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

Abstract

The invention relates to bacillus velezensis, a biocontrol inoculant prepared from the bacillus velezensis and application of the biocontrol inoculant, and belongs to the technical field of biological inoculants. The bacillus velezensis provided by the invention is bacillus velezensis and is named as JK1, 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, West Beichen Road, Chaoyang District, Beijing, the number of the preservation center is CGMCC No.31375, and the preservation time is July 19, 2024. The bacillus JK1 disclosed by the invention has relatively excellent bacteriostatic action on different pathogenic bacteria regardless of living bacteria, supernate or cell lysis solution. The bacillus JK1 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 biocontrol agents, and specifically relates to a strain of Bacillus velez, a biocontrol agent prepared from the Bacillus velez 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 Velezii. Moreover, for fruit and vegetable pathogens such as Colletotrichumfioriniae and Colletotrichum citricola, there are few biocontrol strains found so far, and their inhibition rates are not high; and for the four pathogens of Stemphylium eturmiunum, Clathrospora diplospora, Chaetomium cochliodes and Galactomyces candidus, there are currently no reports of good biocontrol strains.

[0007] Therefore, it is necessary to provide a Bacillus Velezii biocontrol strain having a broader spectrum of antibacterial effects, and a very significant inhibition rate against fruit and vegetable pathogens such as Colletotrichum fioriniae and Colletotrichum citricola, while having a significant inhibition effect on four pathogens, Stemphylium eturmiunum, Clathrospora diplospora, Chaetomium cochliodes 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 antibacterial preservation of fruits and vegetables. The present invention provides a strain of Bacillus Velezii, which can be effectively used for disease control and antibacterial preservation of fruits and vegetables, especially for pathogens such as Colletotrichum fioriniae and Colletotrichum citricola, the antibacterial rate can reach 100%, and has obvious antibacterial effect on four pathogens such as Stemphylium eturmiunum, Clathrospora diplospora, Chaetomium cochliodes and Galactomyces candidus. The present invention also provides a biocontrol agent made from the Bacillus and its application in antibacterial and antibacterial 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 velezensis, named JK1, 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.31375, and the preservation time is: July 19, 2024.

[0010] The gene sequence of the Bacillus JK1 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 JK1.

[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 Colletotrichum fioriniae, Colletotrichumcitricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichumacutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergilluspuulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeriavariabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyceschartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccumparvum, Fusarium proliferatum, Rhizomucor variabilis, Phomaherbarum, Sclerotium hydrophilum, Fusariumincarnatum, Fusarium graminearum and Penicillium oxalicum.

[0014] The concentration of Bacillus JK1 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 JK1 living cells of the present invention have extremely high antibacterial effects on a variety of different pathogens, and their broad-spectrum antibacterial properties are extremely excellent. Among them, the antibacterial effect of the Bacillus JK1 living cells on Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthe fusicola and Pyricularia oryzae is the most significant, with an antibacterial rate of 100%, and can completely inhibit the growth of pathogenic fungi. The JK1 live bacteria had an inhibition rate of more than 90% against Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternariaporri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora and Fusarium decemcellulare, and had a good inhibitory effect on Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomycescandidus, Neofusicoccumparvum, Fusarium proliferatum, Rhizomucor variabilis and Phoma herbarum had moderate antibacterial effects, with an inhibition rate of more than 80%.

[0017] (2) The supernatant of Bacillus JK1 of the present invention has a certain antibacterial effect on different pathogenic fungi. Among them, the supernatant of Bacillus JK1 had the best antibacterial effect against Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthe fusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis and Clathrospora diplospora, with the inhibition rates all greater than 90%. The supernatant of Bacillus JK1 had a certain antibacterial effect on Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccum parvum, Fusarium proliferatum and Rhizomucorvariabilis, and the inhibition rate was greater than 80%.

[0018] (3) The Bacillus JK1 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 JK1 strain supernatant. Among them, the Bacillus JK1 cell lysate has a good antibacterial effect on Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri and Penicillium decumbens, with an antibacterial rate of more than 90%. JK1 cell lysate had certain antibacterial effects on Didymosphaeria variabile, Chaetomiumcochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusariumsolani, Fusarium oxysporum, Galactomyces candidus and Neofusicoccumparvum, and the inhibition rates were all greater than 80%.

[0019] (4) The Bacillus JK1 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 1The antibacterial effect of live Bacillus JK1 on different pathogens; the left side of each group is the control (CK), and the right side is the experimental group containing JK1;

[0021] Figure 2 The turbidity of the suspension of Bacillus JK1 cultured at different temperatures;

[0022] Figure 3 The turbidity of suspension of Bacillus JK1 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 the field according to the above invention content still fall within the scope of protection of the present invention.

[0024] Example 1

[0025] 1. Source of strains

[0026] In 2023, peach leaves were collected from a 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 its classification was named Bacillus velezensis, and it was named Bacillus JK1. 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. 31375, and the deposit time is: July 19, 2024.

[0029] 3. Antibacterial test of strains

[0030] (I) Antibacterial test of live bacteria of Bacillus JK1

[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 JK1 strain was inoculated into LB broth and cultured at 37°C with constant shaking at 150 rpm for 24 h. The cell suspension density was determined as 1 × 10 8 cfu / mL. Add JK1 strain cell suspension to PDA medium, shake and mix to make plates. The cell density of JK1 strain on each plate is 1×10 6 cfu / mL. Use a 6mm sterile puncher to take the Botrytis cinerea cake and place it in the center of the PDA plate containing the JK1 strain. Use the PDA plate containing 100mg / L carbendazim as the pesticide control, and the PDA plate medium without JK1 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 JK1 live bacteria have certain antibacterial effects on different pathogens. JK1 live bacteria have the most significant antibacterial effect on Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthe fusicola and Pyricularia oryzae, with an inhibition rate of 100%, which can completely inhibit the growth of pathogenic fungi. The JK1 live bacteria had an inhibition rate of more than 90% against Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternariaporri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora and Fusarium decemcellulare, and had a good inhibitory effect on Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomycescandidus, Neofusicoccumparvum, Fusarium proliferatum, Rhizomucor variabilis and Phoma herbarum had certain antibacterial effects, and the inhibition rate was greater than 80%.

[0039] Compared with the antibacterial rate of 100 mg / L carbendazim, JK1 viable bacteria were effective against Alternaria alternata, Alternariaporri, Alternaria angustiovoidea, Aspergilluspuulaauensis, Aspergillustubingensis, Colletotrichum acutatum, Colletotrichum citricola, Colletotrichumgloeosporioides, Cladosporium bruhnei, Clathrospora diplospora, Chaetomiumcochliodes, Curvularia lunata, Didymosphaeria variabile, Diaporthefusicola, Fusarium decemcellulare, Fusarium proliferatum, Galactomycescandidus, Lasiodiplodia theobromae, Neofusicoccumparvum, Nigrospora oryzae, Penicillium decumbens, Penicillium Oxalicum,Phytophthora capsici, Pithomyces chartarum, Rhizoctonia solani, Rhizomucor variabilis, 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 Colletotrichum siamense, Fusarium solani, Fusarium graminearum, Fusarium incarnatum, Phoma herbarum and Sclerotium hydrophilum were better, and the antibacterial rates were higher than those treated with live Bacillus JK1. After treatment with live JK1 and 100 mg / L carbendazim, the antibacterial effects on Colletotrichum fioriniae, Fusarium oxysporum, Pyricularia oryzae and Botryosphaeria dothidea were similar.

[0040] Table 1 Antibacterial effects of live Bacillus JK1 cells and carbendazim on different pathogens

[0041]

[0042]

[0043] (II) Antibacterial test of supernatant of Bacillus JK1

[0044] 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.

[0045] 1. Experimental Design

[0046] After culturing for 24 hours, Bacillus JK1 was repeatedly centrifuged to remove live bacteria and prepare JK1 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.

[0047] Calculate the inhibition rate R of mycelial growth

[0048] R(%)=(R1-R2) / R1×100%

[0049] 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.

[0050] 2. Test results

[0051] The antibacterial effects of Bacillus JK1 supernatant on different pathogenic fungi are shown in Table 2. Among them, the supernatant of JK1 had the best antibacterial effect on Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergilluspuulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicilliumdecumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichumsiamense, Curvularia lunata, Aspergillus tubingensis and Clathrospora diplospora, with the inhibition rates all greater than 90%. The supernatant of Bacillus JK1 had a certain antibacterial effect on Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccum parvum, Fusarium proliferatum and Rhizomucor variabilis, and the antibacterial rate was greater than 80%. The supernatant of Bacillus JK1 had a general antibacterial effect on Phoma herbarum, Sclerotiumhydrophilum and Fusariumincarnatum, and the antibacterial rate was greater than 70%.

[0052] Table 2 Antibacterial effect of Bacillus JK1 supernatant on different pathogenic fungi

[0053]

[0054]

[0055] (III) Antibacterial test of Bacillus JK1 cell lysate

[0056] 1. Experimental Design

[0057] Add 10 ml of sterile saline to the live cells of Bacillus JK1 and shake well. Put the shaken solution into a 50 ml centrifuge tube and crush the JK1 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 JK1 cell lysate, and store it at 4°C for later use.

[0058] 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.

[0059] 2. Test results

[0060] As can be seen from Table 3, the Bacillus JK1 cell lysate has a certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the JK1 strain supernatant. Among them, the Bacillus JK1 cell lysate has a good antibacterial effect on Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri and Penicillium decumbens, with an inhibition rate of more than 90%. JK1 cell lysate had certain antibacterial effects on Didymosphaeria variabile, Chaetomiumcochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusariumsolani, Fusarium oxysporum, Galactomyces candidus and Neofusicoccumparvum, with the inhibition rates all greater than 80%. JK1 cell lysate had moderate antibacterial effects on Fusariumproliferatum, Rhizomucorvariabilis and Phoma herbarum, with the inhibition rates greater than 70%.

[0061] Table 3 Antibacterial effect of Bacillus JK1 cell lysate on different pathogenic fungi

[0062]

[0063]

[0064] 4. Life characteristics of biocontrol bacteria

[0065] Bacillus JK1 has strong vitality, which is mainly reflected in its wide applicability in temperature, pH, field nutrient sources and humidity environments.

[0066] 1. Temperature adaptability

[0067] (1) Experimental design

[0068] Inoculate JK1 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 culture medium without bacteria as blank control. Repeat 3 times for each treatment. 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 JK1 has a wide adaptability to temperature and shows good survival ability at 16-37℃. The turbidity of the culture solution is greater than 65% after 18h. Bacillus JK1 can grow at 45℃, but the growth is slow. The turbidity of the culture solution is 48.92% after 18h. It is difficult for Bacillus JK1 to reproduce in vivo at 4℃ or 60℃. The turbidity of Bacillus JK1 is 0 after 18h of culture.

[0071] Table 4 Determination of adaptability of Bacillus JK1 to culture at different temperatures

[0072]

[0073] 2. pH adaptability

[0074] (1) Experimental design

[0075] 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 JK1 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 18 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%.

[0076] (2) Test results

[0077] 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 JK1 is greater than 50% after 18 hours under the condition of pH = 5-8, indicating that Bacillus JK1 has good survival ability under the condition of pH = 5-8. Under the conditions of pH = 9 and pH = 10, the turbidity of the culture solution after 18 hours is 40.87% and 21.25% respectively, indicating that Bacillus JK1 can grow under alkaline conditions, but the growth is relatively slow. When pH = 4, the turbidity of the culture solution is less than 10% after 18 hours, indicating that Bacillus JK1 is basically difficult to grow in a strong acid environment.

[0078] Table 5 Determination of adaptability of Bacillus JK1 culture at different pH

[0079]

[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 JK1 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 JK1 can survive well for more than 60 days. The JK1 strain can survive when the nitrogen, phosphorus, and potassium content is low. In the absence of nitrogen, phosphorus, and potassium, respectively, Bacillus JK1 can survive in the soil for more than 60 days, indicating that the JK1 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 JK1

[0087]

[0088] Note: Among each single factor, the non-variable factor maintains the highest soil nutrient condition formula.

[0089] Table 7 60-day adaptability of Bacillus JK1 to nutrient sources

[0090]

[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 JK1 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 JK1 after 60 days under different field moisture content conditions. The results show that Bacillus JK1 can survive after 60 days when the soil moisture content is ≥10%. When the soil moisture content is 50-70%, Bacillus JK1 shows a strong survival ability.

[0098] This strain has a strong tolerance to high temperature and high humidity environments, and is not adapted to extremely low temperature environments. However, most plants are still dormant in a low temperature environment of 4°C, 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 Bacillus JK1 to different field water contents after 60 days

[0100]

[0101] 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.

[0102] 6. Tolerance to broad-spectrum fungicides

[0103] 1. Experimental Design

[0104] 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 inoculated with the same concentration of JK1 strain as a control. 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 JK1 strain, with 3 replicates per treatment.

[0105] 2. Test results

[0106] The test results are shown in Table 9, and Bacillus JK1 has different resistance to different fungicides. Among them, after the normal dose and 10 times the dose of pyraclostrobin, tebuconazole, fluopyram, carbendazim, azoxystrobin, propiconazole, myclobutanil, mancozeb, difenoconazole, boscalid, fluopyram, isoprodinil, myclobutanil and flusilazole were co-cultured with Bacillus JK1 for 24 hours, Bacillus JK1 could grow on the LB plate, indicating that Bacillus JK1 has good resistance to these 10 commonly used fungicides under low concentration conditions and can survive in the environment of spraying these 10 drugs. Bacillus JK1 can grow live bacteria after co-cultured with normal doses of pyraclostrobin, pyrimidine nucleotide antibiotics and prochloraz for 24 hours, but it cannot survive after co-cultured with 10 times the dose for 24 hours.

[0107] Table 9 Tolerance of Bacillus JK1 to common fungicides

[0108]

[0109] 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 velezensis, named JK1, 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.31375, 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 Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichumsiamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccum parvum, Fusarium proliferatum, Rhizomucor variabilis, Phoma herbarum, Sclerotium hydrophilum, Fusarium incarnatum, Fusarium graminearum and Penicillium oxalicum.

7. The use according to claim 4, characterized in that: The concentration of Bacillus JK1 in the biocontrol agent is 1×10 6-8 cfu / L.

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