CN111073825A - Bacterium with plant soil-borne disease resistance effect and application thereof - Google Patents
Bacterium with plant soil-borne disease resistance effect and application thereof Download PDFInfo
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Abstract
The invention relates to a bacterium Bacillus velezensis (Bacillus velezensis) which has a probiotic effect on plants and can prevent and control plant soil-borne diseases and a microbial inoculum thereof, belonging to the technical field of biological pesticides. The strain is separated from root soil of three-year-old panax notoginseng, is identified as bacillus belgii, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the preservation number of CGMCC No. 15320. The Bacillus belgii, the strain suspension, the strain fermentation liquor extracting solution or the microbial inoculum containing the strain can promote the seed germination of plants and the growth of the plants to a certain extent, and can also obviously reduce the incidence rate of plant soil-borne diseases, particularly the incidence rate of the plant soil-borne diseases on a continuous cropping ground. The microbial inoculum containing the strain can also be mixed with organic fertilizer to prepare bacterial fertilizer for use, thereby bringing wide application prospect to the biological control field of plant diseases.
Description
The technical field is as follows:
the invention relates to a biocontrol bacterium Bacillus velezensis and application thereof, in particular to a Bacillus velezensis bacterium with a probiotic effect on plant growth and application thereof in preventing and treating plant soil-borne diseases, especially in continuous cropping plant soil-borne diseases, and belongs to the technical field of biological pesticides.
Background art:
in recent 20 years, the conversion from traditional agriculture to modern agriculture is gradually realized in China, and the data published in 2015 by the annual book of Chinese agriculture shows that the planting area of the protected area in China exceeds 380 ten thousand hm2High added value vegetable 2035.26 ten thousand hm2. The planting area of important Chinese medicinal materials such as ginseng and pseudo-ginseng all live at the first position of the world in 2015, and the planting area of the ginseng reaches 0.21 kilohm2The planting area of the pseudo-ginseng reaches 3.02 kilohm2. With the enlargement of the cultivation area of a single crop and the successive year cultivation, pathogenic bacteria and ova in soil are accumulated, destructive soil-borne diseases such as root rot, blight, epidemic diseases, bacterial wilt, root knot nematode and the like occur in successive years, the disease is aggravated year by year, the crop yield and quality are seriously affected, the yield is generally reduced by 20-40%, the serious yield is reduced by more than 60%, even the crop is completely harvested, and the successive cropping obstacle is caused. Plant soil-borne diseases refer to diseases caused by pathogens such as fungi, bacteria, nematodes and viruses that live in the soil with disease residues and attack crops from the roots or stems of the crops under appropriate conditions, and fungal pathogens mainly include: the main bacterial pathogens include pseudomonas and ralstonia solanacearum. Therefore, inhibiting the activity of the pathogens of the rhizosphere system becomes the basis for protecting the root system and preventing and treating soil-borne diseases.
The chemical method is still a main measure for preventing and treating soil-borne diseases adopted in field production at the present stage due to high efficiency and simplicity, and although the chemical method can kill microorganisms in soil in a large amount, the chemical method has very little effect on preventing and treating root rot and blight, and the accompanying problems of pesticide residue, excessive heavy metal and the like cannot be ignored, particularly the root rot which breaks out in continuous cropping soil cannot be prevented and treated. The plant rhizosphere inhabits a large number of microorganism groups closely related to the growth of plants, and the biological control means which reasonably regulates and controls the micro-ecological environment of the rhizosphere, improves the rhizosphere environment of the plants and inhibits the propagation of pathogenic microorganisms has great application potential in the aspects of the safety of crops, particularly medicinal plants, and the sustainability of planting by applying rhizosphere growth-promoting bacteria.
The invention content is as follows:
the invention aims to: aiming at the problems that root rot, rust rot, blight, epidemic disease, bacterial wilt and the like in the existing soil-borne diseases seriously occur, the prevention and treatment measures such as pesticides, crop rotation, chemical treatment of soil and the like are not ideal in effect, and environmental pollution is caused, a Bacillus belius (Bacillus velezensis) strain for efficiently preventing and treating the soil-borne diseases of plants and promoting the growth of the plants and application thereof are provided.
The specific technical scheme is as follows:
the invention provides a Bacillus velezensis bacterium which is identified as Bacillus velezensis through morphological and molecular biological research. Culturing the strain on an LB culture medium for 2 days, wherein the colony is integrally grey white, the diameter is 3-5mm, the edge is in a sawtooth shape, the surface is provided with folds, and no pigment is left; the LB culture medium comprises: 3-8 g of yeast extract powder, 6-20 g of peptone, 3-10 g of sodium chloride and 10-20 g of agar, diluting to a constant volume of 1L of distilled water, keeping the pH natural, and sterilizing at 121 ℃ for 30 min. The invention also provides application of the Bacillus belgii bacterium in promoting plant growth and preventing and controlling soil-borne diseases.
In the application of the Bacillus belgii bacteria in promoting plant growth and preventing and controlling root rot, rust rot, wilt, epidemic disease, bacterial wilt and the like, the Bacillus belgii bacteria are used for root irrigation of plants by using bacterial strain suspension, bacterial strain fermentation liquor extracting solution, solid microbial inoculum or the combination of the four.
The plant can be medicinal materials, vegetables, etc.
The bacterial suspension is prepared by the following method: inoculating the strain into an LB culture medium plate, performing inverted culture at the temperature of 20-40 ℃ for 1-3 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer under the wavelength of 600nm, wherein the concentration of the bacterial suspension is as follows: 0.1-0.8.
The strain fermentation liquor is prepared by the following method: inoculating the strain into an LB solid culture medium plate, performing inverted culture at 20-40 ℃ for 1-3 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer under the wavelength of 600nm, wherein the concentration of the bacterial suspension is as follows: 0.1-0.8; inoculating the bacterial suspension into a fermentation culture medium LB, culturing at 20-40 ℃ for 1-5 days at the speed of 120-.
The strain fermentation liquor extract is prepared by the following method: inoculating the strain into an LB solid culture medium plate, performing inverted culture at 20-40 ℃ for 1-3 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer under the wavelength of 600nm, wherein the concentration of the bacterial suspension is as follows: 0.1-0.8; inoculating the bacterial suspension into a fermentation culture medium LB, culturing at 20-40 ℃ for 1-5 days at the speed of 120-. The obtained fermentation liquor is extracted by using equal volume of ethyl acetate, and concentrated extract liquor is collected.
The bacterial strain solid microbial inoculum is prepared by the following method: inoculating the strain into an LB solid culture medium plate, performing inverted culture at 20-40 ℃ for 1-3 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer under the wavelength of 600nm, wherein the concentration of the bacterial suspension is as follows: 0.1-0.8; inoculating the bacterial suspension into a fermentation culture medium LB, culturing at 20-40 ℃ for 1-5 days at the speed of 120-. Inoculating the fermentation liquor into a solid fermentation culture medium, culturing at 20-40 ℃, stirring under aseptic conditions after culturing for 1-3d, and continuing culturing for 1-3d after stirring to obtain a solid fermentation microbial inoculum; placing the obtained solid microbial inoculum in a drying oven at 30-40 deg.C, drying by blowing for 20-30h until the water content is below 10-20%, and the viable count of the microbial inoculum can reach 1.0-5.0 × 109Packaging above one/g.
The LB solid culture medium is: 3-8 g of yeast extract powder, 6-20 g of peptone, 3-10 g of sodium chloride and 10-20 g of agar, diluting to a constant volume of 1L of distilled water, keeping the pH natural, and sterilizing at 121 ℃ for 30 min; the LB fermentation medium is: 3-8 g of yeast extract powder, 6-20 g of peptone and 3-10 g of sodium chloride, fixing the volume to 1L of distilled water, keeping the pH natural, and sterilizing at 121 ℃ for 30 min; the solid fermentation medium comprises: 350g of wheat bran 300-350g, 50-100g of corn flour, 5-10g of bean cake powder and KNO30.5g，K2HPO41.5g，NaCl 1.5g，CaCO34g, adding 400ml of water, and sterilizing at 121 ℃ for 30 min.
The application of the biocontrol bacterium Bacillus belezii bacteria in promoting plant growth and preventing and controlling root rot, rust rot, blight, epidemic disease, bacterial wilt and the like comprises the following steps:
can be used in the seed stage of plants, and the concentration of the bacterial suspension is diluted by using an ultraviolet spectrophotometer under the wavelength of 600nm as follows: 0.1-0.8, then 10 of dilution2-104Double, 1L of the dilution soaks 1 kg of seeds.
The method can be used for irrigating roots in the plant growing period or the seedling period, and an ultraviolet spectrophotometer is used for diluting the concentration of bacterial suspension under the wavelength of 600nm as follows: 0.1-0.8, 10 dilution2-10450ml of the extract was used per plant. The application is continued for 2-3 times, each time at intervals of 30-90 days. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
Can be used by irrigating roots in plant growing period or seedling period, and diluting fermentation liquid or fermentation extract 102-104For double application, 50ml of each strain was used. The application is continued for 2-3 times, each time at intervals of 30-90 days. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
The solid microbial inoculum can be directly spread in the plant growing period or the seedling period, the solid microbial inoculum is spread on a plant seedbed, the dosage of each mu is 10-40kg, or the microbial inoculum is uniformly mixed by fine sand and then is scattered at the plant rhizosphere. The application is continued for 2-3 times, each time at intervals of 30-90 days. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
The invention has the beneficial effects that:
the screened biocontrol bacterium Bacillus velezensis strain suspension or strain fermentation liquor or fermentation liquor extracting solution or solid microbial inoculum can obviously inhibit the growth of soil-borne disease pathogenic bacteria Fusarium solani, Cylindrocarpon, Alternaria alternata, malignant mildew, Ralstonia solani and the like and promote the growth of plants. Because the biological agent is adopted, a series of problems caused by the use of chemical pesticides do not exist, and the biological agent is beneficial to reducing agricultural pollution and effectively preventing and treating root rot, rust rot, blight, epidemic disease, bacterial wilt and the like. The biocontrol bacterium Bacillus beleisi is separated from soil, is harmoniously compatible with soil ecology, and is beneficial to fully exerting the advantages of strains.
The biocontrol bacterial strain has simple culture conditions, stable heredity, easy industrial production expansion and good development and application prospects.
The Bacillus velezensis of the invention is preserved in China general microbiological culture Collection center (CGMCC), and the address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, the preservation time: 29 months 01 and 2018, and the preservation number is CGMCC No. 15320.
Description of the drawings:
FIG. 1 shows colony morphology of Bacillus velezensis (Bacillus velezensis) bacteria.
FIG. 2 shows the disease prevention effect of Bacillus velezensis fermentation broth on Panax notoginseng.
The specific implementation mode is as follows:
for a better understanding of the present invention, the following figures and examples further illustrate, but are not to be construed as limiting, the experimental procedures set forth in the following examples are conventional and are not intended to be limiting.
Example 1 acquisition of Bacillus belgii (Bacillus velezensis) CGMCC No. 15320:
collecting rhizosphere soil of three-year-old radix Notoginseng from Yunnan province, removing stones from the soil sample, weighing 5g, placing into a triangular flask containing 45mL sterile water, and oscillating for 20min to obtain 10-1The soil suspension of (a); diluting to 10 degrees in stages-2、10-3、10-4、10-5Then, each diluent is taken and coated on a separation plate, inverted and cultured for 1-5 days at 26 ℃. And culturing and observing, and transferring the single colony to an LB culture medium for culturing to obtain the strain CGMCC No. 15320.
Example 2 identification of Bacillus velezensis (Bacillus velezensis) CGMCC No.15320
1 morphological feature Observation
The strain Bacillus velezensis CGMCC No.15320 cultured for 1-5 days is opened to the flat dish cover and photographed under a black background. The colonies were pale white, with wrinkles on the surface and no pigment produced, and the outermost periphery was pale white (FIG. 1).
216S rRNA sequence analysis
After extracting the genomic DNA of the strain by a liquid nitrogen grinding method, adopting a primer: 16s F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; 16s R: 5'-AAGGAGGTGATCCAGCCGCA-3', PCR amplification was performed. After gel electrophoresis, the gel is sent to bioengineering (Shanghai) Limited company for sequencing, and the total length of the sequence is 1524bp (shown as SEQ). The obtained sequence is submitted to a GenBank database for BLAST analysis and comparison, and the strain with higher homology with CGMCC No.15320 belongs to Bacillusvelezensis, and the strain serial number MH371021 is submitted to NCBI.
Example 3 promoting action of Bacillus velezensis suspension on seed germination of Panax notoginseng, cucumber, pepper and corn
Inoculating the strain into an LB culture medium plate, performing inverted culture at 26-30 ℃ for 1-5 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and diluting the concentration of the bacterial suspension by using an ultraviolet spectrophotometer under the wavelength of 600nm to obtain: 0.1-0.8, diluting 100 times for later use. Distilled water was used as a control, and each group was replicated three times, each for 100 seeds, and each group was soaked with 50mL of the bacterial suspension diluent for 1-5 h. Filtering with a screen to obtain seeds, performing moisture-preserving culture at 20-25 deg.C for 20 days, and counting the germination rate of Notoginseng radix seeds; performing moisture-preserving culture at 20-25 ℃ for 24 hours, and counting the germination rate of cucumber seeds; and (3) carrying out moisture-preserving culture at 20-25 ℃ for 5 days, carrying out statistics on germination rates of the pepper and corn seeds, wherein the results are shown in table 1, and the results show that the germination rates of the seeds treated by using the Bacillus belgii are higher than those of the control group, and have significant differences, which indicates that the bacterial suspension has a promoting effect on plant seed germination.
TABLE 1 influence of Bacillus belgii suspension on the germination rate (%) of plant seeds
Note: p < 0.05; p <0.001.
Example 4 growth promoting action of Bacillus velezensis fermentation broth on Panax notoginseng, cucumber, pepper and corn
Inoculating the strain into an LB culture medium plate, performing inverted culture at 26-30 ℃ for 1-5 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer under the wavelength of 600nm, wherein the concentration of the bacterial suspension is as follows: 0.1-0.8; inoculating the bacterial suspension into a fermentation culture medium LB, culturing at 26-30 ℃ for 1-5 days at 120-200r/min, and diluting the fermentation liquor by 100 times for later use. Distilled water was used as a control, three replicates each were inoculated with 100 plants, and 50mL of roots were irrigated. Fertilizing time: and (5) 5d after the seedlings of the biennial pseudo-ginseng, the cucumber, the pepper and the corn emerge. Respectively collecting plant samples 30 days after fungus irrigation for whole plant freshness quality and root system enzyme activity determination. The root activity determination method is triphenyltetrazolium chloride (TTC) method, and refers to Zhangyilian 'plant physiology experiment guidance third edition'.
According to the measurement indexes, the Graphpad software is used for performing significance difference analysis, the results are shown in table 2, the results show that the strain Bacillus velezensis fermentation liquor can significantly improve the biomass of plants such as pseudo-ginseng, cucumber, pepper and corn, and the results in table 3 show that the strain Bacillus velezensis fermentation liquor can significantly improve the root activity of the plants, plays a role in promoting the nutrient absorption of the plants and has a growth promoting effect on the plants.
TABLE 2 Bacillus belgii fermentation broth promoting the plant seedling biomass (g/strain)
Note: p < 0.05; p <0.01.
TABLE 3 enhancement of the plant shoot root vigor [ μ g/(g.h) ] by B.belgii fermentation broths
Note: p < 0.05; p <0.01.
Example 5 disease prevention Effect of Bacillus velezensis fermentation broth on Panax notoginseng, Panax ginseng, Cucumis sativus, Capsicum annuum and corn
The fermentation broth was prepared as in example 4, with distilled water as a control, and each group was replicated three times, each was inoculated with 100 seedlings, and 50mL of each seedling was rooted. And (4) respectively supplementing the microbial inoculum for 45 days after the cucumber, the pepper and the corn, and measuring the seedling survival rate of the plants after root irrigation for 90 days.
Compared with a control group (CK group), the fermentation liquor group of Bacillus velezensis bacteria (Bacillus velezensis) has fewer pathogenic plants and higher seedling survival rate, and the control group is shown in Table 4 and is found to have the following diseases under the condition of no application: pseudo-ginseng root rot, pseudo-ginseng blight, ginseng rust rot, cucumber fusarium wilt, cucumber bacterial wilt, pepper fusarium wilt, pepper bacterial wilt and corn root rot, and the healthy seedling survival rate of a Bacillus subtilis (Bacillus velezensis) fermentation broth group is high, so that significant differences exist, and the Bacillus subtilis can effectively prevent pseudo-ginseng root rot, pseudo-ginseng blight, ginseng rust rot, cucumber fusarium wilt, cucumber bacterial wilt, pepper fusarium wilt, pepper bacterial wilt, corn root rot and the like.
TABLE 4 Bacillus beilaisi fermentation broth for plant disease prevention effect (% seedling stock)
Note: p < 0.05; p <0.01.
Example 6 disease prevention Effect of Bacillus velezensis (Bacillus velezensis) solid microbial Agents on continuous cropping of Panax notoginseng
The preparation method of the fermentation liquid is the same as that in the embodiment 4, the fermentation liquid is inoculated into a solid fermentation culture medium, cultured at the temperature of 20-40 ℃, stirred under the aseptic condition after being cultured for 1-3 days, and continuously cultured for 1-3 days after being stirred to obtain the solid fermentation microbial inoculum; and (3) placing the obtained solid microbial inoculum in a drying oven at the temperature of 30-40 ℃, drying for 20-30h by blowing, and packaging. Antagonistic plants spaced for three years are selected to treat the continuous cropping soil of the panax notoginseng, solid microbial inoculum is applied in the seedling transplanting period, clear water is used as a control, the application amount is 0.5-2.0 g/plant, each group is repeated three times, 400 panax notoginseng seedlings are inoculated repeatedly, microbial inoculum addition is carried out after 3 months, 6 months and 12 months respectively, the addition amount is the same as above, the occurrence condition of panax notoginseng root rot is observed after 15 months, the occurrence rate of the root rot of the control group is 76%, the occurrence rate of the root rot of the treatment group is 27%, the result shows that the occurrence rate of the root rot of the treatment group is reduced by 49% compared with that of the control group, and a good disease prevention effect is shown on the continuous cropping soil, which is shown in figure 2.
Example 7 Bacillus belgii bacteria (Bacillus velezensis) fermentation broth ethyl acetate extract bacteriostasis test
The Bacillus velezensis fermentation liquor ethyl acetate extracting solution is used for carrying out antagonism tests on panax notoginseng root rot pathogenic bacteria (Fusarium solani), panax notoginseng blight pathogenic bacteria (Phytophthora cactorum), ginseng rust rot pathogenic bacteria (cyclicarbon destructor), cucumber Fusarium oxysporum pathogenic bacteria (Fusarium oxysporum), cucumber bacterial wilt pathogenic bacteria (Ralstonia solanaceum), pepper bacterial wilt pathogenic bacteria (Fusarium oxysporum), pepper bacterial wilt pathogenic bacteria (Ralstonia solanaceum) and corn root rot pathogenic bacteria (Fusarium moniliforme) by adopting a 96-pore plate turbidimetry. The final concentration of the fermentation liquid extract is 0.5 mg/ml. Washing spores from slant to obtain spore suspension, counting with blood count plate, and diluting to spore concentration of 107cfu/ml, sucking 0.5ml of the diluted fungal spore suspension, adding into 50ml of liquid PDB culture medium, and uniformly mixing to obtain a fungal suspension; bacteria prepared the thallus into OD with sterile water600Taking 0.3 bacterial suspension, sucking 0.5ml of the diluted bacterial suspension, adding the bacterial suspension into 50ml of liquid LB culture medium, and uniformly mixing to obtain bacterial suspension. Blank Control (CK): 198. mu.l suspension + 2. mu.l sterile water; DMSO control group: 198. mu.l suspension + 2. mu.l DMSO; positive control group: 198. mu.l suspension + 2. mu. l G418 or 198. mu.l suspension + 2. mu.l streptomycin sulfate; experimental groups: 198. mu.l suspension + 2. mu.l broth Extract (Extract), three replicates per group. And (3) culturing the 96-well plate in an incubator at 28 ℃, measuring the OD value at 600nm after 48 hours of fungi, and measuring the OD value at 600nm after 24 hours of bacteria. As a result, the fermentation broth extract had an inhibition ratio of 66.41. + -. 0.56% for Fusarium solani, an inhibition ratio of 58.38. + -. 0.61% for Cylindrocarpon destructans, an inhibition ratio of 53.12. + -. 0.72% for Phytophtora cactorum, an inhibition ratio of 73.62. + -. 0.62% for Fusarium oxysporum, and an inhibition ratio of 73.62. + -. 0.62% for Ralstonia solanacearumThe inhibition rate of the fusarium moniliforme is 80.44 +/-0.37%, and the inhibition rate of the fusarium moniliforme is 54.37 +/-0.71%, and the inhibition rate is significantly different from that of a blank control group. The activity screening experiment result shows that the fermentation liquor of the strain Bacillus velezensis has better antagonistic action on pathogenic bacteria of panax notoginseng root rot, pathogenic bacteria of panax notoginseng blight, pathogenic bacteria of ginseng rust rot, pathogenic bacteria of cucumber wilt, pathogenic bacteria of cucumber bacterial wilt, pathogenic bacteria of pepper bacterial wilt and pathogenic bacteria of corn root rot.
The PDB culture medium is as follows: 100 g-200 g of potatoes and 5 g-20 g of glucose, diluting the mixture to 1L of distilled water with a constant volume, naturally adjusting the pH value, and sterilizing the mixture for 30min at 121 ℃.
Claims (10)
1. Bacillus velezensis (Bacillus velezensis) characterized by the deposit number: CGMCC No. 15320.
2. Use of the bacillus belius (bacillus velezensis) according to claim 1 for controlling soil-borne diseases in plants.
3. Use of Bacillus velezensis according to claim 1 for controlling soil-borne diseases in the continuous cropping of plants.
4. The use of claim 2 or 3, wherein the soil-borne disease is root rot, rust rot, wilt, epidemic disease, bacterial wilt.
5. Use of Bacillus velezensis according to claim 1 for promoting plant growth or seed germination.
6. The use according to any one of claims 2 to 5, wherein the plant is treated by root drenching or broadcasting using a suspension or fermentation broth of a strain of Bacillus belgii or an extract of the fermentation broth of the strain or a microbial preparation containing the strain.
7. The use according to claim 6, wherein the suspension of the strain is prepared by: inoculating the Bacillus belgii strain into an LB solid culture medium plate, performing inverted culture at the temperature of 20-40 ℃ for 1-3 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer at the wavelength of 600nm, wherein the concentration of the bacterial suspension is as follows: 0.1-0.8.
8. The use according to claim 5, wherein the strain broth is prepared by: inoculating the strain into an LB solid culture medium plate, performing inverted culture at 20-40 ℃ for 1-3 days, activating the strain, preparing the activated strain into a bacterial suspension by using distilled water, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer under the wavelength of 600nm, wherein the concentration of the bacterial suspension is as follows: 0.1-0.8; inoculating the bacterial suspension into LB fermentation medium, culturing at 20-40 deg.C and 120-9More than one per ml; and extracting the obtained strain fermentation liquor by using equal volume of ethyl acetate, and collecting concentrated extract liquor to obtain a strain fermentation liquor extracting solution for later use.
9. The use of claim 5, wherein the solid microbial inoculum containing the strain is prepared by the following method: inoculating the strain fermentation broth obtained in claim 7 into a solid fermentation culture medium, culturing at 20-40 ℃, stirring under aseptic conditions after culturing for 1-3d, and continuing culturing for 1-3d after stirring to obtain the solid fermentation inoculant.
10. The use according to claim 7, 8 or 9, wherein the LB solid medium is: 3-8 g of yeast extract powder, 6-20 g of peptone, 3-10 g of sodium chloride and 10-20 g of agar, diluting to a constant volume of 1L of distilled water, keeping the pH natural, and sterilizing at 121 ℃ for 30 min; the LB fermentation medium is: 3-8 g of yeast extract powder, 6-20 g of peptone and 3-10 g of sodium chloride, fixing the volume to 1L of distilled water, keeping the pH natural, and sterilizing at 121 ℃ for 30 min; the solid fermentation medium comprises: 350g of wheat bran 300-350g, 50-100g of corn flour, 5-10g of bean cake powder and KNO30.5g，K2HPO41.5g，NaCl 1.5g，CaCO34g, adding 400ml of water, and sterilizing at 121 ℃ for 30 min.
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| CN111690572A (en) * | 2020-07-15 | 2020-09-22 | 西北农林科技大学 | Bacillus belgii and application and preparation thereof |
| CN111807898A (en) * | 2020-05-15 | 2020-10-23 | 云南省微生物发酵工程研究中心有限公司 | Bio-organic fertilizer with function of preventing and treating tobacco soil-borne diseases and preparation method thereof |
| CN112342173A (en) * | 2020-12-02 | 2021-02-09 | 中国农业科学院特产研究所 | Bacillus belgii and application thereof |
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| CN113712047A (en) * | 2021-09-09 | 2021-11-30 | 兰州市大行农业废弃物处理有限公司 | Disease-preventing growth-promoting microbial agent special for Lanzhou lily and preparation method thereof |
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