Bacillus Sweissii MQ20YS04 and application thereof

Abstract

The invention discloses a bacillus stutzeri MQ20YS04 and an application of the bacillus stutzeri MQ20YS04. The preservation number of the bacillus swezeyi MQ20YS04 disclosed by the invention is CCTCC (China Center for Type Culture Collection) NO: M 2024616, and the preservation number of the bacillus swezeyi MQ20YS04 disclosed by the invention is CCTCC NO: M 2024616. The strain has strong secondary metabolite synthesis capability, and can produce siderophore, IAA and the like. The strain MQ20YS04 also has the potential of plant growth promotion and disease control, and the strain MQ20YS04 has a relatively strong bacteriostatic effect on various pathogenic fungi such as botrytis cinerea, fusarium oxysporum, rhizoctonia solani and sclerotinia sclerotiorum, and has a very good control effect on pepper phytophthora blight and codonopsis pilosula root rot. In addition, the strain MQ20YS04 also has nitrogen fixation and phosphorus solubilization capabilities, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Bacillus sweziei MQ20YS04 and applications thereof. Background Art

[0002] Modern agricultural practices rely mainly on industrially produced agrochemicals (e.g., fertilizers and pesticides) to increase food production. Crop diseases caused by pathogenic bacteria affect modern agricultural production, causing approximately 20%-30% yield losses. In the past few decades, chemical pesticides have been widely used to prevent and control crop diseases. However, excessive use of chemical pesticides to protect crops has caused serious environmental problems and reduced the sustainability of agriculture. Therefore, the ideal way to achieve sustainable agricultural production will urgently need to explore other strategies that can maximize crop performance. The use of beneficial microorganisms for biological control is an important alternative to reduce the damage caused by crop diseases to sustainable agriculture. After years of development, microbial live bacterial preparations that can produce active substances or biopesticides containing active ingredients have become a hot topic and focus of research in recent years. Biopesticides have the characteristics of a wide range of sources, safety to non-target organisms, and good environmental compatibility, and have become a new trend in the development of the world's pesticide industry. Biocontrol microorganisms can not only effectively control plant diseases and promote plant growth, but are also safe for humans and animals, have good environmental compatibility, and are not likely to cause plant pathogens to develop resistance, thus meeting consumers' multiple demands for agricultural product yield, quality, and safety. Using biocontrol microorganisms to control plant diseases has become an inevitable trend for sustainable agricultural development.

[0003] Soil microorganisms are considered to be the cornerstone of the next green revolution and play a vital role in plant growth and stress response. Bacteria from the genus Bacillus are considered to be important biocontrol agents and are the first choice in sustainable agricultural systems. To date, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velez, etc. of the genus Bacillus can control bacterial and / or fungal diseases of a variety of crops such as vegetables, fruit trees, and Chinese medicinal materials. However, Bacillus that promote the growth of one crop may not have the same effect on other crops. Some Bacillus have a general growth-promoting effect on several crops, while other Bacillus show strong host crop selectivity and colonize a single crop or a limited number of crops. In addition, biocontrol microorganisms often interact with microbial communities in the soil ecological environment. Exogenous biocontrol microorganisms may not be able to effectively establish themselves in the new soil environment due to competition with the resident bacterial community, so the successful use of biocontrol agents may depend on regional adaptability. At present, some commercial biocontrol agents at home and abroad are not very effective in preventing and controlling some soil-borne diseases of crops in Gansu due to the above reasons. This urgently requires us to base ourselves on Gansu's unique microbial resources and screen out some biocontrol agents suitable for Gansu's soil ecological environment and climatic conditions for the prevention and control of economic crop diseases in Gansu. Summary of the invention

[0004] The invention aims to provide a strain of Bacillus swezii MQ20YS04 and application thereof.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a strain of Bacillus swezeyi MQ20YS04 isolated and purified from salinized soil in Wuwei City, Gansu Province, classified and named Bacillus swezeyi MQ20YS04 (Bacillus swezeyi MQ20YS04), which has been deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postal code 430072, deposit number CCTCC NO: M 2024616, and deposit date April 3, 2024.

[0006] In a second aspect, the present invention provides a bacterial agent, a biological pesticide or a biological fertilizer prepared from the Bacillus swezii, its fermentation broth or a crude extract of the fermentation broth.

[0007] In a third aspect, the present invention provides any of the following uses of the Bacillus swezii or its bacterial agent:

[0008] 1) Used to antagonize plant pathogenic fungi;

[0009] 2) Used to prevent and treat pepper blight;

[0010] 3) Used to prevent and control plant root rot;

[0011] 4) Used for preparing biological pesticides;

[0012] 5) Used for preparing biofertilizer;

[0013] 6) Used for plant growth promotion.

[0014] The plant pathogenic fungi include, but are not limited to, Alternaria azukiae, Botrytis aclada, B. cinerea, B. elliptica, Colletorichum fioriniae, Fusarium graminearum, F. oxysporum, F. solani, F. verticillium, Phytophthora capsici, Pyricularia oryzae, Rhizoctonia solani AG-5, Rhizoctonia solani AG-6, and Sclerotinia sclerotiorum.

[0015] In a fourth aspect, the present invention provides any of the following uses of the Bacillus swezii:

[0016] ①Used to produce siderophore;

[0017] ②Used to produce amylase;

[0018] ③Used to produce protease;

[0019] ④Used for producing pectinase;

[0020] ⑤Used to produce cellulase;

[0021] ⑥Used to produce IAA;

[0022] ⑦Used for dissolving phosphorus (such as inorganic phosphorus);

[0023] ⑧Used for nitrogen fixation;

[0024] ⑨Used to produce NH 3 .

[0025] In a fifth aspect, the present invention provides a fermentation method of the Bacillus swezii, wherein the fermentation medium used is: 3.0-7.0 g / L soluble starch, 12.5 g / L yeast extract, MgSO 4 5.0-9.0g / L.

[0026] The fermentation conditions are as follows: seed liquid inoculation amount 9%, rotation speed 180-220 r / min, fermentation at 30° C. for 60 h, and initial fermentation pH value 5.0-7.0.

[0027] The number of viable Bacillus swezii in the fermentation broth was 4.16×10 11 CFU / mL.

[0028] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0029] The Bacillus swezeyi MQ20YS04 of the present invention has a strong ability to synthesize secondary metabolites and can produce siderophore, IAA and the like. The bacterium also has the potential for plant growth promotion and disease prevention and control. The strain MQ20YS04 has a strong antibacterial effect on various pathogenic fungi such as Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum, and has a good prevention and control effect on pepper blight and Codonopsis pilosula root rot. In addition, the strain MQ20YS04 also has the ability to fix nitrogen and dissolve phosphorus, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of greenhouse seedling trays and potted plants in the preferred embodiment of the present invention are shown in Figure 1. A and E, strain MQ20YS04; B and F, Bacillus subtilis; C and G, fluazinam; D and H, water control.

[0031] Figure 2 The strain MQ20YS04 in the preferred embodiment of the present invention produces disease prevention and growth promotion related characteristic substances. A, protease; B, α-amylase; C, pectinase; D, cellulase; E, phosphate solubilization; F, iron carrier; G, IAA; H, NH 3 .

[0032] Figure 3 The results of field control of pepper blight in a preferred embodiment of the present invention are shown in Figure 1. A, strain MQ20YS04; B, Bacillus subtilis; C, fluazinam; D, clean water control.

[0033] Figure 4 This is a test for screening components of the fermentation medium in a preferred embodiment of the present invention.

[0034] Figure 5 This is a single factor screening test of fermentation conditions in a preferred embodiment of the present invention.

[0035] Figure 6 The field effect of the fermentation liquid of strain MQ20YS04 in the preferred embodiment of the present invention on preventing and controlling Codonopsis pilosula root rot. A, strain MQ20YS04; B, Bacillus subtilis; C, azoxystrobin-methyl; D, clean water control. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0037] Example

[0038] 1. Materials and Methods

[0039] 1.1 Isolation and preservation of bacteria

[0040] Soil samples were collected from salinized plots in Minqin County, Wuwei City, Gansu Province. Five points were randomly selected from the sampling plots, and 0-5 cm salinized soil was taken. After mixing, it was brought back in a sealed bag. The soil and plant residues were removed with a 0.2 cm mesh sieve and air-dried at room temperature. 10 g of soil sample was weighed and placed in a conical flask containing 90 mL of sterile water. It was shaken for 10 min and made into 10 -1 The soil suspension of 10000 concentration was allowed to stand for the soil particles to settle, and then 1 mL of the supernatant was transferred into a test tube containing 9 mL of sterile water to make 10 -2 The soil suspension of concentration was diluted in sequence to make the final concentration of 10 -5 Soil suspension. Select 10 -3 , 10 -4 and 10 -5 200 μL of each suspension of the three concentrations was taken and added to the microbial isolation medium (tryptone 10 g, yeast extract 5 g, NaCl 100 g, agar 18 g, H 2 01000mL) plate, spread evenly with a coating rod, and repeat 3 times. Culture the culture dish at 28℃ for 5 days, select colonies with different morphologies and transfer them to a new microbial separation medium plate for purification and culture, and store at 4℃ for testing. The strains are placed in a -80℃ ultra-low temperature refrigerator for long-term storage in cryopreservation tubes (glycerol content is 20%).

[0041] 1.2 Determination of the ability of biocontrol bacteria to antagonize multiple pathogenic fungi

[0042] The antagonistic ability of biocontrol bacteria against various pathogenic fungi was screened and determined by plate confrontation culture method. The antagonistic ability of biocontrol bacteria against various pathogenic fungi was screened and determined by plate confrontation culture method. The samples of Alternaria azukiae, Botrytis aclada, B. cinerea, B. elliptica, Colletorichum fioriniae, Fusarium graminearum, F. oxysporum, F. solani, F. verticillium, Phytophthora capsici, Pyricularia oryzae, Rhizoctonia solani AG-5, Rhizoctonia solani AG-6 and Sclerotinia sclerotiorum cultured at 25℃ for 7 days were cut with a hole puncher (6.5 mm). sclerotiorum) colony edge cake was inoculated on the culture medium at a point 2 cm away from the edge of the culture dish, and at the same time, the soil isolated strain was inoculated at a point 2 cm away from the edge of the culture dish on the other side. After constant temperature cultivation at 25°C for 10 days, the size of the inhibition zone was measured. Each treatment was repeated 3 times.

[0043] 1.3 Preparation of culture medium of strain MQ20YS04

[0044] After the strain MQ20YS04 was inoculated on LB solid medium for activation, it was transferred to a 100 mL Erlenmeyer flask containing 50 mL LB liquid medium and cultured at 28°C, 150 rpm for 24 h to obtain seed solution. The seed solution was inoculated into the culture medium (1 / 2PDB+1 / 2LB) at a ratio of 1:100 (volume ratio) and cultured at 28°C, 150 rpm for 24 h.

[0045] 1.4 Determination of the control effect of strain MQ20YS04 on pepper blight in seedling trays

[0046] Pepper variety "Changfeng" seeds were surface sterilized in 0.1% sodium hypochlorite for 20 min and then washed four times with sterile water. Seeds were sown in 50-hole seedling trays filled with horticultural soil, with one seed per hole. Seedlings were grown in a greenhouse with no pests and controlled at 20-30°C. After 8 weeks, 20 mL of the culture solution of strain MQ20YS04 (viable bacteria concentration 10 8 CFU / mL) were poured into the horticultural soil at the root of the plant to prevent excess liquid from flowing out from the bottom of the seedling tray. 11spores / g, WP, Deqiang Biological Co., Ltd., Harbin, China) and fluazinam (ai50%, SC, Jiangyin Suli Chemical Co., Ltd., Jiangyin, China) were used as blank control and positive control, respectively, and the plants were treated with the same treatment method as above. Each treatment was repeated 3 times, with a total of 150 plants. 10 mL of P. capsici inoculum (1×10 5 Sporangium / mL) was poured into the horticultural soil at the root of each plant. The inoculated plants were placed in a growth chamber to maintain humidity above 90% and temperature at 20-30°C. The disease level was investigated on the 7th day after inoculation of the pathogen, and the prevention and control effect was calculated. Disease grading standard: Level 0: healthy and symptom-free; Level 1: The rhizomes of seedlings slightly blacken, and the leaves do not wilt or wilt recoverably; Level 2: The rhizomes of seedlings turn black by 1 to 2 cm, the leaves wilt irreversibly, and the lower leaves occasionally fall off; Level 3: The rhizomes of seedlings turn black by more than 2 cm, and the leaves wilt or fall off obviously; Level 4: The rhizomes of seedlings turn black and constrict, and all leaves fall or the plants wilt except the growth point; Level 5: The whole plant dies.

[0047] 1.5 Determination of the potted control effect of strain MQ20YS04 on pepper blight

[0048] Pepper seeds were treated as described above. Seeds were sown in 72-well seedling trays filled with horticultural soil, with one seed per well. Seedlings were grown in a pest-free greenhouse at a temperature of 20-30°C. After 6 weeks, seedlings were transplanted into plastic pots (10 cm in diameter) filled with horticultural soil and continued to grow in a pest-free greenhouse at a temperature of 20-30°C for 4 weeks. 50 mL of the fermentation liquid of strain MQ20YS04 (viable bacteria concentration 10 8 CFU / mL) were poured into the horticultural soil at the root of the plant to prevent excess liquid from flowing out of the bottom of the plastic pot. Water, Bacillus subtilis and fluazinam were used as blank control and positive control respectively to treat the plants with the same treatment method as above. Each treatment was repeated 3 times, with a total of 90 plants. 10mL P.capsici inoculum (1×10 5 Sporangium / mL) was poured into the horticultural soil at the root of each plant. The inoculated plants were placed in a growth chamber to maintain humidity above 90% and temperature at 20-30°C. The disease level was investigated on the 14th day after inoculation of the pathogen and the control effect was calculated. The disease grading standard was the same as above.

[0049] 1.6 Identification of strain MQ20YS04 using gene sequence analysis

[0050] The whole genomic DNA of the biocontrol bacterium MQ20YS04 was extracted using the Fast TIANamp Bacteria DNA Kit (Tiangen, Beijing). The 16S rRNA gene of the strain was amplified by PCR using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') of the bacterial 16S rRNA gene. The gyrA gene of the strain was amplified by PCR using the primers gyrA-F (5'-CAGTCAGGAAATGCGTACGTCCTT-3') and gyrA-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3').

[0051] The gyrB gene of the strain was amplified by PCR using primers UP1 (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP2r (5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'). The cheA gene of the strain was amplified by PCR using primers cheA-F (5'-AGGGCTGCCCATACATTAAAAGGAATGAGC-3') and cheA-R (5'-AGTTTCAACCGGGACCATTCTCATATTCAG-3'). The mdh gene of the strain was amplified by PCR using primers mdh86F (5'-CCCAGCTTCCTTCAGGTTCAGA-3') and mdh628R (5'-CTGCATTCTGAATACGTTTGGTCA-3'). The rpoB gene of the strain was amplified by PCR using primers rpoB-F (5'-AGGTCAACTAGTTCAGTATGGAC-3') and rpoB-R (5'-AGGAACCGTAACCGGCAACTT-3'). The rpoD gene of the strain was amplified by PCR using primers rpoD-F (5'-GAAGGCGAAATCGACATCGC-3') and rpoD-R (5'-ATGCTCATGCGRCGGTTGAT-3'). 25 μL of the reaction mixture was amplified on a PCR thermal cycler. The PCR products were purified and sent to Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) for sequencing. The sequencing results were compared in the NCBI database, and the 16S rRNA, gyrA, gyrB, cheA, mdh, rpoB and rpoD gene sequences of related species were downloaded from the NCBI gene bank database, and phylogenetic analysis was performed using MEGA 10.0. The 16S rRNA, gyrA, gyrB, cheA, mdh, rpoB, and rpoD gene sequences of strain MQ20YS04 were submitted to the NCBI database.

[0052] 1.7 In vitro detection of disease prevention and growth-promoting related substances

[0053] 1.7.1 Production of siderophore

[0054] Select the strain and inoculate it onto CAS medium, place it in a 30℃ incubator for 14 days, and observe whether a yellow-orange circle appears. If so, it means that the strain has the ability to produce siderophore.

[0055] 1.7.2 Production of amylase

[0056] The strain was selected and inoculated onto NA medium containing 1% soluble starch, and cultured in a 30°C incubator for 5 days to observe whether a transparent circle appeared. If so, it indicated that the strain had the ability to produce amylase.

[0057] 1.7.3 Protease production

[0058] The strain was selected and inoculated onto a casein yeast medium containing 7% skimmed milk powder (5 g casein, 2.5 g yeast extract, 1 g glucose, 1000 mL water), and cultured in a 30°C incubator for 5 days. The appearance of a transparent circle was observed. If so, it indicated that the strain had the ability to produce protease.

[0059] 1.7.4 Production of pectinase

[0060] The strain was selected and inoculated onto a basic medium containing 1% pectin (1 g sodium nitrate, 1 g dipotassium hydrogen phosphate, 1 g potassium chloride, 0.5 g magnesium sulfate, 0.5 g yeast powder, 1 g glucose, 18 g agar, and 1000 mL water), and cultured in a 30°C incubator for 7 days. Gram's iodine solution (1.0 g iodine, 2.0 g potassium iodide, and 300 mL water) was poured into the pectin medium, and the appearance of a transparent circle was observed under a dark blue background. If so, it indicated that the strain had the ability to produce pectinase.

[0061] 1.7.5 Production of cellulase

[0062] The strain was selected and inoculated onto a basic culture medium containing 1% cellulose, and cultured in a 30°C incubator for 5 days. The cellulose culture medium was submerged in 0.01% Congo red solution for 15 minutes, and the culture medium was rinsed with water 3 times to observe whether a transparent circle appeared. If so, it indicated that the strain had the ability to produce cellulase.

[0063] 1.7.6 Generation of IAA

[0064] The strain was cultured in a TSB medium containing 0.1% L-tryptophan at 30°C in a shaking incubator for 72 h. The bacterial suspension was centrifuged to remove the bacteria. 2.0 mL of Salkawski colorimetric agent (50 mL, 35% perchloric acid, 1 mL 0.5 M ferric chloride) was added to 1.0 mL of sterile filtrate and color was developed at room temperature for 30 min. If a pink color was produced, it indicated that IAA was produced. The culture medium was used as a control.

[0065] 1.7.7 Phosphorus dissolution

[0066] Pick the strain and inoculate it onto PVK medium (0.5 g yeast powder, 10.0 g glucose, 5.0 g calcium phosphate, 0.5 g ammonium sulfate, 0.2 g potassium chloride, 0.1 g magnesium chloride, 0.0001 g manganese sulfate, 0.0001 g ferrous sulfate, 18.0 g agar, 1000 mL water), place it in a 30°C incubator for 14 days, and observe whether a transparent circle appears. If so, it means that the strain has the ability to dissolve phosphate.

[0067] 1.7.8 Potassium dissolution

[0068] The strain was selected and inoculated onto Aleksandrov medium (5.0 g of glucose, 0.5 g of magnesium sulfate heptahydrate, 0.005 g of ferric chloride, 2.0 g of calcium carbonate, 2.5 g of yeast powder, 18.0 g of agar, and 1000 mL of water) containing 0.2% potassium feldspar, and cultured in a 30°C incubator for 14 days. The appearance of a transparent circle was observed, and if so, it indicated that the strain had the ability to solubilize potassium.

[0069] 1.7.9 Nitrogen fixation

[0070] The strain was selected and inoculated onto Agar's nitrogen-free medium (10.0 g glucose, 0.2 g potassium dihydrogen phosphate, 0.5 g calcium carbonate, 0.2 g magnesium sulfate heptahydrate, 0.2 g sodium chloride, 0.1 g calcium sulfate, 18.0 g agar, 1000 mL water), and cultured in a 30°C incubator for 14 days to observe whether it grew normally. If so, it indicated that the strain had the ability to fix nitrogen.

[0071] 1.7.10NH 3 Production

[0072] Inoculate 50 μL of bacterial suspension into 30 mL of peptone ammoniation medium (5.0 g of peptone, 0.5 g of potassium hydrogen phosphate, 0.25 g of sodium chloride, 0.5 g of magnesium sulfate heptahydrate, 0.01 g of ferrous sulfate heptahydrate, 1000 mL of water), place in a 30°C incubator for 3 days, add 1.0 mL of Nessler color developer (6.0 g of mercuric chloride, 12.4 g of potassium iodide, 30 mL of 20% sodium hydroxide, 70 mL of water), if the yellow color turns to brown, it means that NH 3 The culture medium was used as a control.

[0073] 1.8 Determination of the field control effect of strain MQ20YS04 on pepper blight

[0074] During the 2022 pepper growing season, an artificial inoculation field trial was conducted at the Lanzhou Experimental Station of the Gansu Academy of Agricultural Sciences to determine the efficacy of the biocontrol bacterial strain MQ20YS04 against pepper blight. The strain MQ20YS04 biocontrol agent and pepper seedlings were prepared as described above. On May 17, pepper seedlings (9 weeks old) were transplanted to 300m 2The row and plant spacings were kept at 50 cm and 30 cm respectively. The treatments were set with the biocontrol bacterial strains MQ20YS04 and Bacillus subtilis (ai1×10 11 spores / g, WP, Deqiang Biological Co., Ltd., Harbin, China), fluazinam (ai50%, SC, Jiangyin Suli Chemical Co., Ltd., Jiangyin, China) and water (blank control) were randomly divided into two groups, and each treatment was repeated 3 times. On August 1, 8 and 15, 100 mL of the culture medium of strain MQ20YS04 (viable bacteria concentration 10 8 CFU / mL), Bacillus subtilis and fluazinam (according to the dosage recommended by the supplier) and water were poured into the roots of the plants. On August 15, 10 mL of P. capsici inoculum (1×10 5 Sporangium / mL) was poured into the soil around the roots of each plant. The disease level was investigated on the 15th day after the pathogen was inoculated, and the control effect was calculated. Disease grading standard: Level 0: healthy and symptom-free; Level 1: only leaves and fruits above ground have lesions; Level 2: above-ground stems and branches have brown rot spots; Level 3: brown rot spots at the base of the stems; Level 4: above-ground stems, branches and stem bases have brown rot spots, and some branches die; Level 5: the whole plant dies.

[0075] 1.9 Optimization of fermentation medium components and conditions

[0076] 1.9.1 Optimization of fermentation medium components of strain MQ20YS04

[0077] 1.9.1.1 Preparation of fermentation seed solution of strain MQ20YS04

[0078] The strain MQ20YS04 was inoculated on LB medium, placed in a constant temperature incubator, and cultured at 28°C for 48 hours. A single colony was picked up with a transfer loop and added to a 250mL Erlenmeyer flask containing 100mL LB culture solution. The culture was shaken in a shaker at 28°C and 180r / min for 24 hours to obtain the seed solution.

[0079] 1.9.1.2 Live bacteria count of MQ20YS04 fermentation broth

[0080] In an ultra-quiet workbench, use a pipette to draw 1 mL of fermentation liquid into a test tube containing 9 mL of distilled water that has been sterilized at high temperature. After shaking, draw 1 mL of diluent and add it to the next test tube. Perform gradient dilutions in sequence to make a final concentration of 10 -7 Take 100 μL of the bacterial suspension in the last test tube and add it to the LB plate. Use a sterilized applicator to spread it evenly. Each group has 9 plates (repeated 3 times). Incubate in a 28℃ constant temperature incubator for 24 hours and use the Xunshu MF3 series microscopic colony counter to count the number of live bacteria.

[0081] 1.9.1.3 Selection of carbon sources

[0082] The number of viable bacteria in the fermentation broth was taken as the response value. LB medium was used as the basic medium. Sucrose, glucose, lactose, maltose and soluble starch at a concentration of 5 g / L were used to replace the carbon source in the medium. LB without any carbon source was used as a control. The shaking fermentation culture conditions were set as follows: the seed liquid inoculation amount was 5%, and the shaking fermentation was carried out at 180 r / min and 28 °C for 36 h. Each treatment was repeated 3 times.

[0083] 1.9.1.4 Selection of nitrogen sources

[0084] The number of viable bacteria in the fermentation broth was taken as the response value. On the basis of screening out the carbon source as soluble starch, 10 g / L NH 4 HCO 3 、NaNO 2 NH 4 Cl, (NH 4 ) 2 SO 4 , yeast extract and peptone to replace the nitrogen source of the fermentation medium. The shaking fermentation culture conditions were consistent with those for carbon source screening, and 3 replicates were set for each treatment.

[0085] 1.9.1.5 Screening of inorganic salts

[0086] The number of viable bacteria in the fermentation broth was taken as the response value. On the basis of screening out the carbon and nitrogen sources as soluble starch and yeast extract, MgSO with a concentration of 5 g / L was used. 4 7H 2 O、CuSO 4 7H 2 O, FeSO 4 7H 2 O.KH 2 PO 4 , NaCl and KCl to replace the inorganic salts in the fermentation medium. The shaking fermentation culture conditions were consistent with those for carbon source screening, and 3 replicates were set for each treatment.

[0087] 1.9.1.6 Screening of fermentation medium component concentrations

[0088] The number of viable bacteria in the fermentation broth was the response value. After screening out the nitrogen source as 10 g / L yeast extract and the inorganic salt as 5 g / LMgSO 4 7H 2 O, the concentrations of soluble starch were 1g / L, 3g / L, 5g / L, 7g / L and 9g / L, and the shaking fermentation culture conditions were the same as those for carbon source screening, with 3 replicates for each treatment. 4 7H 2O, the concentrations of yeast extract were 5g / L, 7.5g / L, 10g / L, 12.5g / L and 15g / L, and the shaking fermentation culture conditions were the same as those for carbon source screening, with 3 replicates for each treatment. On the basis of screening out 5g / L soluble starch as the carbon source and 10g / L yeast extract as the nitrogen source, MgSO 4 7H 2 The concentrations of O were 1g / L, 3g / L, 5g / L, 7g / L and 9g / L for screening. The shaking fermentation culture conditions were consistent with the carbon source screening, and 3 replicates were set for each treatment.

[0089] 1.9.2 Optimization of shake flask fermentation conditions

[0090] The above-screened soluble starch 0.5%, yeast extract 1.0%, MgSO 4 7H 2 O0.7% was used as the basic culture medium component, and the number of viable bacteria in the fermentation broth was used as the response value to optimize the shake flask fermentation speed, inoculation amount, temperature, time, and initial pH of strain MQ20YS04. Three replicates were set for each experimental group. During the optimization of one factor, other conditions were kept unchanged, and the number of viable bacteria in the shake flask fermentation broth under different conditions was counted. The speed was set to 140, 160, 180, 200, and 220r / min; the inoculation amount was set to 1%, 3%, 5%, 7%, and 9%; the temperature was set to 26℃, 28℃, 30℃, 32℃, and 34℃; the time was set to 24, 36, 48, 60, and 72h; the initial pH was set to 5.0, 6.0, 7.0, 8.0, and 9.0.

[0091] 1.9.3 Plackett-Burman test

[0092] Based on the analysis of single-factor fermentation medium components and the optimization of shake flask fermentation conditions, the viable count of strain MQ20YS04 was used as the response value to screen eight factors, including carbon source, nitrogen source, inorganic salt, rotation speed, inoculation amount, temperature, time and initial pH, to identify the main effect factors. Each factor was set at the lowest (-1) and highest (1) levels. The experimental factors and level values ​​are shown in Table 1.

[0093] Table 1 Factor levels and coding of Plackett-Burman experimental design

[0094] factor coding Low level (-1) High level (1) Soluble starch (%) <![CDATA[X 1 ]]> 0.3 0.7 Yeast extract (%) <![CDATA[X 2 ]]> 0.75 1.25 Magnesium sulfate (%) <![CDATA[X 3 ]]> 0.5 0.9 Speed ​​(r / min) <![CDATA[X 4 ]]> 180 220 Temperature(℃) <![CDATA[X 5 ]]> 26 30 Time (h) <![CDATA[X 6 ]]> 36 60 Initial pH <![CDATA[X 7 ]]> 5.0 7.0 Inoculation amount (%) <![CDATA[X 8 ]]> 5 9

[0095] 1.9.4 Response Surface Methodology for Optimizing Experimental Design

[0096] According to the results of Plackett-Burman experimental design, four factors, namely yeast extract, temperature, time and inoculation amount, were selected, and the number of viable bacteria in the fermentation broth was used as the response value to conduct a four-factor three-level Box-Behnken experimental design (Table 2). A fitting regression model was established and analyzed for the results of Box-Behnken experimental design.

[0097] Table 2 Box-Behnken test design factors and level coding

[0098]

[0099]

[0100] 1.10 Field test of fermentation broth of strain MQ20YS04

[0101] The fermentation medium components and shake flask fermentation conditions optimized above were used to prepare the fermentation liquid of strain MQ20YS04 for use. The experiment was conducted in the terraced fields of Lijiawan Village, Xinzhai Town, Weiyuan County, Dingxi City, Gansu Province, with the previous crop being Codonopsis pilosula. The cultivation mode of Codonopsis pilosula was open-field furrow sowing, with a row spacing of 30 cm and a plant spacing of 2-3 cm. There were 4 treatments in the experiment, namely T1 (strain MQ20YS04, 1×10 8 CFU / mL), T2 (0.8% azoxystrobin granules, Shaanxi Biaozheng Crop Science Co., Ltd.), T3 (commercial product: 100 billion spores / g Bacillus subtilis, Langfang Pesticide Pilot Plant, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 500 times dilution) and T4 (blank control, clean water), each treatment was repeated 3 times, each plot was 48m 2 (6m×8m), with random block arrangement. In April 2023 and 2024, transplant one-year-old Codonopsis pilosula seedlings, dig furrows and place the Codonopsis pilosula seedlings on the furrow slope at an angle of 15°, with the buds 3-5cm from the ground surface. According to the experimental design, evenly spread the prepared pesticides on the Codonopsis pilosula seedlings and the furrows. Other agricultural operations in each plot are unified. After the seedlings emerged neatly in June, the seedling emergence survey was conducted. In October, the incidence of Codonopsis root rot (the pathogenic bacteria Fusarium spp. that cause Codonopsis root rot include F.oxysporum, F.solani and F.verticillium) was investigated, and the disease index and control effect were calculated. The disease classification standard is: 0: no disease; 1: the diseased area accounts for less than 5% of the whole plant; 3: the diseased area accounts for 6%-25% of the whole plant; 5: the diseased area accounts for 26%-50% of the whole plant; 7: the diseased area accounts for 51%-75% of the whole plant; 9: the diseased area accounts for more than 76% of the whole plant. Disease index = ∑ (disease level × number of plants with this disease level) / (total number of plants investigated × highest disease level) × 100; control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0102] 2. Results and Analysis

[0103] 2.1 Isolation, purification and antagonistic ability determination

[0104] Saline soil was collected from salinized land in Minqin County, Wuwei City, Gansu Province. A total of 40 bacterial isolates were isolated by directly observing the colony morphology and color of bacteria. After screening and testing the antagonistic ability by plate confrontation culture method, 10 bacterial strains could antagonize 14 pathogenic fungi, among which strain MQ20YS04 showed a strong antagonistic ability (Table 3).

[0105] Table 3 Antagonistic ability of strain MQ20YS04 against various plant pathogens

[0106] Plant pathogens Antibacterial zone (mm) Antibacterial rate (%) Alternaria azukiae 8.09±0.77 61.87±0.40 Botrytis aclada 5.90±0.12 61.03±0.43 Botrytis cinerea 5.04±0.24 54.36±1.74 Botrytis elliptica 6.58±0.46 45.21±0.18 Colletorichum fioriniae 13.41±0.51 53.86±0.72 Fusarium graminearum 5.59±0.30 53.57±1.09 Fusarium oxysporum 5.90±0.74 67.10±0.67 Fusarium solani 5.67±0.21 52.44±0.62 Fusarium verticillioides 4.19±0.19 68.14±2.07 Phytophthora capsici 8.92±0.25 47.70±1.86 Pyricularia oryzae 9.12±0.72 59.44±1.46 Rhizoctonia solani AG-5 9.52±0.29 54.97±0.77 Rhizoctonia solani AG-6 9.41±0.40 52.17±1.78 Sclerotinia sclerotiorum 8.44±0.27 54.27±0.59

[0107] 2.2 Greenhouse disease prevention effect

[0108] The greenhouse of the Institute of Plant Protection, Gansu Academy of Agricultural Sciences, conducted a test on the effectiveness of greenhouse seedling trays and potted plants in preventing and controlling pepper blight. The disease of each treatment was investigated 7 and 14 days after inoculation of the pathogen, and the prevention and control effect was calculated. The prevention and control effect of strain MQ20YS04 on pepper blight was 66.67% and 69.33% ( Figure 1 ). The greenhouse pot experiment repeatedly verified that the strain MQ20YS04 had a stable protective effect against pepper blight and could be further tested in the field.

[0109] 2.3 Gene sequence analysis and identification of strain MQ20YS04

[0110] The genome of strain MQ20YS04 was extracted, and the primer combinations 27F and 1492R, gyrA-F and gyrA-R, UP1 and UP2r, cheA-F and cheA-R, mdh86F and mdh628R, rpoB-F and rpoB-R, rpoD-F and rpoD-R were used to perform PCR amplification of 16SrRNA, gyrA, gyrB, cheA, mdh, rpoB and rpoD genes, respectively, to obtain the target fragments, which were sent to Sangon Biotech (Shanghai) Co., Ltd. for base sequence determination. The obtained 16S rRNA, gyrA, gyrB, cheA, mdh, rpoB and rpoD gene sequences of strain MQ20YS04 were 1438 bp, 951 bp, 1167 bp, 884 bp, 457 bp, 558 bp and 329 bp, respectively. The obtained sequences were compared with the 16S Homology comparison analysis of rRNA, gyrA, gyrB, cheA, mdh, rpoB and rpoD gene sequences showed that the similarities between the 16S rRNA, gyrA, gyrB, cheA, mdh, rpoB and rpoD gene sequences of strain MQ20YS04 and those of Bacillus swezeyi were 99.72%, 99.79%, 99.66%, 99.32%, 99.78%, 100.00% and 98.78%, respectively. Based on the results of homology comparison analysis, strain MQ20YS04 was identified as Bacillus swezeyi, and the accession numbers of 16S rRNA, gyrA, gyrB, cheA, mdh, rpoB and rpoD gene sequences were PQ451477, PQ487785, PQ487786, PQ487787, PQ487788, PQ487789 and PQ487790, respectively. It was deposited in the China Center for Type Culture Collection (CCTCC) on April 3, 2024, with the deposit number CCTCC NO: M 2024616.

[0111] 2.4 Determination of in vitro disease prevention and growth promotion related characteristic substances

[0112] The results of in vitro disease prevention and growth promotion related characteristic substances detection showed that strain MQ20YS04 can produce α-amylase, protease, pectinase, cellulase, siderophore, IAA and NH 3 , has the ability to fix nitrogen and dissolve phosphorus, but no ability to dissolve potassium ( Figure 2 ).

[0113] 2.5 Field control effect of strain MQ20YS04 on pepper blight

[0114] In 2022, an artificial inoculation field test of strain MQ20YS04 against pepper blight was conducted at the Lanzhou Experimental Station of Gansu Academy of Agricultural Sciences. The disease of each treatment was investigated on the 15th day after inoculation with the pathogen (P. capsici), and the control effect was calculated. The control effect of strain MQ20YS04 on pepper blight was 65.16±1.88% ( Figure 3 ).

[0115] 2.6 Single factor screening of fermentation medium components

[0116] Different carbon sources had a significant effect on the viable count of strain MQ20YS04. The viable count per unit volume of the medium with soluble starch as the carbon source was the largest, which was 1.50×10 9 CFU / mL, the influence of other carbon sources on the number of viable bacteria was sucrose>maltose>glucose>lactose, so soluble starch was selected as the carbon source of the culture medium for subsequent experiments. When different nitrogen sources were used as the culture medium components, the number of viable bacteria per unit volume of strain MQ20YS04 was yeast extract, NH 4 Cl, peptone, (NH 4 ) 2 SO 4 NH 4 HCO 3 、NaNO 2 The number of viable bacteria in the medium with yeast extract as the nitrogen source was significantly higher than that in the medium with other nitrogen sources (P<0.05), so yeast extract was selected as the nitrogen source for the subsequent experiments. Different inorganic salt mediums had a significant effect on the number of viable bacteria of strain MQ20YS04 (P<0.05). 4 7H 2 O and CuSO 4 7H 2 When O was an inorganic salt, strain MQ20YS04 hardly grew. 4 7H 2 When MgSO is used as a culture medium component, the number of viable bacteria per unit volume reaches the maximum. Therefore, MgSO 4 7H 2 O as the inorganic salt of the culture medium to optimize the composition ratio ( Figure 4 ). The different concentrations of soluble starch had significant effects on the number of viable bacteria per unit volume of strain MQ20YS04 (P<0.05). At 5 g / L, the number of viable bacteria per unit volume reached the maximum value of 6.38×10 10CFU / mL, so the soluble starch concentration of 5g / L was selected as the optimal fermentation concentration of strain MQ20YS04. The yeast extract concentrations of 10g / L and 15g / L had no significant effect on the number of viable bacteria per unit volume of strain MQ20YS04 (P<0.05), while the other three concentrations had significant effects on the number of viable bacteria per unit volume of strain MQ20YS04 (P<0.05), so the yeast extract concentration of 10g / L was selected as the optimal fermentation concentration of strain MQ20YS04. MgSO 4 7H 2 The effects of different concentrations of O on the number of viable bacteria per unit volume of strain MQ20YS04 were significant (P<0.05). At 7 g / L, the number of viable bacteria per unit volume reached the maximum value of 2.28×10 10 CFU / mL, so MgSO 4 7H 2 O concentration of 7 g / L was the optimal fermentation concentration for strain MQ20YS04 ( Figure 4 ).

[0117] 2.7 Single factor test of fermentation conditions

[0118] The effects of different rotation speeds on the number of viable bacteria per unit volume of strain MQ20YS04 were significant (P<0.05). At 200 r / min, the number of viable bacteria per unit volume reached the maximum value of 1.29×10 10 CFU / mL, so the speed of 200r / min was selected as the optimal fermentation speed of strain MQ20YS04. The effects of different fermentation inoculation amounts on the number of viable bacteria were significantly different (P<0.05). When the inoculation amount was 9%, the number of viable bacteria in the fermentation broth reached the maximum value of 3.79×10 10 CFU / mL, so 9% was selected as the optimal fermentation inoculum of strain MQ20YS04. The effects of different temperatures on the number of viable bacteria in the fermentation broth were significantly different (P<0.05). The number of viable bacteria in the fermentation broth at 28°C was significantly higher than that at other temperatures, which was 1.69×10 10 CFU / mL, therefore, 28℃ was selected as the optimal fermentation temperature for strain MQ20YS04. The effects of different culture times on the number of viable bacteria in the fermentation broth were significantly different (P<0.05). The maximum number of viable bacteria per unit volume in the fermentation broth was 2.96×10 at 48h. 10 CFU / mL, so 48h was selected as the optimal fermentation time for strain MQ20YS04. The effects of different initial pH values ​​on the number of viable bacteria in the fermentation broth were significantly different (P<0.05). When the initial pH was 6, the number of viable bacteria in the fermentation broth reached the maximum value, which was 3.92×10 10CFU / mL, so the initial pH 6 was selected as the optimal initial pH value for fermentation culture of strain MQ20YS04 ( Figure 5 ).

[0119] 2.8 Plackett-Burman test (Table 4 and Table 5)

[0120] Table 4 Plackett-Burman test design and response values

[0121]

[0122] Table 5 Evaluation of the effects of each factor in the Plackett-Burman experimental design

[0123] factor effect coefficient Standard error of coefficient T-value P-value constant 2.242 0.009 263.68 0.000 <![CDATA[X 1 ]]> 0.020 0.010 0.009 1.200 0.316 <![CDATA[X 2 ]]> 0.113 0.056 0.009 6.640 0.007 <![CDATA[X 3 ]]> 0.011 0.005 0.009 0.650 0.562 <![CDATA[X 4 ]]> -0.014 -0.007 0.009 -0.830 0.467 <![CDATA[X 5 ]]> 0.098 0.049 0.009 5.790 0.010 <![CDATA[X 6 ]]> 0.082 0.041 0.009 4.810 0.017 <![CDATA[X 7 ]]> -0.044 -0.022 0.009 -2.590 0.081 <![CDATA[X 8 ]]> -0.072 -0.036 0.009 -4.230 0.024

[0124] Minitab 17 software was used to perform multiple regression analysis on the data in Table 4, and the regression equation with the number of viable bacteria as the response value was obtained. 1 +0.056X 2 +0.006X 3 -0.007X 4 +0.049X 5 +0.041X 6 -0.022X 7 -0.036X 8 , and the effect evaluation of each factor was obtained. According to the effect evaluation table, the four factors of yeast extract, temperature, time and inoculation amount had a significant effect on the number of viable bacteria per unit volume of fermentation broth (P<0.05), while the other four factors did not have a significant effect on the number of viable bacteria per unit volume of fermentation broth. Therefore, yeast extract, temperature, time and inoculation amount were selected as key factors for response surface optimization design.

[0125] 2.9 Response surface analysis and optimization

[0126] According to the four key factors screened out by Plackett-Burman test, Box-Behnken test design was carried out. The variance analysis of the test design factors, response values ​​and regression equations are shown in Tables 6 and 7.

[0127] Table 6 Box-Behnken response surface experimental design and results

[0128]

[0129]

[0130] Table 7 Results of variance analysis of regression model

[0131] Sources of variance Degrees of Freedom sum of squares Mean Square F-number P-value <![CDATA[X 1 ]]> 1 64.40 64.40 10.00 0.0069 <![CDATA[X 2 ]]> 1 128.71 128.71 19.98 0.0005 <![CDATA[X 3 ]]> 1 203.36 203.36 31.57 0.0001 <![CDATA[X 4 ]]> 1 30.40 30.40 4.72 0.0475 <![CDATA[X 2 ]]> 1 225 225 34.94 0.0001 <![CDATA[X 2 ]]> 1 6.25 6.25 0.97 0.3413 <![CDATA[X 2 ]]> 1 10.89 10.89 1.69 0.2145 <![CDATA[X 2 ]]> 1 272.25 272.25 42.28 0.0001 <![CDATA[X 1 X 2 ]]> 1 11.22 11.22 1.74 0.2080 <![CDATA[X 1 X 3 ]]> 1 13.69 13.69 2.12 0.1669 <![CDATA[X 1 X 4 ]]> 1 3.22 3.22 0.50 0.4913 <![CDATA[X 2 X 3 ]]> 1 8.70 8.70 1.35 0.2645 <![CDATA[X 2 X 4 ]]> 1 67.64 67.64 10.50 0.0059 <![CDATA[X 3 X 4 ]]> 1 8.33 8.33 1.29 0.2744 Model 14 1071.97 76.57 11.89 0.0001 Residual 14 90.16 6.44 Lack of Fit 10 75.18 7.52 2.01 0.2620 Pure Error 4 14.98 3.74 sum 28 1162.13

[0132] Design-Expert 10.0.3 was used to perform quadratic linear fitting regression on the test data in Table 6, and the quadratic polynomial regression equation was obtained:

[0133] Y=20.50+2.32X 1 +3.28X 2 +4.12X 3 -1.59X 4 +7.50X 1 X 2 +1.25X 1 X 3 -

[0134] 1.65X 1 X 4 +8.25X 2 X 3 +1.68X 2 X 4 -1.85X 3 X 4 -0.70X 1 2 +1.16X 2 2 -3.23X 3 2 +1.13X 4 2 .

[0135] The regression model was subjected to variance analysis. As shown in Table 7, the model P < 0.01, indicating that the regression equation is extremely significant, and the lack of fit term P = 0.2620 > 0.05 (not significant), indicating that the regression model is reliable and can be used to optimize the number of viable bacteria in the fermentation of strain MQ20YS04. 2 =0.92, indicating that the regression equation has a high degree of fit; the correction coefficient Adj.R 2 =0.845, indicating that the model prediction value is highly correlated with the actual value, so the model can be used for the prediction and analysis of the number of viable bacteria in the fermentation of strain MQ20YS04.

[0136] In summary, the key factors and optimal conditions for the number of live bacteria in the fermentation of strain MQ20YS04 are: yeast extract 12.5g / L, temperature 30℃, time 60h, inoculation volume 9%. The other four factors did not reach a significant level in the Plackett-Burman test for the number of live bacteria per unit volume of the fermentation broth, so the optimal conditions for the other four factors are: soluble starch 3.0-7.0g / L, magnesium sulfate 5.0-9.0g / L, speed 180-220r / min, initial pH 5.0-7.0. According to the experimental design and actual production conditions, the optimal adjustment conditions are soluble starch 3.0g / L, yeast extract 12.5g / L, magnesium sulfate 5.0g / L, speed 200r / min, inoculation volume 9%, temperature 30℃, time 60h and initial pH 7.0. Under these conditions, the model predicts that the maximum number of live bacteria is 4.21×10 11 CFU / mL. The validation test was carried out under this optimal condition and the number of viable bacteria was 4.16×10 11 CFU / mL, which was close to the model prediction value (98.82%), indicating that the model had high credibility and could be applied to the fermentation of strain MQ20YS04.

[0137] 2.10 Field test of the efficacy of the fermentation liquid of strain MQ20YS04 against Codonopsis pilosula root rot

[0138] In 2023 and 2024, the effect of the fermentation liquid of strain MQ20YS04 on the prevention and control of Codonopsis root rot was tested in the terraced fields of Lijiawan Village, Xinzhai Town, Weiyuan County, Dingxi City, Gansu Province. The control effects of the fermentation liquid of strain MQ20YS04 on Codonopsis root rot were 65.35±2.57% and 66.03±3.12% ( Figure 6 ).

[0139] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Bacillus swezii ( Bacillus swezeyi ) MQ20YS04, the deposit number is CCTCC NO: M2024616.

2. A microbial agent, biological pesticide or biological fertilizer prepared from the Bacillus swezii according to claim 1, its fermentation liquid or a crude extract of the fermentation liquid.

3. A biocontrol agent or antibacterial agent prepared from the Bacillus swezii according to claim 1.

4. Any of the following uses of the Bacillus swezii or its bacterial agent according to claim 1: 1) Used to antagonize plant pathogenic fungi; 2) Used to prevent and control pepper blight; 3) Used to prevent and treat plant root rot; 4) Used for the preparation of biological pesticides; 5) Used to prepare biofertilizer; 6) Used for plant growth promotion.

5. The use according to claim 4, characterized in that: The plant pathogenic fungi include Alternaria alternata ( Alternaria azukiae )、Botrytis cinerea( Botrytis aclada ), Botrytis cinerea ( B . cinerea ), Botrytis cinerea ( B . elliptica ) Colletorichum fioriniae graminearum ( Fusarium graminearum ), Fusarium oxysporum ( F . oxysporum ) F. solani )、Verticillium spp.( F. verticillium )、Phytophthora capsici( Phytophthora capsici ), Pyricularia oryzae Pyricularia oryzae ) and Rhizoctonia solani AG-5 ( Rhizoctonia solani AG-5), Rhizoctonia solani AG-6 ( Rhizoctonia solani AG-6) and Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ).

6. Any of the following uses of the Bacillus swezii of claim 1: ①Used to produce siderophore; ②Used to produce amylase; ③Used to produce protease; ④Used for producing pectinase; ⑤Used to produce cellulase; ⑥Used to produce IAA; ⑦Used for dissolving phosphorus; ⑧Used for nitrogen fixation; ⑨Used to produce NH3.

7. The use according to claim 6, characterized in that: The phosphorus is inorganic phosphorus.

8. The fermentation method of Bacillus swezii according to claim 1, characterized in that: The fermentation medium is: soluble starch 3.0-7.0 g / L, yeast extract 12.5 g / L, MgSO4 5.0-9.0 g / L; The fermentation conditions were as follows: seed liquid inoculation amount 9%, rotation speed 180-220 r / min, fermentation at 30 °C for 60 h, and initial fermentation pH value of 5.0-7.0; The viable count of Bacillus swezii in the fermentation broth was 4.16 × 10 11 CFU / mL.

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