CN115305222B - Bacillus strain and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of microorganism separation culture, and particularly relates to a bacillus strain, which is named as bacillus belicus (Bacillus velezensis) GNZY01 and is preserved in the Guangdong province microorganism strain preservation center with the preservation number of: GDMCC No:62694. the invention also discloses application of the bacillus strain in biological control of sugarcane root rot. The bacillus strain can antagonize the sugarcane root rot and realize effective control of the sugarcane root rot.
Description
Technical Field
The invention belongs to the technical field of microbial isolation and culture, and particularly relates to a bacillus strain and application thereof.
Background
Sugarcane is an important sugar crop in the south of China, and the sugar yield of the sugarcane is about 90% of the national sugar yield. The sugarcane is a perennial root crop of Gramineae, and the successive year planting in the same land block can cause accumulation of some pathogenic bacteria in plant bodies or soil year by year, so that the diseases are serious, the seed property is degenerated, and serious threat is formed to the yield and the sugar yield of the sugarcane. Sugarcane root rot is a serious soil-borne disease in sugarcane production, which can cause brown or rotted root systems of infected plants, weak growth vigor and poor vigor, and causes large-area reduction. The main pathogens that can cause the occurrence of sugarcane root rot are Jiang Xiong Pythium (Pythium arrhenomane), pachymetra chaunorhiza, wuling ginseng (Xylariasp.) and sesame seed stem blight (Macrophomina phaseolina), fusarium moniliforme and Pythum gramicolase, fusarium sharpleasure (Fusarium communication), xylaria arbuscula.
So far, the means of sugarcane root rot is less in practical production and application. In many cases, chemical agents are used, and the prevention effect is good, but the problems of food safety, ecological environment and the like are brought at the same time, and a series of serious problems of soil microecological unbalance, salinization, acidification and the like occur. But the physical control has slow effect and high cost, and is not suitable for large-scale planting. Therefore, the existing control means are more and more difficult to meet the requirements of healthy development of the sugarcane industry, and it is very important to find ways for effectively controlling the occurrence of sugarcane root rot.
Biological control is safe to people and livestock, is environment-friendly and is not easy to cause drug resistance, and in recent years, a plurality of reports about antagonistic bacteria for controlling plant root rot exist, for example, liao Hailang and other disease inhibition tests of isolated roots show that a large number of antagonistic bacteria exist in healthy coptis root systems, so that coptis root rot can be effectively controlled; xu Shiyang and the like show that 6 microbial inoculum combinations can effectively prevent and treat the capsicum root rot through measuring the nutrient content and the activity of soil enzymes in the root surrounding soil; zhang Manman and the like find that trichoderma asperellum FJ035 (Trichoderma asperellum) separated from rhizosphere soil of cucumbers can effectively prevent and treat root rot of the cucumbers. However, screening for antagonistic bacteria against sugarcane root rot and biological control thereof have been rarely studied.
Disclosure of Invention
The invention aims to solve the technical problems and provides a bacillus strain which can antagonize sugarcane root rot and realize effective control of the sugarcane root rot.
The technical scheme of the invention is as follows:
a bacillus strain designated bacillus belicus (Bacillus velezensis) GNZY01 deposited with the cantonese collection of microorganism strain under accession number: GDMCCNo:62694.
preferably, the bacillus strain of the present invention is derived from sugarcane rhizosphere soil suffering from root rot.
The invention also provides application of the bacillus strain in biological control of sugarcane root rot, and application experiments show that the bacillus belicus (Bacillus velezensis) GNZY01 can antagonize sugarcane root rot and realize effective control of sugarcane root rot.
Preferably, the bacillus strain of the invention has the following specific steps in biological control of sugarcane root rot:
(1) Preparing a sterile fermentation liquid: beating 5 bacillus cakes, inoculating to NB culture medium, culturing at 37deg.C and 180r/min for 24 hr, centrifuging at 8000r/min for 20min, and filtering supernatant with a filter to obtain sterile fermentation liquid;
(2) Preparation of conidium suspensions: inoculating 3 pathogenic bacteria cakes of sugarcane root rot disease of 5mm into 100mL of PDB culture medium, and carrying out shake culture for 5d at 28 ℃ and 130r/min to obtain a conidium suspension;
(3) The sterile fermentation filtrate is respectively and uniformly mixed with the conidium suspension and the PDA culture medium, and is cultured for 3d at 28 ℃ to observe the antibacterial effect.
In the step (3) of the invention, when the sterile fermentation filtrate is mixed with the conidium suspension: mixing uniformly according to the volume ratio of 1:9, 2:8 and 3:7 respectively; when the sterile fermentation filtrate is mixed with PDA culture medium: mixing uniformly according to the volume ratio of 1:9, 2:8 and 3:7 respectively.
The PDA culture medium comprises the following raw materials: 200g of potato, 20g of glucose, 15g of agar powder and 1000mL of distilled water; the PDB culture medium comprises the following raw materials: 200g of potato, 20g of glucose and 1000mL of distilled water.
As another preferred scheme, the application of the bacillus strain disclosed by the invention comprises the following specific steps:
(1) Bacillus bacterial suspension: inoculating bacillus colony into 100ml NB culture medium, placing in a constant temperature shaking table, and shake culturing at 28deg.C and 180r/min for 24 hr to obtain bacillus suspension;
(2) Adding vermiculite into sterile soil according to the weight ratio of 11:1, adding sterile water with the same weight as the sterile soil, stirring uniformly, filling into a PVC pipe, adding bacillus bacterial suspension with the weight of 3.6-4.0% of the sterile soil, implanting the sterilized black skin sugarcane tubers, inoculating sugarcane root rot pathogen after culturing for 7d, and observing effects after inoculating for 45 d.
Preferably, the pathogenic bacteria of sugarcane root rot disease according to the invention are Fusarium shared (Fusarium communication). The bacillus beijerinus (Bacillus velezensis) GNZY01 has remarkable control effect on sugarcane root rot caused by Fusarium sharing.
Preferably, in the step (1), NB medium raw materials are: 10g of peptone, 3g of beef powder, 5g of NaCl and 1000mL of distilled water.
Preferably, the OD of the Bacillus bacterial suspension of the present invention 600 The bacillus suspension with the concentration is 0.5, and has the best control effect on sugarcane root rot.
By adopting the technical scheme, the invention has the beneficial effects that:
1. the bacillus (Bacillus velezensis) GNZY01 obtained by screening has good antibacterial activity on shared fusarium.
2. The bacillus (Bacillus velezensis) GNZY01 can antagonize the sugarcane root rot and realize the effective control of the sugarcane root rot.
Strain preservation description: the bacillus bescens strain (Bacillus velezensis) GNZY01 of the invention was deposited at the collection of microbiological strains (GDMCC) in the cantonese province, 8.11, 2022, at the deposit address: guangzhou city, first, middle road 100, guangdong academy of sciences of microorganisms, accession number GDMCCNo:62694.
drawings
FIG. 1 is a graph showing the effect of Bacillus belicus GNZY01 against shared Fusarium bacteria in example 1 of the present invention.
FIG. 2 is a diagram showing morphological characteristics of Bacillus bailii GNZY01 in example 1 of the present invention.
FIG. 3 is a phylogenetic tree of Bacillus bailii GNZY01 in example 1 of the present invention.
FIG. 4 is a graph showing the inhibitory effect of Bacillus belicus GNZY01 fermentation broth on growth of mycelia of Fusarium shared in example 2 of the present invention.
FIG. 5 is a graph showing the effect of Bacillus bailii GNZY01 fermentation broth in example 2 of the present invention on spore germination inhibition of Fusarium shared.
FIG. 6 is a graph showing the effect of Bacillus belicus GNZY01 on the overground part of sugarcane growth in example 3 of the present invention.
FIG. 7 is a graph showing the effect of Bacillus belicus GNZY01 in example 3 of the present invention on the growth of root system in the lower part of sugarcane growth.
Reference numerals in fig. 4 and 5: CK-1 was 10% by volume, CK-1 was 20% by volume, and CK-3 was 30% by volume.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following examples:
potato dextrose agar medium (Potato Dextrose Agar, PDA) composition: 200g of potato, 20g of glucose, 15g of agar powder and 1000mL of distilled water;
potato dextrose water medium (Potato Dextrose Broth, PDB) composition: 200g of potato, 20g of glucose and 1000mL of distilled water;
beef extract peptone medium (Nutrient Agar, NA): 10g of peptone, 3g of beef powder, 5g of NaCl, 15g of agar and 1000mL of distilled water;
beef extract peptone culture (Nutrient Broth, NB) composition: 10g of peptone, 3g of beef powder, 5g of NaCl and 1000mL of distilled water.
Fusarium sharable (Fusarium comosum) was isolated and stored by the university of Guangxi, university, sugar cane biology key laboratory.
Example 1: acquisition and identification of Bacillus bailii GNZY01
(1) Rhizosphere soil sample collection: dividing the sugarcane into four typical fields according to the severity of the sugarcane with root rot in regions such as Guangzhou southern sand region, qingshun southern village and the like, randomly selecting five sugarcane plants for collecting rhizosphere soil samples by adopting a five-point sampling method in each field, taking the samples back to a laboratory, and storing the samples in a refrigerator at the temperature of minus 20 ℃;
(2) Preparation of an agrobacteria suspension: 10g of a conservation rhizosphere soil sample is taken and placed in a 250ml triangular flask, 90ml of sterile water and a small amount of glass beads are added, shake culture is carried out for 20min at 28 ℃ and 200r/min, and the mixture is placed at room temperature for 20min in a stable environment, thus obtaining soil fungus suspension;
(3) Separating and purifying bacillus belicus GNZY 01: separating Bacillus bailii by plate dilution gradient method, sucking 1mL of soil fungus suspension, adding into a test tube containing 9mL of sterile water, and diluting with another test tube to obtain 10 -1 、10 -2 、10 -3 And 10 -4 4 concentration gradient soil bacterial suspensions, absorbing 20 mul bacterial suspension in each gradient, uniformly coating on an NA flat plate, repeating for five times, culturing for 3d at 28 ℃, observing colony formation, and picking single colonies with different culture characteristics such as morphology, color and the like for purification and preservation;
(4) Bacterial strain primary screening: performing primary screening of bacillus beleiensis by adopting a plate counter method, selecting activated and cultured shared fusarium colonies for 3d, beating a bacterial cake by using a sterile puncher with the diameter of 5mm, placing the bacterial cake in the center of a PDA plate, inoculating the single colony selected in the step (3) to 3 points which are in triangular symmetry at the position 20mm away from the shared fusarium, culturing at the constant temperature of 28 ℃ for 3d, observing the formation of a bacteriostasis ring, and selecting 7 strains with the bacteriostasis ring for re-screening;
(5) Strain re-screening: the shared fusarium cakes are made by adopting the same method as the primary screening in the step (4), placed in the center of a PDA flat plate, 7 strains obtained by the primary screening in the step (4) are accessed at the same point, repeated for five times, the PDA flat plate without inoculating the antibacterial strain is used as a control, the colony diameter (mm) of the shared fusarium is measured after the constant temperature culture for 3d at 28 ℃, and the antibacterial rate is calculated as follows:
(results are shown in Table 1)
By re-screening, antagonistic strain GNZY01 with the highest bacteriostatic rate was obtained, which had the opposite effect on shared fusarium, as shown in fig. 1.
Table 1 7 inhibition effect of strains on Fusarium shared
| Strain numbering | Bacteriostatic rate/% | Diameter/mm of inhibition zone |
| GNZY01 | 72.83a | 18.87±0.87b |
| GNZY02 | 59.98b | 18.96±0.86b |
| GNZY03 | 18.49g | 12.07±0.29d |
| GNZY04 | 24.04f | 15.58±0.49c |
| GNZY05 | 54.75c | 18.66±0.82b |
| GNZY06 | 31.39e | 24.98±2.16a |
| GNZY07 | 46.83d | 19.56±0.68b |
| CK | -- | 41.85.+ -. 0.18 (colony diameter) |
Note that: the different lowercase letters after the same column of data indicate significant differences at P <0.05 levels (Duncan's new complex polar difference method). The following is the same.
The strain GNZY01 is identified by morphological characteristics, physiological and biochemical experiments and 16SrDNA sequence analysis, and the results are as follows:
(1) Morphological features: reference is made to the handbook of the identification of the common bacterial System and the handbook of the identification of Berger's bacteria: the strain GNZY01 is inoculated on a PDA culture medium, and the growth condition is good after the strain is cultured for 24 hours at the temperature of 28 ℃. Gram staining is positive, bacterial cells are short rods, colony morphology is regular round, edges are neat, light yellow and matt, and the bacterial cells are opaque, as shown in figure 2.
(2) Physiological and biochemical characteristics: reference is made to the manual for identification of the specific bacteria Bai Jie and the manual for identification of the common bacterial system: the strain GNZY01 can be grown in 3%, 5%, 7%, 10% NaCl solution using 10 carbon sources (glucose, sucrose, lactose, maltose, fructose, xylose, mannose, inositol, sorbitol, mannitol) tested. At H 2 The results of the S production test were positive, the strain was positive in the starch hydrolysis test, the V-P assay test, the hydrolyzed cellulose test, the indole test, the nitrate test, the methyl red staining test, the contact enzyme test, the milk decomposition test, the citrate test, and the motility test were shown in Table 2.
TABLE 2 physiological and biochemical characteristics of Bacillus strain GNZY01
Note that: "+" and "-" denote positive and negative, respectively.
(3) 16SrDNA gene amplification and sequence analysis:
bacterial 16SrDNA universal Primer was used as Primer27F:5'-AGAGTTTGATCCTGGCTCAG-3' and Primer1492R 5'-TACGGYTACCTTGTTACGACTT-3' PCR amplification was performed on the DNA of strain GNZY01. The total volume of the PCR reaction system was 25. Mu.L: 1. Mu.L of DNA template, 1. Mu.L of primer 27F/1492R each, 12.5. Mu.L of 2 XTaq PCR Master mix, ddH 2 O9.5. Mu.L. PCR reaction conditions: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 50℃for 35s, elongation at 72℃for 45s,35 cycles; extending at 72℃for 10min. The PCR products were detected by 0.8% agarose gel electrophoresis and sent to the engineering (Shanghai) Limited for sequencing, after which BLAST alignment at NCBI was performed after sequencing, sequence analysis was performed using MEGAX software to construct a phylogenetic tree (see FIG. 3).
The sequencing result shows that the sequence length of the strain GNZY01 is 1407bp, and the sequencing result carries out BLAST homology alignment in NCBI. The strain GNZY01 is subjected to BLAST comparison on NCBI by using a 16SrDNA sequence, the similarity between the strain GNZY01 and bacillus (Bacillus velezensis MT 649755.1) is found to be highest, the similarity reaches 82%, more than 90% of similarity is achieved by downloading the sequences of 15 strains of different bacillus species, serratia marcescens (Serratia marcescens) is selected as an outer group strain, and a phylogenetic evolutionary tree is constructed by MEGAX. The strain GNZY01 was found to be on the same branch as bacillus (Bacillus velezensis SRCM 101368).
In summary, the identified strain GNZY01 strain was bacillus belicus (Bacillus velezensis), which was named bacillus belicus GNZY01.
(4) Preservation of Bacillus GNZY01
Based on the above identification results, bacillus belicus GNZY01 was deposited at the Cantonese microorganism strain collection (GDMCC) at 2022, 8 and 11, at the deposit address: guangzhou city, first, middle road 100, guangdong academy of sciences of microorganisms, accession number GDMCCNo:62694.
example 2: test of Bacillus Strain (Bacillus velezensis) GNZY01 fermentation broth against Fusarium shared
(1) Preparing bacillus fermentation liquid: beating 5 bacillus cakes with a sterile puncher (phi=5mm), inoculating to NB medium, culturing at 37deg.C and 180r/min for 24 hr, centrifuging at 8000r/min for 20min, and filtering supernatantFiltering to obtain bacillus fermentation liquor;
(2) Preparation of conidium suspensions: inoculating 3 pathogenic bacteria cakes of sugarcane root rot disease of 5mm into 100mL of PDB culture medium, and carrying out shake culture for 5d at 28 ℃ and 130r/min to obtain a conidium suspension;
(3) Uniformly mixing bacillus fermentation liquor and conidium suspension containing a PDB culture medium according to the volume ratio of 1:9, 2:8 and 3:7 to prepare conidium suspension containing the fermentation liquor, wherein the volume content of the conidium suspension containing the fermentation liquor is 10%, 20% and 30%, inoculating 1 pathogenic bacteria cake of 5mm in the center of a plate containing the fermentation liquor, and inoculating the conidium suspension containing the bacillus fermentation liquor;
(4) Dripping the conidium suspension containing the bacillus fermentation liquor onto a single-sided concave glass slide, taking sterile water as a control to replace the conidium suspension containing the bacillus fermentation liquor, respectively culturing for 3d and 24h at constant temperature of 28 ℃ for 3 times, repeating for 3 times, counting the germination rate of spores under a microscope, measuring the germination rate of spores, and calculating the germination inhibition rate of spores, wherein the calculation formula of the germination inhibition rate of spores is as follows:
the method comprises the steps of carrying out a first treatment on the surface of the (the results are shown in Table 3 and FIG. 5)
(5) Taking out the prepared PDA culture medium after high-temperature high-pressure sterilization, cooling to 50 ℃, mixing and shaking the bacillus fermentation liquor and the PDA culture medium uniformly according to the volume ratio of 1:9, 2:8 and 3:7, immediately pouring the mixture into a flat plate to prepare a PDA flat plate containing 10%, 20% and 30% of fermentation liquor, inoculating 1 pathogenic bacteria cake with the thickness of 5mm in the center of the flat plate containing the bacillus fermentation liquor, taking sterile water to replace the bacillus fermentation liquor as a reference, culturing for 3d at constant temperature of 28 ℃, repeating for 6 times, measuring the colony diameter, and calculating the hypha growth inhibition rate:
. (the results are shown in Table 3 and FIG. 4)
TABLE 3 Effect of fermentation broths of different concentrations on shared Fusarium hypha growth and spore germination
From table 3, the concentration of the fermentation broth of bacillus (Bacillus velezensis) GNZY01 strain had a significant effect on the growth and growth inhibition of hyphae, the germination rate of spores, and the germination inhibition of fusarium shared. Increasing the concentration of bacillus (Bacillus velezensis) GNZY01 strain broth reduced the hyphal growth and spore germination rate of fusarium shared, with average 13.62% and 10.13% decrease in C (20%) treated hyphal growth and spore germination rate and average 33.00% and 39.26% decrease in C (30%) treated hyphal growth and spore germination rate compared to C (10%) treated; increasing the concentration of bacillus (Bacillus velezensis) GNZY01 strain broth increased both the shared fusarium hypha growth inhibition and spore germination inhibition, which increased by 52.83% and 2.78% on average for C (20%) and by 94.43% and 42.49% on average for C (30%) compared to C (10%). In conclusion, the fermentation liquor of the bacillus (Bacillus velezensis) GNZY01 strain has good effect of inhibiting the spore germination rate of shared fusarium. The results show that the inhibition rate of bacillus (Bacillus velezensis) GNZY01 strain fermentation broth on hyphal growth and spore germination of shared fusarium is increased with increasing fermentation broth concentration.
Example 3: bacillus strain (Bacillus velezensis) GNZY01 biocontrol effect test
(1) Bacillus bacterial suspension: inoculating a bacillus GNZY01 colony into 100ml of NB culture medium, placing into a constant-temperature shaking table, and carrying out shaking culture for 24 hours at 28 ℃ and 180r/min to obtain bacillus suspension;
(2) Adding vermiculite into sterile soil at a weight ratio of 11:1, adding sterile water with the same weight as the sterile soil, stirring uniformly, filling into a PVC pipe, and adding 3.8% bacillus bacterial suspension (OD) with the weight of the sterile soil 600 =0.5), black skin sugarcane tubers sterilized with 1% naclo solution were planted, inoculated with shared fusarium after 7d of cultivation, inoculated with 45d (managed in 45d according to conventional management methods) and the sugarcane root system was taken out for scanning and analysis of the results, the effect of bacillus strain (Bacillus velezensis) GNZY01 on the overground part of sugarcane was shown in fig. 6, and the effect on the growth of the root system at the underground part of sugarcane was shown in fig. 7.
The control group (CK) mentioned in fig. 6 and 7 is specifically: adding vermiculite into sterile soil according to the weight ratio of 11:1, adding sterile water with the same weight as the sterile soil, stirring uniformly, filling into a PVC pipe, adding sterile water with the weight of 3.8% of the sterile soil, implanting black skin sugarcane tubers sterilized by using a 1% NaClO solution, culturing for 7d, inoculating shared Fusarium, inoculating for 45d (the management method and the conditions are the same as above), and taking out sugarcane root systems for scanning and analyzing the results. (i.e., without addition of Bacillus suspension)
As can be seen from the results of FIG. 6, the effect of Bacillus (Bacillus velezensis) GNZY01 on the growth of the diseased sugarcane aerial parts is evident. After 45d, the growth vigor of the treated group was significantly better than that of the control group, with the most obvious effect on the sugarcane plant height and leaf area.
As can be seen from the results of FIG. 7, the Bacillus (Bacillus velezensis) GNZY01 has a significant effect on the growth of the root system of the lower part of the diseased sugarcane. From the total root length, the effect of bacillus (Bacillus velezensis) GNZY01 for inhibiting shared fusarium to promote the root length of sugarcane is very remarkable, and the root length (778.16 dm) of sugarcane treated by bacillus (Bacillus velezensis) GNZY01 is remarkably longer than that of diseased plants (36.75)dm). From the total root surface area, the total root surface area after the biocontrol bacteria treatment is increased by 306.61dm 2 The method comprises the steps of carrying out a first treatment on the surface of the From the total root volume, the total root volume of the treatment group is increased by 7.28dm compared with the control group 3 . It is comprehensively considered that after the bacillus (Bacillus velezensis) GNZY01 is used for treating the diseased plant, the length of roots can be increased, the surface area and the volume of the plant can be increased, the resistance and the disease resistance of the plant are enhanced, and the growth and the development of the plant are promoted.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (8)
1. A bacillus strain, characterized in that: the bacillus strain is named as bacillus belicus (Bacillus velezensis) GNZY01, and is deposited in the Guangdong province microorganism strain collection, and the deposit number is: GDMCC No:62694.
2. the use of the bacillus strain of claim 1, wherein: the bacillus belicus GNZY01 strain is applied to biological control of sugarcane root rot caused by shared fusarium.
3. The use of the bacillus strain of claim 2, wherein: the specific steps of biological control of sugarcane root rot caused by shared fusarium by the bacillus belicus GNZY01 strain are as follows:
(1) Preparing a sterile fermentation liquid: beating 5 bacillus bailii GNZY01 cakes, inoculating to NB culture medium, culturing at 37deg.C and 180r/min for 24 hr, centrifuging at 8000r/min for 20min, and filtering supernatant with a filter to obtain sterile fermentation liquid;
(2) Preparation of conidium suspensions: inoculating 3 pathogenic bacteria cakes of sugarcane root rot disease of 5mm into 100mL of PDB culture medium, and carrying out shake culture for 5d at 28 ℃ and 130r/min to obtain a conidium suspension;
(3) The sterile fermentation filtrate is respectively and uniformly mixed with the conidium suspension and the PDA culture medium, and is cultured for 3d at 28 ℃ to observe the antibacterial effect.
4. The use of a bacillus strain according to claim 2, characterized by the specific steps of:
(1) Bacillus bailii GNZY01 bacterial suspension: picking bacillus beleiensis GNZY01 bacterial colony, inoculating the bacillus beleiensis GNZY01 bacterial colony into 100ml of NB culture medium, placing the bacillus beleiensis GNZY01 bacterial colony into a constant temperature shaking table, and carrying out shaking culture for 24 hours at 28 ℃ and 180r/min to obtain bacillus beleiensis GNZY01 bacterial suspension;
(2) Adding vermiculite into sterile soil according to the weight ratio of 11:1, adding sterile water with the same weight as the sterile soil, uniformly stirring, filling into a container, adding bacillus belicus GNZY01 bacterial suspension with the weight of 3.6-4.0% of the sterile soil, implanting the sterilized black skin sugarcane tubers, inoculating sugarcane root rot pathogen after culturing for 7d, and observing effects after inoculating for 45 d.
5. The use of the bacillus strain of claim 3 or 4, wherein: the NB medium comprises the following raw materials: 10g of peptone, 3g of beef powder, 5g of NaCl and 1000mL of distilled water.
6. The use of the bacillus strain of claim 3, wherein: the PDB culture medium comprises the following raw materials: 200g of potato, 20g of glucose and 1000mL of distilled water; the PDA culture medium comprises the following raw materials: 200g of potato, 20g of glucose, 15g of agar powder and 1000mL of distilled water.
7. The use of the bacillus strain of claim 3, wherein: in the step (3), when the sterile fermentation filtrate is mixed with the conidium suspension: mixing uniformly according to the volume ratio of 1:9, 2:8 and 3:7 respectively; when the sterile fermentation filtrate is mixed with PDA culture medium: mixing uniformly according to the volume ratio of 1:9, 2:8 and 3:7 respectively.
8. The use of the bacillus strain according to claim 4, wherein: OD of the Bacillus bailii GNZY01 bacterial suspension 600 0.5.
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