Bacillus subtilis, biocontrol inoculant prepared from bacillus subtilis and application of biocontrol inoculant

Abstract

The invention relates to bacillus subtilis, a biocontrol inoculant prepared from the bacillus subtilis and application of the biocontrol inoculant, and belongs to the technical field of biological inoculants. The bacillus provided by the invention is bacillus subtilis and is named as KC2, the preservation unit is China General Microbiological Culture Collection Center, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the number of the preservation center is CGMCC No.31372, and the preservation time is July 19, 2024. The bacillus KC2 disclosed by the invention has a certain bacteriostatic effect on different pathogenic bacteria regardless of living bacteria, supernate or cell lysis solution. The bacillus KC2 is high in living ability and wide in application range of nutrient sources, temperature, humidity and pH value. The compound has no pathogenicity to fruits and vegetables, has good tolerance to broad-spectrum bactericides, and has a great application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological microbial agents, and specifically relates to a bacillus subtilis, a biocontrol agent made from the bacillus, and applications of the biocontrol agent in antibacterial and antibacterial preservation of fruits and vegetables. Background Art

[0002] Fruits and vegetables are easily attacked by soil-borne pathogens during the planting process, and are often threatened by bacterial and fungal infections, especially fungal diseases, with common symptoms including necrosis (leaf spots, leaf blight), rot (root rot, fruit rot) and wilting (root, stem base, vascular tissue invasion), etc. Traditionally, most methods of disease prevention and control are chemical fungicides, breeding of disease-resistant varieties, intercropping, grafting, etc., but these prevention and control measures have their own advantages and disadvantages.

[0003] On the other hand, fresh fruits and vegetables will suffer from different degrees of infectious diseases (pathogenic microorganism infection) due to changes in their physiological and biochemical characteristics after harvesting and during long-term storage. Post-harvest infectious diseases of fruits and vegetables are caused by pathogenic microorganisms infecting hosts during the growth and development period of fruits and vegetables in the field. In addition, cross-infection during transportation and storage is also a cause of infectious diseases. The presence of pores between the epidermal tissues of most fruits and vegetables and wounds caused by improper transportation facilitates pathogenic microorganisms to invade the internal flesh tissue through the epidermis of fruits and vegetables, aggravating the degree of post-harvest infectious diseases of fruits and vegetables. At present, the main method for controlling post-harvest infectious diseases of fruits and vegetables is to spray pesticides before harvesting and lower the storage temperature after harvesting, but excessive use of pesticides will cause serious excessive pesticide residues, endangering people's health, and long-term excessive use of pesticides will cause pathogenic microorganisms to develop drug resistance, greatly reducing the effect of disease prevention and control.

[0004] Biological control is a technology that uses living biocontrol bacteria and their metabolically active substances to control the occurrence of diseases. Biocontrol bacteria can colonize and grow in crop plants and rhizosphere, forming a biological barrier to protect crops from pathogens. Their metabolically active substances can inhibit and kill pathogenic fungi on the one hand, and induce plants to improve disease resistance on the other.

[0005] Biological control methods have more advantages than chemical control methods. In addition to the ease of production and use of biological agents, mixed bacterial agents made from biocontrol bacteria and their metabolites are more environmentally friendly, pollution-free and pesticide-free, and meet the strategic requirements of my country's sustainable development. Moreover, biocontrol agents are less sensitive to environmental changes and have stable effects. In addition, the nutrients decomposed and transformed by biocontrol bacteria can be used by crops, thereby improving crop quality and increasing yields.

[0006] However, although the currently developed biocontrol strains and biocontrol agents have shown certain effects, and some have broad-spectrum antibacterial properties, the antibacterial rates of existing Bacillus biocontrol agents are generally low, especially the types and antibacterial rates of Bacillus subtilis, which still need to be improved. Moreover, for fruit and vegetable pathogens such as Actinomucor elegans, Aspergillus pseudoglaucus, Botrytis sp., and Colletotrichum coffeanum, there are currently few biocontrol strains found, and their antibacterial rates are not high. In addition, for the two pathogens Dimorphosporafolicola and Galactomyces candidus, no reports of biocontrol strains with antibacterial effects have been found.

[0007] Therefore, there is a need to provide a Bacillus subtilis biocontrol strain that has a broader spectrum of antibacterial effects and a very significant inhibition rate against fruit and vegetable pathogens such as Actinomucor elegans, Aspergillus pseudoglaucus, Botrytis sp., and Colletotrichum coffeanum, while also showing an inhibition effect against two pathogens, Dimorphosporafolicola and Galactomyces candidus. Summary of the invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a biocontrol strain that can be used for disease control and bacteriostasis and preservation of fruits and vegetables. The present invention provides a Bacillus subtilis strain that can be used for disease control and bacteriostasis and preservation of fruits and vegetables with high efficiency, especially for Actinomucor elegans, Aspergillus pseudoglaucus, Botrytis sp., Colletotrichum coffeanum and other fruit and vegetable pathogens. The bacteriostasis rate is extremely significant, and the two pathogens Dimorphosporafolicola and Galactomyces candidus are also shown to have an antibacterial effect. The present invention also provides a biocontrol agent made from the Bacillus and its application in antibacterial and bacteriostasis preservation of fruits and vegetables.

[0009] After a lot of experimental research and exploration, the inventors of the present invention obtained a strain of Bacillus subtilis, which is Bacillus subtilis, named KC2, and the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation center number is: CGMCC No.31372, and the preservation time is: July 19, 2024.

[0010] The gene sequence of the above-mentioned Bacillus provided by the present invention is shown in SEQ NO.1.

[0011] The present invention further provides a biocontrol agent comprising the above-mentioned Bacillus.

[0012] The present invention further provides application of the biocontrol agent in the antibacterial treatment of fruit and vegetable pathogens and application of the bacillus in the preparation of a fruit and vegetable preservative.

[0013] Specifically, the fruit and vegetable pathogenic bacteria include Acremonium sclerotigenum, Actinomucorelegans, Aspergillus pseudoglaucus, Botrytis sp., Colletotrichum coffeanum, Clathrospora diplospora, Cladosporium tenuissimum, Bjerkandera adusta, Dimorphosporafolicola, Colletotrichum acutatum, Alternaria brassicae, Aspergillus sp., Talaromyces sp., Aspergillus aculeatus, Colletotrichumfioriniae, Diaporthe sp., Penicillium crustosum, Alternaria alternata, Curvularia lunata, Fusarium decemcellulare, Diaporthe pseudophoenicicola, Aspergilus niger, Fusarium falciforme, Monilinia fructicola, Aspergillus flavus, Galactomycescandidus, Fusarium solani, Aspergillus tubingensis, Stagonosporopsiscucurbtacearum, Fusariumproliferatum, Cladosporium colombiae, Colletotrichumgloeosporioides, Fusarium circinatum and Fusariumincarnatum.

[0014] The concentration of Bacillus KC2 in the biocontrol agent of the present invention is 1×10 6-8 cfu / mL.

[0015] The beneficial effects of the present invention are:

[0016] (1) The Bacillus KC2 living cells of the present invention have certain antibacterial effects on different pathogens. Among them, the antibacterial effect of the Bacillus KC2 living cells on Acremonium sclerotigenum, Actinomucor elegans, Aspergillus pseudoglaucus, Botrytis sp., Colletotrichum coffeanum, Clathrosporadiplospora and Cladosporium tenuissimum is the most significant, with an antibacterial rate of 100%, which can completely inhibit the growth of pathogenic fungi. Among them, the inhibition rate of KC2 viable bacteria on Bjerkandera adusta, Dimorphosporafolicola, Colletotrichum acutatum, Alternaria brassicae, Aspergillus sp., Talaromyces sp., Aspergillus aculeatus, Colletotrichumfioriniae, Diaporthe sp., Penicilliumcrustosum, Alternaria alternata, Curvularia lunata, Fusarium decemcellulare and Diaporthepseudophoenicicola was greater than 90%, showing a good inhibitory effect; the antibacterial effect on Aspergilusniger, Fusarium falciforme, Monilinia fructicola, Aspergillus flavus, Galactomyces candidus, Fusarium solani, Aspergillus tubingensis, Stagonosporopsis cucurbtacearum and Fusariumproliferatum was average, with an inhibition rate of greater than 80%.

[0017] (2) The supernatant of Bacillus KC2 of the present invention has a certain antibacterial effect on different pathogenic fungi. Among them, the supernatant of Bacillus KC2, Acremonium sclerotigenum, Actinomucorelegans, Aspergilluspseudoglaucus, Botrytis sp., Colletotrichum coffeanum, Clathrosporadiplospora, Cladosporium tenuissimum, Bjerkandera adusta, Dimorphosporafolicola, Colletotrichum acutatum, Alternaria brassicae, Aspergillus sp., Talaromyces p., Aspergillus aculeatus, Colletotrichumfioriniae and Diaporthe sp. have better antibacterial effects, and the antibacterial rates are all greater than 90%. The supernatant of Bacillus KC2 had a general antibacterial effect on Penicillium crustosum, Alternaria alternata, Curvularia lunata, Fusarium decemcellulare, Diaporthepseudophoenicicola, Aspergilus niger, Fusariumfalciforme, Moniliniafructicola, Aspergillus flavus, Galactomyces candidus, Fusarium solani, Aspergillustubingensis, Stagonosporopsis cucurbtacearum and Fusariumproliferatum, with the inhibition rates all greater than 80%.

[0018] (3) The Bacillus KC2 cell lysate of the present invention has a certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the KC2 strain supernatant. Among them, the Bacillus KC2 cell lysate has a good antibacterial effect on Acremoniumsclerotigenum, Actinomucor elegans, Aspergilluspseudoglaucus, Botrytis sp., Colletotrichum coffeanum, Clathrospora diplospora, Cladosporium tenuissimum, Bjerkandera adusta, Dimorphospora folicola, Colletotrichum acutatum, Alternariabrassicae, Aspergillus sp. and Talaromyces sp., and the antibacterial rate is greater than 90%. The KC2 cell lysate had a general antibacterial effect on Aspergillus aculeatus, Colletotrichum fioriniae, Diaporthe sp., Penicillium crustosum, Alternaria alternata, Curvularia lunata, Fusariumdecemcellulare, Diaporthepseudophoenicicola, Aspergilus niger, Fusariumfalciforme, Monilinia fructicola, Aspergillus flavus, Galactomyces candidus, Fusarium solani and Aspergillus tubingensis, with the inhibition rates all greater than 80%.

[0019] (4) The Bacillus KC2 provided by the present invention has a wide antibacterial spectrum, involving 34 fruit and vegetable pathogens. Although the prior art (CN114369556B-a strain of Bacillus, a biocontrol agent prepared from the Bacillus and its application) also provides a strain of Bacillus biocontrol strain, it only involves 32 fruit and vegetable pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the inhibition rate of KC2 live bacteria against different pathogens;

[0021] Figure 2 The turbidity of suspension of Bacillus KC2 cultured at different temperatures;

[0022] Figure 3 The turbidity of suspension of Bacillus KC2 cultured at different pH values. DETAILED DESCRIPTION

[0023] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0024] Example

[0025] 1. Source of strains

[0026] In 2023, healthy peach leaves were collected from the peach planting base in Shijia Town, Jianyang City, Chengdu City, Sichuan Province, placed in sterile sealed bags, and brought back to the Institute of Agricultural Products of Chengdu Academy of Agricultural and Forestry Sciences to isolate and purify antagonistic bacteria.

[0027] 2. Identification of strains

[0028] The DNA of the bacteria was extracted, and the 16S rDNA sequence was analyzed by PCR. The gene sequence is shown in the sequence table SEQ NO.1. The obtained sequence was compared on the NCBI website and determined to be Bacillus subtilis, and it was named Bacillus KC2. The depository unit is: General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit center number is: CGMCC No. 31372, and the deposit time is: July 19, 2024.

[0029] 3. Antibacterial test of strains

[0030] (I) Antibacterial test of live bacteria of Bacillus KC2

[0031] The strain has rich antibacterial types and has a good preventive and therapeutic effect on pathogens on strawberries, blueberries, grapes, apples, mangoes, cherries, kiwis, winter dates, peppers, wax apples, eggplants, garlic, and loofahs.

[0032] 1. Experimental Design

[0033] A single colony of the KC2 strain was inoculated into LB broth and cultured at 28 °C with constant shaking at 150 rpm for 24 h. The cell suspension density was determined as 1 × 10 8 cfu / mL. Add KC2 strain cell suspension to PDA medium, shake and mix to make plates. The cell density of KC2 strain on each plate is 1×10 6cfu / mL. Use a 6mm sterile puncher to take the Botrytis cinerea cake and place it in the center of the PDA plate containing the KC2 strain. Use the PDA plate containing 100mg / L carbendazim as the pesticide control, and the PDA plate medium without KC2 bacteria as the blank control. Each treatment is repeated 3 times. Seal the plate with the cake with a ziplock bag and place it in a 26℃ constant temperature incubator for culture. When the mycelium of the control plate is fully grown, measure the width of the inhibition zone.

[0034] Calculate the inhibition rate R of mycelial growth:

[0035] R(%)=(R1-R2) / R1×100%

[0036] Where R is the percentage of inhibition of radial hyphae growth, R1 is the hyphae growth of the blank control, and R2 is the hyphae growth of the treated group.

[0037] 2. Test results

[0038] The test results are shown in Table 1 and Figure 1 As shown. Figure 1As shown in Table 1, Bacillus KC2 live bacteria have certain antibacterial effects on different pathogens. KC2 live bacteria have the most significant antibacterial effect on Acremonium sclerotigenum, Actinomucor elegans, Aspergillus pseudoglaucus, Botrytis sp., Colletotrichumcoffeanum, Clathrosporadiplospora and Cladosporium tenuissimum, with an inhibition rate of 100%, which can completely inhibit the growth of pathogenic fungi. Among them, the inhibition rate of KC2 viable bacteria against Bjerkandera adusta, Dimorphosporafolicola, Colletotrichum acutatum, Alternaria brassicae, Aspergillus sp., Talaromyces sp., Aspergillus aculeatus, Colletotrichum fioriniae, Diaporthe sp., Penicilliumcrustosum, Alternaria alternata, Curvularia lunata, Fusarium decemcellulare and Diaporthepseudophoenicicola was greater than 90%, showing a good inhibitory effect; it had a certain antibacterial effect on Aspergilusniger, Fusariumfalciforme, Monilinia fructicola, Aspergillusflavus, Galactomycescandidus, Fusarium solani, Aspergillus tubingensis, Stagonosporopsiscucurbtacearum and Fusariumproliferatum, with an inhibition rate of greater than 80%.

[0039] Compared with the antibacterial rate of 100 mg / L carbendazim, KC2 viable bacteria were effective against Acremonium sclerotigenum, Actinomucor elegans, Alternaria brassicae, Aspergillus aculeatus, Aspergillus flavus, Aspergillus pseudoglaucus, Aspergillus sp., Aspergillus tubingensis, Aspergilus niger, Bjerkandera adusta, Botrytis sp., Cladosporiumcolombiae, Cladosporium tenuissimum, Clathrospora diplospora, Colletotrichumacutatum, Colletotrichumcoffeanum, Curvularia lunata, Dimorphosporafolicola, Fusariumproliferatum, Galactomyces candidus, Monilinia fructicola, Penicilliumcrustosum, Stagonosporopsis cucurbtacearum and Talaromyces sp., and the inhibition rates were significantly higher than those of 100 mg / L carbendazim treatment. After 100 mg / L carbendazim treatment, the antibacterial effect on Alternaria alternata, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthe pseudophoenicicola, Fusarium circinatum, Fusarium decemcellulare, Fusarium falciforme, Fusarium incarnatum and Fusarium solani was better, and the inhibition rates were higher than those of Bacillus KC2 live bacteria treatment. KC2 live bacteria and 100 mg / L carbendazim had similar antibacterial effects on Diaporthe sp.

[0040] Table 1 Antibacterial effects of live Bacillus KC2 cells and carbendazim on different pathogens

[0041]

[0042] (II) Antibacterial test of supernatant of Bacillus KC2

[0043] The supernatant antibacterial test mainly tests the antibacterial effect of extracellular antibacterial substances. This method mainly excludes nutritional competition factors and is an important indicator for evaluating the biocontrol effect of biocontrol bacteria.

[0044] 1. Experimental Design

[0045] After culturing for 24 hours, the live cells of Bacillus KC2 were removed by repeated centrifugation filtration to prepare KC2 supernatant. 200 μL of supernatant was evenly spread on the PDA plate, and different pathogen cakes were placed in the center of the PDA plate surface using a 6mm sterile puncher. An equal amount of sterile water was used as a control, and each treatment was repeated three times. The prepared plates were placed in a 26°C constant temperature incubator for incubation, and the width of the inhibition zone was measured when the mycelium of the control plate was fully grown.

[0046] Calculate the inhibition rate R of mycelial growth:

[0047] R(%)=(R1-R2) / R1×100%

[0048] Where R is the percentage of inhibition of radial hyphae growth, R1 is the hyphae growth of the blank control, and R2 is the hyphae growth of the treated group.

[0049] 2. Test results

[0050] The antibacterial effects of Bacillus KC2 supernatant on different pathogenic fungi are shown in Table 2. Among them, KC2 supernatant has the best antibacterial effect on Acremoniumsclerotigenum, Actinomucorelegans, Aspergillus pseudoglaucus, Botrytissp., Colletotrichumcoffeanum, Clathrosporadiplospora, Cladosporiumtenuissimum, Bjerkanderaadusta, Dimorphosporafolicola, Colletotrichumacutatum, Alternariabrassicae, Aspergillussp., Talaromycessp., Aspergillusaculeatus, Colletotrichumfioriniae and Diaporthesp., and the inhibition rates are all greater than 90%. The supernatant of Bacillus KC2 had a certain antibacterial effect on Penicilliumcrustosum, Alternaria alternata, Curvularialunata, Fusariumdecemcellulare, Diaporthe pseudophoenicicola, Aspergilusniger, Fusariumfalciforme, Moniliniafructicola, Aspergillusflavus, Galactomycescandidus, Fusariumsolani, Aspergillus tubingensis, Stagonosporopsiscucurbtacearum and Fusariumproliferatum, and the antibacterial rate was greater than 80%. The supernatant of Bacillus KC2 had a general antibacterial effect on Cladosporiumcolombiae, Colletotrichumgloeosporioides, Fusariumcircinatum and Fusariumincarnatum, and the antibacterial rate was greater than 70%.

[0051] Table 2 Antibacterial effect of Bacillus KC2 supernatant on different pathogenic fungi

[0052]

[0053]

[0054] (III) Antibacterial test of Bacillus KC2 cell lysate

[0055] 1. Experimental Design

[0056] Add 10 ml of sterile physiological saline to the live cells of Bacillus KC2 and shake well. Put the shaken solution into a 50 ml centrifuge tube and crush the KC2 strain cells in an ultrasonic cell crusher. Filter the crushed cell solution once with a microporous membrane filter with a pore size of 0.22 μm to obtain a liquid, which is the Bacillus KC2 cell lysate, and store it at 4°C for later use.

[0057] 200 μL of cell lysate was evenly spread on the PDA plate. Different pathogenic fungi cakes were placed in the center of the PDA plate surface using a 6 mm sterile punch. An equal amount of sterile water was used as a control. Each treatment was repeated three times. The prepared plate was placed in a 26°C constant temperature incubator for incubation. When the mycelium of the control plate was fully grown, the width of the inhibition zone was measured. The formula for the inhibition rate R of mycelium growth is the same as above.

[0058] 2. Test results

[0059] As can be seen from Table 3, Bacillus KC2 cell lysate has a certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of KC2 strain supernatant. Among them, Bacillus KC2 cell lysate has a good antibacterial effect on Acremonium sclerotigenum, Actinomu corelegans, Aspergillus pseudoglaucus, Botrytis sp., Colletotrichum coffeanum, Clathrosporadiplospora, Cladosporium tenuissimum, Bjerkandera adusta, Dimorphosporafolicola, Colletotrichum acutatum, Alternaria brassicae, Aspergillus sp. and Talaromyces sp., with an antibacterial rate of more than 90%. KC2 cell lysate had a certain antibacterial effect on Aspergillus aculeatus, Colletotrichum fioriniae, Diaporthesp., Penicillium crustosum, Alternaria alternata, Curvularia lunata, Fusarium decemcellulare, Diaporthe pseudophoenicicola, Aspergilus niger, Fusarium falciforme, Monilinia fructicola, Aspergillus flavus, Galactomyces candidus, Fusarium solani and Aspergillus tubingensis, and the antibacterial rate was greater than 80%. KC2 cell lysate had a general antibacterial effect on Stagonosporopsis cucurbtacearum, Fusarium proliferatum, Cladosporium colombiae and Colletotrichum gloeosporioides, and the antibacterial rate was greater than 70%.

[0060] Table 3 Antibacterial effect of Bacillus KC2 cell lysate on different pathogenic fungi

[0061]

[0062]

[0063] 4. Life characteristics of biocontrol bacteria

[0064] Bacillus KC2 has strong vitality, which is mainly reflected in its wide applicability in temperature, pH, field nutrient sources and humidity environments.

[0065] 1. Temperature adaptability

[0066] (1) Experimental design

[0067] Inoculate KC2 strain into LB broth and culture at 4℃, 16℃, 28℃, 37℃, 60℃, 150r / min in a shaker. Repeat 3 times for each treatment. After 18h, measure the absorbance and transmittance at 600nm with a UV-visible spectrophotometer. Use LB liquid medium without bacteria as blank control. Repeat 3 times for each treatment. Suspension turbidity = (100-transmittance) × 100%.

[0068] (2) Test results

[0069] The test results are shown in Table 4 and Figure 2 As shown in the figure, Bacillus KC2 showed good survival ability at 16-37℃, and the turbidity of the culture solution was greater than 50% after 18h. Bacillus KC2 could grow at 45℃, but the growth was slow, and the turbidity of the culture solution was 41.12% after 18h. It was difficult for Bacillus KC2 to reproduce alive at 4℃ or 60℃, and the turbidity of Bacillus KC2 was 0 after 18h of culture.

[0070] Table 4 Determination of adaptability of Bacillus KC2 culture at different temperatures

[0071]

[0072] 2. pH adaptability

[0073] (1) Experimental design

[0074] Use 1mol / LHCl or 1mol / LNaOH to adjust the pH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively, sterilize and inoculate 1mL of Bacillus KC2 seed culture solution into 100mL LB liquid medium adjusted to different pH, culture at 37℃, 150r / min in a shaker, and measure its absorbance and transmittance at 600nm wavelength with UV-visible spectrophotometer after 18h. Take the LB liquid medium without bacteria as blank control, and repeat 3 times for each treatment. Suspension turbidity = (100-transmittance) × 100%.

[0075] (2) Test results

[0076] The test results are shown in Table 5 and Figure 3 From Table 5 and Figure 2It can be seen that the turbidity of the culture solution of Bacillus KC2 was greater than 50% after 18 hours under the condition of pH = 5-9, indicating that Bacillus KC2 showed good survival ability under the condition of pH = 5-9. When the pH was <5 or >9, the turbidity of the culture solution was less than 10% after 18 hours, indicating that Bacillus KC2 was basically difficult to grow in a strong acid or strong base environment.

[0077] Table 5 Determination of adaptability of Bacillus KC2 culture at different pH

[0078]

[0079] 3. Adaptability of field nutrient sources

[0080] (1) Experimental design

[0081] According to the nutrient classification standards of the second national soil survey and the current soil conditions in Sichuan, the following soil nutrient conditions were formulated: total nitrogen 2g / kg, total phosphorus 1g / kg, total potassium 20g / kg, and water conditions field water holding capacity 70%. Based on the above conditions, the following single-factor variable design was performed for N, P, and K element nutrients, with 5 different gradients set for each factor (see Table 6):

[0082] (2) Test results

[0083] As can be seen from Table 7, Bacillus KC2 showed strong survival ability after being cultured for 60 days under different nutrient source environments. Under the conditions of 2g / kg total nitrogen, 1g / kg total phosphorus, and 20g / kg total potassium, Bacillus KC2 can survive well for more than 60 days. The KC2 strain can survive when the nitrogen, phosphorus, and potassium content is low. In the absence of nitrogen, phosphorus, and potassium, respectively, Bacillus KC2 can survive in the soil for more than 60 days, indicating that the KC2 strain does not rely on a specific nutrient element to survive, and can provide itself with nutrient sources through other elements.

[0084] Table 6 Single factor level table of field nutrient sources of Bacillus KC2

[0085]

[0086] Note: Among each single factor, the non-variable factor maintains the highest soil nutrient condition formula.

[0087] Table 760d Bacillus KC2 nutrient source adaptability

[0088]

[0089]

[0090] Note: “++” means the number of viable cells is ≥10 8cfu / mL, "+" means the number of viable bacteria is ≥10 6 cfu / mL, “-” means no viable bacteria.

[0091] 4. Field humidity adaptability

[0092] (1) Experimental design

[0093] In a sterile sand tube, add a sterile nutrient solution with 2g / kg total nitrogen, 1g / kg total phosphorus, and 20g / kg total potassium, and control the moisture at 10%, 30%, 50%, and 70%. Inoculate 1mL of KC2 seed culture solution, cover with sealing film, and culture at 37℃. Repeat each treatment 3 times. After 60 days, take a soil sample and dissolve it in physiological saline, take a small amount of sand and spread it on the LB plate to observe whether there are live bacteria and their number.

[0094] (2) Test results

[0095] The survival of Bacillus KC2 after 60 days under different field moisture content conditions can be seen from Table 8. The results show that Bacillus KC2 can show good survival ability after 60 days under the condition of soil moisture content ≥10%.

[0096] This strain has a strong tolerance to high temperature environment and a wide adaptability to humidity environment, so the vitality of this strain is suitable for most crop soils. When the air humidity is high, this strain can be mixed with inorganic nutrient solution and sprayed on the surface of crops as foliar fertilizer and protective agent.

[0097] Table 860d Adaptability of Bacillus KC2 to different field moisture contents

[0098]

[0099] Note: “++” means the number of viable cells is ≥10 8 cfu / mL, "+" means the number of viable bacteria is ≥10 6 cfu / mL, “-” means no viable bacteria.

[0100] 6. Tolerance to broad-spectrum fungicides

[0101] 1. Experimental Design

[0102] A total of 17 chemical fungicides, including pyraclostrobin, tebuconazole, epoxiconazole, carbendazim, azoxystrobin, propiconazole, oxazolidinone, myclobutanil, mancozeb, difenoconazole, boscalid, fluopyram, isoprodinil, myclobutanil, pyrimidine nucleotide antibiotics, prochloraz and flusilazole, were added to the LB liquid culture medium, and the pesticide concentration was the normal field dosage concentration of the above drugs and 10 times the dosage concentration; the LB liquid culture medium without fungicides and biocontrol bacteria was used as a control to inoculate the same concentration of KC2 strains. The culture was cultured in a shaking incubator at 37°C and 120r / min. After 24 hours, the LB liquid culture medium was dipped with an inoculation loop and streaked on the LB plate to observe the growth of the KC2 strain, and 3 replicates were used for each treatment.

[0103] 2. Test results

[0104] The test results are shown in Table 9. Bacillus KC2 has different resistance to different fungicides. Among them, after the normal dose and 10 times the dose of pyraclostrobin, tebuconazole, fluopyram, carbendazim, propiconazole, myclobutanil, difenoconazole, boscalid, fluopyram, isoprodinil and myclobutanil were co-cultured with Bacillus KC2 for 24 hours, Bacillus KC2 could grow on the LB plate, indicating that Bacillus KC2 had good resistance to these 10 commonly used fungicides under low concentration conditions and could survive in the environment of spraying these 10 drugs. Bacillus KC2 could grow live bacteria after co-cultured with normal doses of myclobutanil, prochloraz and flusilazole for 24 hours, but could not survive after co-cultured with 10 times the dose for 24 hours. Bacillus KC2 could not survive after being treated with normal doses and 10 times the dose of thiazolin, mancozeb and pyrimidine nucleotide antibiotics.

[0105] Table 9 Tolerance of Bacillus KC2 to common fungicides

[0106]

[0107] Note: “+” indicates the presence of live bacteria, “-” indicates the absence of live bacteria.

[0108] KC2 strain 16S sequence list

[0109] <211> 1417

[0110] <212> DNA

[0111] <213> Bacillus KC2

[0112] CCACCTTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTT

[0113] ACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTAT

[0114] TCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGT

[0115] CGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTT

[0116] AACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAG

[0117] CCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGG

[0118] TTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAA

[0119] GATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACG

[0120] AGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGT

[0121] CCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCG

[0122] TTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAAT

[0123] TCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATG

[0124] CGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCAT

[0125] CGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCT

[0126] TTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTG

[0127] TTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCC

[0128] TCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCG

[0129] GGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCA

[0130] ATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCAC

[0131] GTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTC

[0132] GAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTT

[0133] CATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATT

[0134] CCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTG

[0135] GCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTA

[0136] CCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGA

[0137] AGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAG

[0138] CCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGT

Claims

1. A Bacillus strain, characterized in that The Bacillus is Bacillus subtilis, named KC2, and the preservation unit is: General Microbiology Center of China Culture Collection Administration, the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation center number is: CGMCC No. 31372, and the preservation time is: July 19, 2024.

2. The Bacillus according to claim 1, characterized in that The gene sequence of the Bacillus is shown in SEQ NO.

1.

3. A biocontrol agent, characterized in that: The biocontrol agent comprises the Bacillus as claimed in claim 1 or 2.

4. Use of the biocontrol agent according to claim 3 in the fight against pathogens of fruits and vegetables.

5. Use of the bacillus according to claim 1 or 2 in preparing a fruit and vegetable preservative.

6. The use according to claim 4, characterized in that: The fruit and vegetable pathogenic bacteria include Acremoniumsclerotigenum, Actinomucor elegans, Aspergillus pseudoglaucus, Botrytis sp., Colletotrichum coffeanum, Clathrospora diplospora, Cladosporium tenuissimum, Bjerkandera adusta, Dimorphospora folicola, Colletotrichum acutatum, Alternariabrassicae, Aspergillus sp., Talaromyces sp., Aspergillus aculeatus, Colletotrichumfioriniae, Diaporthe sp., Penicillium crustosum, Alternariaalternata, Curvularia lunata, Fusarium decemcellulare, Diaporthepseudophoenicicola, Aspergilus niger,Fusariumfalciforme,Moniliniafructicola,Aspergillusflavus,Galactomyces candidus, Fusarium solani, Aspergillustubingensis, Stagonosporopsis cucurbtacearum, Fusarium proliferatum, Cladosporiumcolombiae, Colletotrichum gloeosporioides, Fusarium circinatum and Fusarium incarnatum.

7. The use according to claim 4, characterized in that: The concentration of Bacillus KC2 in the biocontrol agent is 1×10 6-8 cfu / L.

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