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

Abstract

The invention relates to bacillus amyloliquefaciens, a biocontrol inoculant prepared from the bacillus amyloliquefaciens and application of the biocontrol inoculant, and belongs to the technical field of biological inoculants. The bacillus amyloliquefaciens provided by the invention is bacillus amyloliquefaciens and is named as JK2, 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 CMCC No.31376, and the preservation time is July 19, 2024. The bacillus amyloliquefaciens JK2 disclosed by the invention has a certain bacteriostatic effect on different pathogenic bacteria regardless of living bacteria, supernate or cell lysis solution. The bacillus amyloliquefaciens JK2 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 strain of Bacillus amyloliquefaciens, a biocontrol agent prepared 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 inhibition rate of existing Bacillus biocontrol agents is generally low, especially the lack of Bacillus amyloliquefaciens. Moreover, for fruit and vegetable pathogens such as Alternaria angustiovoidea, Colletotrichum citricola, and Colletotrichum fioriniae, there are few biocontrol strains found so far, and their inhibition rates are not high.

[0007] Therefore, there is a need to provide a biocontrol strain of Bacillus amyloliquefaciens with a broader spectrum of antibacterial effects and a very significant inhibition rate against fruit and vegetable pathogens such as Alternaria angustiovoidea, Colletotrichum citricola, and Colletotrichum fioriniae. 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 amyloliquefaciens strain that can be used for disease control and bacteriostasis and preservation of fruits and vegetables with high efficiency, especially for Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichumfioriniae and other fruit and vegetable pathogens. 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, which is Bacillus amyloliquefaciens, named JK2. 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: CGMCCNo.31376, 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 pathogens include Alternaria angustiovoidea, Colletotrichumcitricola, Colletotrichum fioriniae, Colletotrichumgloeosporioides, Diaporthe fusicola, Pyricularia oryzae, Cladosporium bruhnei, Colletotrichum acutatum, Nigrospora oryzae, Talaromyces verruculosus, Stemphylium eturmiunum, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Clathrosporadiplospora, Fusarium decemcellulare, Pithomyces chartarum, Alternaria porri, Aspergillus puulaauensis, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Botryosphaeria dothidea, Fusarium solani, Alternariaalternata, Fusarium oxysporum, Galactomyces candidus, Neofusicoccum parvum, Fusarium proliferatum, Phoma herbarum, Aspergillus tubingensis, Rhizomucorvariabilis, Sclerotium hydrophilum, Fusarium incarnatum, Fusarium graminearum and Penicillium oxalicum.

[0014] The concentration of Bacillus JK2 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 JK2 living cells of the present invention have certain antibacterial effects on different pathogens. Among them, the antibacterial effect of the Bacillus JK2 living cells on Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthe fusicola and Pyricularia oryzae 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 JK2 viable bacteria against Cladosporium bruhnei, Colletotrichum acutatum, Nigrosporaoryzae, Talaromyces verruculosus, Stemphylium eturmiunum, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Clathrospora diplospora, Fusarium decemcellulare, Pithomyces chartarum, Alternariaporri and Aspergilluspuulaauensis was greater than 90%, showing a good inhibitory effect; the antibacterial effect on Arthrinium urticae, Aspergillusflavus, Talaromyces variabilis, Microdochium nivale, Fusarium sp., Penicillium sp. and Cladosporium bruhnei was average, with an inhibition rate of greater than 80%.

[0017] (2) The supernatant of Bacillus JK2 of the present invention has a certain antibacterial effect on different pathogenic fungi. Among them, the supernatant of Bacillus JK2 has a good antibacterial effect on Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthe fusicola, Pyricularia oryzae, Cladosporium bruhnei, Colletotrichum acutatum, Nigrosporaoryzae, Talaromyces verruculosus, Stemphylium eturmiunum, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata and Clathrospora diplospora, and the antibacterial rate is greater than 90%. The supernatant of Bacillus JK2 had a general antibacterial effect on Fusarium decemcellulare, Pithomyces chartarum, Alternariaporri, Aspergillus puulaauensis, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Botryosphaeria dothidea, Fusarium solani, Alternariaalternata, Fusarium oxysporum, Galactomyces candidus, Neofusicoccumparvum, Fusariumproliferatum and Phoma herbarum, with the inhibition rates all greater than 80%.

[0018] (3) The Bacillus JK2 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 JK2 strain supernatant. Among them, the Bacillus JK2 cell lysate has a good antibacterial effect on Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthe fusicola, Pyricularia oryzae, Cladosporium bruhnei, Colletotrichum acutatum, Nigrospora oryzae, Talaromyces verruculosus, Stemphylium eturmiunum, Penicillium decumbens and Didymosphaeria variabile, with an antibacterial rate of more than 90%. The antibacterial effect of JK2 cell lysate on Chaetomiumcochliodes, Colletotrichum siamense, Curvularia lunata, Clathrospora diplospora, Fusariumdecemcellulare, Pithomyces chartarum, Alternaria porri, Aspergilluspuulaauensis, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthoracapsici, Botryosphaeria dothidea and Fusarium solani was moderate, and the inhibition rates were all greater than 80%.

[0019] (4) The Bacillus JK2 provided by the present invention has a wide antibacterial spectrum, involving 38 fruit and vegetable pathogens, while 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, but it only involves 32 fruit and vegetable pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the inhibition rate of live Bacillus JK2 against different pathogens;

[0021] Figure 2 The turbidity of the suspension of Bacillus JK2 cultured at different temperatures;

[0022] Figure 3The turbidity of suspension of Bacillus JK2 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 Dazhu County, Dazhou 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 amyloliquefaciens, and was named Bacillus JK2. 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. 31376, and the deposit time is: July 19, 2024.

[0029] 3. Antibacterial test of strains

[0030] (I) Antibacterial test of live bacteria of Bacillus JK2

[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 JK2 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 JK2 strain cell suspension to PDA medium, shake and mix to make plates. The cell density of JK2 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 JK2 strain. Use the PDA plate containing 100mg / L carbendazim as the pesticide control, and the PDA plate medium without JK2 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 JK2 live bacteria have certain antibacterial effects on different pathogens. JK2 live bacteria have the most significant antibacterial effect on Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthefusicola and Pyricularia oryzae, with an inhibition rate of 100%, which can completely inhibit the growth of pathogenic fungi. The JK2 live bacteria had an inhibition rate of more than 90% against Cladosporium bruhnei, Colletotrichumacutatum, Nigrospora oryzae, Talaromyces verruculosus, Stemphylium eturmiunum, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Clathrospora diplospora, Fusariumdecemcellulare, Pithomyces chartarum, Alternariaporri and Aspergilluspuulaauensis, and had a good inhibitory effect on Rhizoctonia solani, Lasiodiplodiatheobromae, Phytophthora capsici, Botryosphaeria dothidea, Fusarium solani, Alternaria alternata, Fusarium oxysporum, Galactomyces candidus, Neofusicoccumparvum, Fusarium proliferatum, Phoma herbarum and Aspergillus tubingensis had certain antibacterial effects, and the inhibition rate was greater than 80%.

[0039] Compared with the antibacterial rate of 100mg / L carbendazim, JK2 viable bacteria were effective against Alternaria angustiovoidea, Aspergillus puulaauensis, Aspergillus tubingensis, Chaetomium cochliodes, Cladosporium bruhnei, Clathrospora diplospora, Colletotrichum acutatum, Colletotrichum citricola, Colletotrichum gloeosporioides, Curvularia lunata, Diaporthefusicola, Didymosphaeria variabile, Fusarium decemcellulare, Fusariumproliferatum, Galactomyces candidus, Lasiodiplodia theobromae, Neofusicoccumparvum, Nigrospora oryzae, Penicillium decumbens, Penicillium oxalicum, Phytophthora capsici, Pithomyces chartarum, Rhizoctonia solani, Rhizomucorvariabilis, Stemphylium eturmiunum and Talaromyces verruculosus had more prominent antibacterial effects, and the antibacterial rates were significantly higher than those treated with 100 mg / L carbendazim. After treatment with 100 mg / L carbendazim, the antibacterial effects on Alternaria alternata, Alternaria porri, Botryosphaeria dothidea, Colletotrichum siamense, Fusarium graminearum, Fusarium incarnatum, Fusarium solani, Phoma herbarum and Sclerotium hydrophilum were better, and the antibacterial rates were higher than those treated with live Bacillus JK2. After treatment with live JK2 and 100 mg / L carbendazim, the antibacterial effects on Colletotrichum fioriniae, Fusarium oxysporum and Pyricularia oryzae were similar.

[0040] Table 1 Antibacterial effect of live Bacillus JK2 cells and carbendazim on different pathogens

[0041]

[0042] (II) Antibacterial test of supernatant of Bacillus JK2

[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, Bacillus JK2 was repeatedly centrifuged to remove live bacteria and prepare JK2 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 culture, 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 the supernatant of Bacillus JK2 on different pathogenic fungi are shown in Table 2. Among them, the supernatant of JK2 had the best antibacterial effect on Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthe fusicola, Pyricularia oryzae, Cladosporium bruhnei, Colletotrichum acutatum, Nigrospora oryzae, Talaromycesverruculosus, Stemphylium eturmiunum, Penicillium decumbens, Didymosphaeriavariabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata and Clathrospora diplospora, and the inhibition rates were all greater than 90%. The supernatant of Bacillus JK2 had a certain antibacterial effect on Fusarium decemcellulare, Pithomyces chartarum, Alternaria porri, Aspergillus puulaauensis, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthoracapsici, Botryosphaeria dothidea, Fusarium solani, Alternaria alternata, Fusariumoxysporum, Galactomyces candidus, Neofusicoccumparvum, Fusariumproliferatum and Phoma herbarum, and the antibacterial rate was greater than 80%. The supernatant of Bacillus JK2 had a general antibacterial effect on Aspergillus tubingensis, Rhizomucor variabilis, Sclerotium hydrophilum and Fusariumincarnatum, and the antibacterial rate was greater than 70%.

[0051] Table 2 Antibacterial effect of Bacillus JK2 supernatant on different pathogenic fungi

[0052]

[0053]

[0054] (III) Antibacterial test of Bacillus JK2 cell lysate

[0055] 1. Experimental Design

[0056] Add 10 ml of sterile saline to the live cells of Bacillus JK2 and shake well. Put the shaken solution into a 50 ml centrifuge tube and crush the JK2 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 JK2 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, the Bacillus JK2 cell lysate has a certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the JK2 strain supernatant. Among them, the Bacillus JK2 cell lysate has a good antibacterial effect on Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthe fusicola, Pyricularia oryzae, Cladosporium bruhnei, Colletotrichum acutatum, Nigrospora oryzae, Talaromyces verruculosus, Stemphylium eturmiunum, Penicillium decumbens and Didymosphaeria variabile, with an inhibition rate of more than 90%. JK2 cell lysate had a certain antibacterial effect on Chaetomiumcochliodes, Colletotrichum siamense, Curvularia lunata, Clathrospora diplospora, Fusariumdecemcellulare, Pithomyces chartarum, Alternaria porri, Aspergilluspuulaauensis, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthoracapsici, Botryosphaeria dothidea and Fusarium solani, and the inhibition rate was greater than 80%. JK2 cell lysate had a general antibacterial effect on Alternaria alternata, Fusarium oxysporum, Galactomycescandidus, Neofusicoccum parvum, Fusarium proliferatum, Phoma herbarum, Aspergillus tubingensis, Rhizomucor variabilis and Sclerotium hydrophilum, and the inhibition rate was greater than 70%.

[0060] Table 3 Antibacterial effect of Bacillus JK2 cell lysate on different pathogenic fungi

[0061]

[0062]

[0063] 4. Life characteristics of biocontrol bacteria

[0064] Bacillus JK2 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] The JK2 strain was inoculated into LB broth and cultured in a shaker at 4°C, 16°C, 28°C, 37°C, and 60°C at 150 r / min. Each treatment was repeated 3 times. After 12 hours, the absorbance and transmittance were measured at a wavelength of 600 nm using a UV-visible spectrophotometer. The LB liquid medium without bacteria was used as a blank control, and each treatment was repeated 3 times.

[0068] Suspension turbidity = (100-transmittance) × 100%.

[0069] (2) Test results

[0070] The test results are shown in Table 4 and Figure 2 As shown in the figure, Bacillus JK2 showed good survival ability at 28-37℃, and the turbidity of the culture solution was greater than 50% after 12h. Bacillus JK2 can grow at 16℃ and 45℃, but the growth is slow. The turbidity of the culture solution after 12h is 35.43% and 17.85%, respectively. It is difficult for Bacillus JK2 to reproduce in vivo at 4℃ or 60℃, and the turbidity of Bacillus JK2 is 0 after 12h of culture.

[0071] Table 4 Determination of adaptability of Bacillus JK2 culture at different temperatures

[0072]

[0073]

[0074] 2. pH adaptability

[0075] (1) Experimental design

[0076] Use 1mol / L HCl or 1mol / L NaOH to adjust the pH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively. After sterilization, inoculate 1mL of Bacillus JK2 seed culture solution in 100mL LB liquid medium adjusted to different pH values, and culture in a shaker at 37°C and 150r / min. After 12 hours, measure the absorbance and transmittance at a wavelength of 600nm with a UV-visible spectrophotometer. Use the LB liquid medium without bacteria as the blank control, and repeat 3 times for each treatment. Suspension turbidity = (100-transmittance) × 100%.

[0077] (2) Test results

[0078] The test results are shown in Table 5 and Figure 3 From Table 5 and Figure 2 It can be seen that the turbidity of the culture solution of Bacillus JK2 was greater than 50% after 12 hours under the condition of pH = 5-8, indicating that Bacillus JK2 showed good survival ability under the condition of pH = 6-8. After culturing for 12 hours under the condition of pH = 9, the turbidity of the culture solution was 18.78%, indicating that Bacillus JK2 can grow under alkaline conditions, but the growth is relatively slow. When pH = 4 or = 10, the turbidity of the culture solution was less than 10% after 12 hours, indicating that Bacillus JK2 is basically difficult to grow in a strong acid or strong base environment.

[0079] Table 5 Determination of adaptability of Bacillus JK2 culture at different pH

[0080]

[0081] 3. Adaptability of field nutrient sources

[0082] (1) Experimental design

[0083] 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):

[0084] (2) Test results

[0085] As can be seen from Table 7, Bacillus JK2 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 JK2 can survive well for more than 60 days. The JK2 strain can survive when the nitrogen, phosphorus, and potassium content is low. In the absence of nitrogen, phosphorus, and potassium, respectively, Bacillus JK2 can survive in the soil for more than 60 days, indicating that the JK2 strain does not rely on a specific nutrient element to survive, and can provide itself with nutrient sources through other elements.

[0086] Table 6 Single factor level table of field nutrient sources of Bacillus JK2

[0087]

[0088]

[0089] Note: Among each single factor, the non-variable factor maintains the highest soil nutrient condition formula.

[0090] Table 760d Bacillus JK2 nutrient source adaptability

[0091]

[0092] Note: “++” means the number of viable cells is ≥10 8 cfu / L, "+" means the number of viable bacteria is ≥10 6 cfu / L, “-” means no viable bacteria.

[0093] 4. Field humidity adaptability

[0094] (1) Experimental design

[0095] 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 JK2 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.

[0096] (2) Test results

[0097] Table 8 shows the survival of Bacillus JK2 after 60 days under different field moisture content conditions. The results show that Bacillus JK2 can show good survival ability after 60 days under the condition of soil moisture content ≥30%. When the moisture content is 10%, Bacillus JK2 cannot survive for more than 60 days.

[0098] This strain has a strong tolerance to high temperature and high humidity environments, and is not adapted to extremely low temperature and humidity environments. However, soil moisture content below 10% does not occur in normal crop production areas, 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.

[0099] Table 8 Adaptability of 60d Bacillus JK2 to different field water contents

[0100]

[0101]

[0102] Note: “++” means the number of viable cells is ≥10 8 cfu / L, "+" means the number of viable bacteria is ≥10 6 cfu / L, “-” means no viable bacteria.

[0103] 6. Tolerance to broad-spectrum fungicides

[0104] 1. Experimental Design

[0105] 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 JK2 strain. 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 JK2 strain, with 3 replicates per treatment.

[0106] 2. Test results

[0107] The test results are shown in Table 9, and the resistance of Bacillus JK2 to different fungicides is different. Among them, after the normal dose and 10 times the dose of pyraclostrobin, tebuconazole, fluopyram, carbendazim, azoxystrobin, propiconazole, myclobutanil, mancozeb, difenoconazole, boscalid, fluopyram, isoproconazole, myclobutanil, prochloraz, and flusilazole were co-cultured with Bacillus JK2 for 24 hours, Bacillus JK2 could grow on the LB plate, indicating that Bacillus JK2 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 JK2 could grow live bacteria after co-cultured with normal doses of pyraclostrobin and pyrimidine nucleotide antibiotics for 24 hours, but could not survive after co-cultured with 10 times the dose for 24 hours.

[0108] Table 9 Tolerance of Bacillus JK2 to common fungicides

[0109]

[0110]

[0111] Note: “+” indicates the presence of live bacteria, “-” indicates the absence of live bacteria.

Claims

1. A Bacillus strain, characterized in that: The Bacillus is Bacillus amyloliquefaciens, named JK2, 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.31376, 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 pathogens include Alternaria angustiovoidea, Colletotrichum citricola, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Colletotrichum acutatum, Nigrospora oryzae, Talaromyces verruculosus, Stemphylium eturmiunum, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Clathrospora diplospora, Fusarium decemcellulare, Pithomyces chartarum, Alternaria porri, Aspergillus puulaauensis, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Botryosphaeria dothidea, Fusarium solani, Alternaria alternata, Fusarium oxysporum, Galactomyces candidus, Neofusicoccumparvum, Fusarium proliferatum, Phoma herbarum, Aspergillustubingensis, Rhizomucor variabilis, Sclerotium hydrophilum, Fusarium incarnatum, Fusarium graminearum and Penicillium oxalicum.

7. The use according to claim 4, characterized in that: The concentration of Bacillus JK2 in the biocontrol agent is 1×10 6-8 cfu / L.

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