the center of another PDA plate, and then spot the activated Bacillus subtilis HMB28948 in step (1) of Example 1 at a distance of 2.0 cm from the indicator bacteria sheet, and set a blank control (not spot the cotton Rhizoctonia solani of HMB28948 strain) growing situation). Incubate at a constant temperature of 25°C, and when the blank control is about to cover the entire petri dish, measure the control growth (colony radius) and treated growth (inhibition growth radius after inoculation with HMB28948) of Rhizoctonia solani, and use the inhibition rate for antagonism Said.

[0060] Bacteriostatic rate (%)=(control growth amount-treatment growth amount)/control growth amount×100.

[0061] Results (see Table 1) The inhibitory rate of Bacillus subtilis HMB28948 on cotton Rhizoctonia solani is 74.4%; it shows that Bacillus subtilis HMB28948 has obvious inhibitory effect on cotton Rhizoctonia solani and has the biological control of preventing cotton wilt. potential.

[0062] Table 1 Test results of the antagonistic effect of the HMB28948 strain of the present invention on Rhizoctonia solani

[0063] Strain name Control growth (mm) Processing growth (mm) Antibacterial rate (%) HMB28948 39.0 10.0 74.4

[0064] Example 3 Comparative test of the control effect of the HMB28948 strain of the present invention on cotton blight

[0065] (1) Experimental treatment:

[0066] (1) HMB28948 microbial agent: The HMB28948 liquid preparation prepared in Example 1 was diluted 50 times with water.

[0067] (2) Blank control: clear water

[0068] (2) Test method:

[0069] First, mix the vermiculite and cotton field soil in a ratio of 1:1, sterilize with high pressure at 121°C for 1 hour, and sterilize again with the same conditions the next day. Inoculate a suspension of Rhizoctonia solani hyphae (approximately 10 7 Pieces/ml) 10ml, mix well and fill it in a plastic flowerpot with a diameter of 20 cm, and compact it. The cotton variety was Jifeng 106, and the HMB28948 liquid formulation prepared in Example 1 was used for soaking seeds for half an hour before soaking in 50-fold water dilution; the seeds were soaked in clear water for half an hour as a blank control. After soaking, 10 seeds were sown in each pot, covered with sterilized soil, and cultured normally in a greenhouse. After emergence, investigate and record the number of plants with wilt disease in the treatment and the blank control on a daily basis. When the disease in the blank control is sufficient, calculate the incidence and control effect.

[0070] Pot experiment results (see Table 2) Bacillus subtilis HMB28948 has a control effect of 94.82% against cotton wilt. It shows that the HMB28948 strain and its microbial agent of the present invention have a good control effect on cotton wilt.

[0071] Table 2 Test results of the prevention effect of the HMB28948 strain of the present invention on cotton wilt

[0072] deal with Number of plants investigated Number of diseased plants Incidence rate (%) Prevention effect (%) HMB28948 liquid formulation 53 1 1.9b 94.82 Blank control 49 18 36.7a --