Bacillus strain, biocontrol agent prepared from the bacillus strain and application thereof

Abstract

The present application relates to a bacillus, a biocontrol agent made from the bacillus and application, and belongs to the technical field of biological bacterial agents. The bacillus provided by the present application is Bacillus subtilis, named ZZ1, and the preservation unit is the General Microbiological Center of China Microorganism Bacterial Strain Preservation Management Committee, the preservation address is the Institute of Microbiology of Chinese Academy of Sciences, No. 3, Xili, Beichen, Chaoyang District, Beijing, the preservation center number is CGMCC No. 31374, and the preservation time is July 19, 2024. The bacillus ZZ1 has certain inhibition effect on different pathogenic bacteria, whether it is a living bacterium, a supernatant or a cell lysate. The bacillus ZZ1 has strong living ability, and is suitable for a wide range of nutrition sources, temperature and humidity, and pH. The bacillus ZZ1 has no pathogenicity to fruits and vegetables, and has good tolerance to broad-spectrum bactericides, and has great application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological bacterial agent, and particularly relates to a bacillus, a biocontrol bacterial agent made of the bacillus and application of the bacillus in fruit and vegetable antibacterial and bacteriostatic preservation. BACKGROUND

[0002] Fruits and vegetables are easily invaded by soil-borne pathogens during planting, and are often threatened by bacteria and fungi, especially fungal diseases, common symptoms of which include necrosis (leaf spots, leaf withering), rot (root rot, fruit rot) and wilting (root, stem base, vascular bundle tissue invasion) and the like. Traditionally, chemical fungicides, cultivation of disease-resistant varieties, interplanting, grafting and other methods are used for disease control, but these control measures have advantages and disadvantages.

[0003] On the other hand, fresh fruits and vegetables are subjected to varying degrees of infectious diseases (pathogenic microorganism infection) during postharvest and long-term storage due to changes in their physiological and biochemical characteristics. Postharvest infectious diseases of fruits and vegetables are caused by pathogenic microorganisms that infect the host during the growth and development period of the fruits and vegetables in the field, and 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, as well as wounds caused by improper transportation, facilitate the entry of pathogenic microorganisms into the internal pulp tissue through the epidermis of the fruits and vegetables, aggravating the degree of postharvest infectious diseases of the fruits and vegetables. At present, the main methods for controlling postharvest infectious diseases of fruits and vegetables are preharvest spraying of pesticides and postharvest reduction of storage temperature, but excessive use of pesticides can cause serious pesticide residue, endangering human health, and long-term excessive use of pesticides can cause pathogenic microorganisms to develop drug resistance, greatly reducing the effect of disease control.

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

[0005] Biological control methods have more advantages than chemical control methods. In addition to easy production and convenient use of biological agents, mixed bacterial agents made of biocontrol agents and their metabolites are more environmentally friendly, causing no pollution and no pesticide residues, which meets the strategic requirements of sustainable development in China. Moreover, biocontrol agents are less sensitive to environmental changes, and their effects are stable. In addition, the nutrients decomposed and transformed by biocontrol agents can be utilized by crops, thereby improving crop quality and increasing yield.

[0006] However, although the existing developed biocontrol strains and biocontrol agents show certain effects, and some have broad-spectrum antibacterial properties, the existing biocontrol agents of Bacillus have generally low inhibition rates. Moreover, for Actinomucor elegans, Alternaria sp., Aspergillus sp., and other fruit and vegetable pathogenic bacteria, fewer biocontrol strains have been found, and their inhibition rates are not high.

[0007] Therefore, it is necessary to provide a Bacillus subtilis biocontrol strain with a wider spectrum of antibacterial effects and a very significant inhibition rate against Actinomucor elegans, Alternaria sp., Aspergillus sp., and other fruit and vegetable pathogenic bacteria. SUMMARY

[0008] In view of the deficiencies in the prior art, the purpose of the present application is to provide a biocontrol strain that can be applied to fruit and vegetable disease control and antibacterial preservation. The present application provides a Bacillus strain that can be efficiently used for fruit and vegetable disease control and antibacterial preservation, especially the inhibition effect of Actinomucor elegans, Alternaria sp., Aspergillus sp., and other pathogenic bacteria is extremely significant. The present application also provides a biocontrol agent made from the Bacillus strain and its application in fruit and vegetable antibacterial and antibacterial preservation.

[0009] The inventors of the present application have obtained a Bacillus strain through extensive experimental research and exploration. The Bacillus strain is Bacillus subtilis, named ZZ-1, and is preserved at the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Beijing City, with the accession number CGMCC No. 31374, and the preservation date is July 19, 2024.

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

[0011] The present application further provides a biocontrol agent containing the above-mentioned Bacillus strain.

[0012] The present application further provides the application of the biocontrol agent in fruit and vegetable disease resistance and the application of the Bacillus strain in the preparation of fruit and vegetable preservatives.

[0013] Specifically, the fruit and vegetable pathogenic bacteria include Acremonium sclerotigenum, Actinomucorelegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthefusicola, Diaporthephoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides, Talaromycesverruculosus, Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusariumincarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichum gloeosporioides, Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternariaalstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsiscucurbitacearum, Stemphylium eturmiunum, Fusariumfujikuroi, Aspergillusjaponicus, Lasiodiplodia pseudotheobromae, Talaromyces sp.Fusarium solani, Fusarium falciforme, Fusarium tricinctum, Penicillium polonicum, Fusarium proliferatum, Gibberella fujikuroi, Fusarium avenaceum and Cladosporium colombiae.

[0014] The concentration of the biocontrol agent Bacillus ZZ-1 in the present application is 1 x 10 6-8 cfu / mL.

[0015] The present application has the following beneficial effects:

[0016] (1) The living bacteria of the bacillus ZZ-1 of the present application has certain inhibition effect on different pathogenic bacteria. The inhibition effect of the living bacteria of the bacillus ZZ-1 on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthefusicola, Diaporthephoenicicola, Didymosphaeriavariabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides and Talaromyces verruculosu is the most significant, the inhibition rate is 100%, and the growth of pathogenic fungi can be completely inhibited.Among them, the viable cell of ZZ-1 has an inhibition rate of more than 90% on Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichum gloeosporioides, Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp. and Fusarium oxysporum, and has a good inhibitory effect; the inhibitory effect on Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme, Fusarium tricinctum and Penicilliumpolonicum is general, and the inhibition rate is more than 80%.

[0017] (2) The supernatant of the Bacillus ZZ-1 has certain bacteriostatic effect on different pathogenic fungi. The bacteriostatic effect of the supernatant of the Bacillus ZZ-1 on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichum gloeosporioides, Penicillium rubens, Pestalotia subcuticularis and Acremonium sp. is the best, and the bacteriostatic rate is greater than 90%.The supernatant of Bacillus ZZ-1 has general antibacterial effect on Alternaria alstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonospora cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme and Fusarium tricinctum, and the inhibition rate is greater than 80%.

[0018] (3) The cell lysate of the Bacillus ZZ-1 of the present application has certain bacteriostatic effect on different pathogenic fungi, but the bacteriostatic effect is slightly lower than that of the supernatant of the ZZ-1 strain. Among them, the cell lysate of the Bacillus ZZ-1 has better bacteriostatic effect on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii and Colletotrichum gloeosporioides, and the bacteriostatic rate is greater than 90%.The ZZ-1 cell lysate has a general antibacterial effect on Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae and Talaromyces sp., and the antibacterial rate is greater than 80%.

[0019] (4) The bacillus ZZ-1 provided by the present application has a wide antibacterial spectrum, and involves 45 kinds of fruit and vegetable pathogens, while the prior art (CN114369556B-A strain of bacillus, a biocontrol agent made of the bacillus and application thereof) also provides a bacillus biocontrol strain, but only involves 32 kinds of fruit and vegetable pathogens. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The antibacterial rate of the ZZ-1 live bacteria to different pathogens;

[0021] Figure 2 The turbidity of the bacillus ZZ-1 cultured at different temperatures;

[0022] Figure 3 The turbidity of the bacillus ZZ-1 cultured at different pH values. DETAILED DESCRIPTION

[0023] The present application will be specifically described below through examples, and it is necessary to point out here that the following examples are only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application. Some non-essential improvements and adjustments made by skilled persons in the art according to the above application content still belong to the protection scope of the present application.

[0024] EMBODIMENT

[0025] I. Strain source

[0026] The soil was collected in a sterile sealed bag from a pine planting base in Hongya County, Leshan City, Sichuan Province in 2002, and then taken back to the Institute of Agricultural Products, Chengdu Academy of Agricultural and Forestry Sciences for separation and purification of antagonistic bacteria.

[0027] II. Strain identification

[0028] The DNA of the bacteria was extracted, and 16S rDNA sequence analysis was performed by PCR method, and the gene sequence is shown in SEQ ID NO. 1. The obtained sequence is compared on the NCBI website, and it is determined as Bacillus subtilis, named ZZ-1, and the preservation unit is: China General Microbiological Culture Collection Center, the preservation address is: No. 1, Beichen West Road, Beijing City, Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences, the preservation center number is: CGMCC No. 31374, and the preservation time is: July 19, 2024.

[0029] III. Bacteriostatic test of the strain

[0030] The strain has rich antibacterial type, and has good control effect on pathogenic bacteria on strawberry, blueberry, grape, apple, mango, cherry, kiwi, winter jujube, pepper, rice, eggplant, garlic and sponge gourd.

[0031] 1. Test design

[0032] ZZ-1 strain single colony was inoculated into LB broth, and cultured at 28℃ with constant shaking at 150rpm for 24h, and the cell suspension density was determined as 1×10 8 cfu / mL with a hemocytometer. Add ZZ-1 strain cell suspension to PDA medium, shake and mix to make plates, and the cell density of ZZ-1 strain in each plate is 1×10 6 cfu / mL. Use a 6mm sterile punch to take the gray botrytis cake and place it in the center of the PDA plate containing ZZ-1 strain, use the PDA plate containing 100mg / L carbendazim as pesticide control, and use the PDA plate without ZZ-1 strain as blank control, each treatment has 3 replicates. Put the plate with the cake in a self-sealing bag and place it in a 26℃ constant temperature incubator for culture, and measure the inhibition zone width when the control plate is full of mycelium.

[0033] Calculate the inhibition rate R of mycelial growth

[0034] R(%)=(R1-R2) / R1x100%

[0035] Where R is the percentage of inhibition of radial mycelial growth, R1 is the mycelial growth of the blank control, and R2 is the mycelial growth of the treatment group.

[0036] 2. Test results

[0037] The test results are shown in Table 1 and Figure 1 Figure 1 ​It can be seen from Table 1 that the live Bacillus ZZ-1 has certain inhibition effect on different pathogenic fungi. The live Bacillus ZZ-1 has the most significant inhibition effect on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthefusicola, Diaporthephoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides and Talaromyces verruculosus, and the inhibition rate is 100%, which can completely inhibit the growth of pathogenic fungi.Among them, the viable ZZ-1 bacteria have a bacteriostatic rate of more than 90% on Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusariumincarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichum gloeosporioides, Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp. and Fusarium oxysporum, and have a good inhibitory effect; have a certain bacteriostatic effect on Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme, Fusarium tricinctum and Penicilliumpolonicum, and have a bacteriostatic rate of more than 80%.

[0038] Compared with the inhibition rate of 100 mg / L carbendazim, ZZ-1 live bacteria had more prominent inhibition effect on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia abyssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Fusarium oxysporum, Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Penicillium polonicum, Gibberella fujikuroi, Fusarium avenaceum and Cladosporium colombiae, and the inhibition rates were significantly higher than that of 100 mg / L carbendazim.The 100 mg / L carbendazim treatment had better antibacterial effect on Botrytis sp., Fusarium solani, Fusarium falciforme and Fusarium tricinctum, and the inhibition rate was higher than that of Bacillus ZZ-1 live bacteria treatment. After ZZ-1 live bacteria and 100 mg / L carbendazim treatment on Colletotrichum fioriniae, Colletotrichum spaethianum, Fusarium incarnatum, Colletotrichum gloeosporioides, Fusarium fujikuroi and Fusarium proliferatum, the antibacterial effect was similar.

[0039] Table 1 Antibacterial effect of Bacillus ZZ-1 live bacteria and carbendazim on different pathogens

[0040]

[0041]

[0042] (II) Antibacterial test of supernatant of Bacillus ZZ-1

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

[0044] 1. Test design

[0045] After 24 h of culture, Bacillus ZZ-1 was removed by repeated centrifuge filtration to prepare ZZ-1 supernatant. 200 μL of supernatant was evenly coated on PDA plates, and different pathogenic bacteria were placed on the surface of the PDA plates with a 6 mm 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℃ constant temperature incubator for culture, and the inhibition zone width was measured when the control plate was full of mycelium.

[0046] Calculate the inhibition rate R of mycelial growth

[0047] R(%) = (R1-R2) / R1 x 100%

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

[0049] 2. Test results

[0050] The antibacterial effects of the supernatant of Bacillus ZZ-1 on different pathogenic fungi are shown in Table 2. Among them, the antibacterial effect of the supernatant of ZZ-1 on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconiabyssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichum gloeosporioides, Penicillium rubens, Pestalotia subcuticularis and Acremonium sp. is the best, and the inhibition rate is more than 90%.The supernatant of Bacillus ZZ-1 has certain bacteriostatic effect on Alternaria alstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme and Fusarium tricinctum, and the inhibition rate is greater than 80%. The bacteriostatic effect of the supernatant of Bacillus ZZ-1 on Penicillium polonicum, Fusarium proliferatum, Gibberella fujikuroi, Fusarium avenaceum and Cladosporium colombiae is general, and the inhibition rate is greater than 70%.

[0051] Table 2 Bacteriostatic effect of the supernatant of Bacillus ZZ-1 on different pathogenic fungi

[0052]

[0053]

[0054] (III) Bacteriostatic test of Bacillus ZZ-1 cell lysate

[0055] 1. Test design

[0056] 10 ml of sterile normal saline was added to the live Bacillus ZZ-1 cells, and the solution was shaken, then the shaken solution was placed in a 50 ml centrifuge tube, and the ZZ-1 strain cells were crushed in an ultrasonic cell crusher. The cell-crushed solution was filtered once with a micropore filter with a pore size of 0.22 μm, and the obtained liquid was the Bacillus ZZ-1 cell lysate, which was stored at 4°C for use.

[0057] 200 μL of cell lysate was evenly coated on a PDA plate, and different pathogenic fungal cakes were placed on the surface of the PDA plate in the center with a 6 mm sterile puncher, and an equal amount of sterile water was used as a control, and each treatment was repeated three times. The prepared plate was placed in a 26°C constant temperature incubator for culture, and the width of the bacteriostatic band was measured when the mycelium of the control group was full. The inhibition rate R of mycelial growth was calculated according to the above formula.

[0058] 2. Test results

[0059] As can be seen from Table 3, the cell lysate of Bacillus ZZ-1 had certain inhibitory effect on different pathogenic fungi, but the inhibitory effect was slightly lower than that of the supernatant of ZZ-1 strain. Among them, the cell lysate of Bacillus ZZ-1 had better inhibitory effect on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii and Colletotrichum gloeosporioides, and the inhibitory rate was greater than 90%.ZZ-1 cell lysate had certain antibacterial effect on Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae and Talaromyces sp., and the inhibition rate was greater than 80%. The antibacterial effect of ZZ-1 cell lysate on Fusarium solani, Fusarium falciforme, Fusarium tricinctum, Penicillium polonicum, Fusarium proliferatum, Gibberella fujikuroi and Fusarium avenaceum was general, and the inhibition rate was greater than 70%.

[0060] Table 3 Inhibition effect of Bacillus ZZ-1 cell lysate on different pathogenic fungi

[0061]

[0062]

[0063] Four, life characteristics of biocontrol bacteria

[0064] Bacillus ZZ-1 has strong life ability, which is mainly manifested in the wide adaptability of temperature, pH, field nutrient source and humidity environment.

[0065] 1. Temperature adaptability

[0066] (1) Test design

[0067] ZZ-1 strain was inoculated into LB broth, and was cultured in a shaking bed at 4°C, 16°C, 28°C, 37°C and 60°C, respectively, at 150 r / min, and each treatment was repeated 3 times. After 12 h, the absorbance and transmittance were measured at 600 nm wavelength by ultraviolet-visible spectrophotometer. The LB liquid medium without inoculation was used as blank control, and each treatment was repeated 3 times. Suspensibility = (100-transmittance) x 100%.

[0068] (2) Test results

[0069] The test results are shown in Table 4 and Figure 2As shown, Bacillus ZZ-1 showed good survivability at 16-37°C, and the suspension density of the culture solution was greater than 50% after 12 hours. Bacillus ZZ-1 could grow at 45°C, but the growth was slow, and the suspension density of the culture solution was 32.97% after 12 hours. It was difficult for Bacillus ZZ-1 to reproduce in vivo at 4°C or 60°C, and the suspension density of the culture solution was 0 after 12 hours.

[0070] Table 4: Determination of the culture adaptability of Bacillus ZZ-1 at different temperatures

[0071]

[0072] 2. Acid-base adaptability

[0073] (1) Test design

[0074] The pH was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 using 1 mol / L HCl or 1 mol / L NaOH, respectively. After sterilization, 1 mL of Bacillus ZZ-1 seed culture solution was inoculated into 100 mL of LB liquid medium adjusted to different pH values, and cultured at 37°C and 150 r / min on a shaker. After 12 hours, the absorbance and transmittance were measured at 600 nm using a UV-visible spectrophotometer. The LB liquid medium without inoculation was used as a blank control, and each treatment was repeated three times. The suspension density = (100-transmittance) x 100%.

[0075] (2) Test results

[0076] The test results are shown in Table 5 and Figure 3 . As shown in Table 5 and Figure 2 , Bacillus ZZ-1 showed good survivability at pH = 6-8, and the suspension density of the culture solution was greater than 45% after 12 hours. Bacillus ZZ-1 could grow at pH = 5 and pH = 9, but the growth was slow, and the suspension density of the culture solution was 16.05% and 22.23%, respectively, after 12 hours. When pH < 5 or > 9, the suspension density of the culture solution was less than 10% after 12 hours, indicating that Bacillus ZZ-1 was difficult to grow in a strong acid or strong base environment.

[0077] Table 5: Determination of the culture adaptability of Bacillus ZZ-1 at different pH values

[0078]

[0079] 3. Field nutrient adaptability

[0080] (1) Test design

[0081] According to the national second soil survey nutrient classification standards and the existing soil conditions in Sichuan, the following soil nutrient conditions are proposed: total nitrogen 2 g / kg, total phosphorus 1 g / kg, total potassium 20 g / kg, and moisture condition field water capacity 70%. According to the above conditions, the N, P, and K element nutrients are designed as follows:

[0082] (2) Test results

[0083] As can be seen from Table 7, Bacillus ZZ-1 showed strong survival ability after 60 days of culture in different nutrient environments. Under the conditions of total nitrogen 2 g / kg, total phosphorus 1 g / kg, and total potassium 20 g / kg, Bacillus ZZ-1 could survive for more than 60 days. ZZ-1 strain could survive in the case of low nitrogen, phosphorus, and potassium element content. In the case of lack of nitrogen and potassium elements, Bacillus ZZ-1 could survive in soil for more than 60 days, but in the case of lack of phosphorus element, Bacillus ZZ-1 could not survive in soil for more than 60 days.

[0084] Table 6 Single factor level table of Bacillus ZZ-1 field nutrient source

[0085] Note: In each single factor, the non-variable factor maintains the highest soil nutrient condition formula.

[0086] Table 760d Bacillus ZZ-1 nutrient source adaptability

[0087]

[0088] Note: “++” indicates that the number of viable bacteria is ≥10 8 cfu / mL, “+” indicates that the number of viable bacteria is ≥10 6 cfu / mL, “-” indicates no viable bacteria.

[0089] 4. Field humidity adaptability

[0090] (1) Test design

[0091] In sterile sand soil tubes, sterile nutrient solution with nutrient conditions of organic carbon 23.59%, total nitrogen 0.98%, total phosphorus 0.85%, and total potassium 2.75% was added, and the moisture was controlled at 10%, 30%, 50%, and 70%. 1 mL of ZZ-1 seed culture was inoculated, and the sealing film was covered. The culture was incubated at 37°C, and each treatment was repeated 3 times. After 60 days, the soil sample was dissolved in physiological saline, and a small amount of sand was spread on LB plates to observe the presence or absence of viable bacteria and their number.

[0092] (2) Test results

[0093] From Table 8, it can be seen that the survival of Bacillus ZZ-1 after 60 days under different field water content conditions. The results show that Bacillus ZZ-1 can exhibit good survival ability after 60 days under the condition that the soil water content is ≥30%. When the water content is 10%, Bacillus ZZ-1 cannot survive for more than 60 days.

[0094] The strain has strong tolerance to high humidity environment, and is not adapted to extremely low temperature and humidity environment, but soil water content below 10% does not occur in normal crop production areas, so the viability of the strain is suitable for most crop soils. When the air humidity is high, the strain can be mixed with inorganic nutrient solution and sprayed on the surface of crops as foliar fertilizer and protective agent.

[0095] Table 8 60d Bacillus ZZ-1 adaptability under different field water content

[0096]

[0097] Note: “++” indicates that the number of viable bacteria ≥10 8 cfu / mL, “+” indicates that the number of viable bacteria ≥10 6 cfu / mL, “-” indicates no viable bacteria.

[0098] Six, tolerance to broad-spectrum fungicides

[0099] 1. Test design

[0100] Pyraclostrobin, tebuconazole, epoxiconazole, carbendazim, azoxystrobin, propiconazole, azoxystrobin, thiophanate-methyl, mancozeb, difenoconazole, boscalid, fluopyram, iprodione, pyrimethanil, pyrimidine nucleotide antibiotic, prochloraz and flusilazole, a total of 17 kinds of chemical fungicides were added to LB liquid medium, so that the concentration of pesticides was 10 times the normal use concentration and 10 times the use concentration of the above-mentioned drugs; LB liquid medium without fungicides and biocontrol bacteria was used as a control and inoculated with ZZ-1 strain at the same concentration. Incubate at 37°C, 120 r / min on a shaker, after 24h, use a loop to dip LB liquid medium on LB plate and observe the growth of ZZ-1 strain, 3 replicates per treatment.

[0101] 2. Test results

[0102] The test results are shown in Table 9, and the resistance of Bacillus ZZ-1 to different fungicides is different. Among them, after the normal dose and 10 times dose of pyraclostrobin, tebuconazole, epoxiconazole, carbendazim, azoxystrobin, propiconazole, zoxam, thiram, myclobutanil, boscalid, fluopyram, dimethirimol and metsulfun were co-cultured with Bacillus ZZ-1 for 24 hours, Bacillus ZZ-1 could grow on LB plate, indicating that Bacillus ZZ-1 had good resistance to the 10 commonly used fungicides at low concentration and could survive in the environment sprayed with the 10 fungicides. Bacillus ZZ-1 could grow after co-culturing with normal dose of mancozeb, pyrimidine nucleotide antibiotics and flusilazole for 24 hours, but could not survive after co-culturing with 10 times dose for 24 hours. Bacillus ZZ-1 could not survive after being treated with normal dose and 10 times dose of propanil.

[0103] Table 9 Tolerance of Bacillus ZZ-1 to common fungicides

[0104]

[0105]

[0106] Note: "+" indicates viable bacteria, and "-" indicates no viable bacteria.

Claims

1. A Bacillus strain, characterized in that, The bacillus is Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ), named ZZ-1, and the depositing unit is China General Microbiological Culture Collection Center, the depositing address is No. 3, Yuan 1, Beichen West Road, Chaoyang District, Beijing, the depositing center number is CGMCC No. 31374, and the depositing time is July 19, 2024.

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

3. The use of the biocontrol agent of claim 2 in the aspect of fruit and vegetable pathogen antibiosis, characterized in that, The fruit and vegetable pathogen is a Cladosporium herbarum Acremonium sclerotigenum ) 、 The fruit and vegetable pathogen is a Mucor circinelloides Actinomucor elegans ) 、 The fruit and vegetable pathogen is a Cladosporium oxysporum Cladosporium colombiae ) 、 The fruit and vegetable pathogen is a Cladosporium tenuissimum Cladosporium tenuissimum ) 、 The fruit and vegetable pathogen is a Guignardia tsugae Colletotrichum fioriniae ) 、 The fruit and vegetable pathogen is a Colletotrichum gloeosporioides Colletotrichum gloeosporioides ) 、 The fruit and vegetable pathogen is a Colletotrichum siamense Colletotrichum siamense ) 、 The fruit and vegetable pathogen is a Diaporthe phaseolorum Diaporthe phoenicicola ) 、 The fruit and vegetable pathogen is a Dendryphium minimum Didymosphaeria variabile ) 、 The fruit and vegetable pathogen is a Fusarium decemellum Fusarium falciforme ) 、 The fruit and vegetable pathogen is a Fusarium roseum Fusarium incarnatum ) 、 The fruit and vegetable pathogen is a Fusarium oxysporum Fusarium oxysporum ) 、 The fruit and vegetable pathogen is a Fusarium proliferatum Fusarium proliferatum ) 、 The fruit and vegetable pathogen is a Fusarium solani Fusarium solani ) 、 The fruit and vegetable pathogen is a Geotrichum candidum Galactomyces candidum ) 、 The fruit and vegetable pathogen is a Gibberella fujikuroi Gibberella fujikuroi ) 、 The fruit and vegetable pathogen is a Monilinia fructicola Monilinia fructicola ) 、 The fruit and vegetable pathogen is a Penicillium chrysogenum Penicillium chrysogenum ) 、 The fruit and vegetable pathogen is a Penicillium polonicum Penicillium polonicum ) 、 The fruit and vegetable pathogen is a Penicillium rubrum Penicillium rubens ) 、 The fruit and vegetable pathogen is a Penicillium sclerotiorum Penicillium sclerotiorum ) 、 The fruit and vegetable pathogen is a Plectosphaerella cucumerina Stagonosporopsis cucurbitacearum ) and Talaromyces verruculosus Talaromyces verruculosus ).

4. Use according to claim 3, characterized in that, The concentration of the biocontrol agent Bacillus ZZ-1 is 1 x 10 6-8 cfu / L.

5. Use of the Bacillus as claimed in claim 1 for the preparation of a fruit and vegetable preservative.

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