Bacillus strain, biocontrol inoculant prepared by bacillus strain, and use thereof

Abstract

Provided in the present invention are a Bacillus strain, a biocontrol inoculant prepared by the Bacillus strain, and the use thereof. The Bacillus strain provided by the present invention is Bacillus subtilis and is named ZZ-1, is preserved in the preservation authority: the China General Microbiological Culture Collection Center at the preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is assigned with the accession number CGMCC No. 31374 on 19 July 2024. The Bacillus strain ZZ-1 of the present invention, regardless of being in the form of live bacterial cells, a supernatant or a cell lysate, has certain antimicrobial effects on various pathogenic fungi. The Bacillus strain ZZ-1 has strong living ability, has a wide application range in terms of nutrient sources, temperature, humidity and pH value, has no pathogenicity to fruits and vegetables, and has relatively good tolerance to broad-spectrum fungicides.

Description

Bacillus, biocontrol agent prepared from the bacillus and application TECHNICAL FIELD

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

[0002] Fruits and vegetables are prone to be invaded by soil-borne pathogens during planting, and are often threatened by bacteria and fungi, especially fungal diseases, which are common symptoms such as necrosis (leaf spot, 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, thereby increasing the degree of postharvest infectious diseases of the fruits and vegetables. Currently, 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 result in serious pesticide residue exceeding the standard, which is harmful to 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 technique that uses living biocontrol agents and their metabolically active substances to control disease occurrence. Biocontrol agents can colonize and grow in the rhizosphere of crops, 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 prepared from biocontrol agents and their metabolites are more environmentally friendly, have no pollution and no pesticide residues, and meet the strategic requirements of sustainable development in China. Moreover, biocontrol agents are less sensitive to environmental changes, have stable effects, and 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, 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, Acremonium sp., Alternaria alstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, 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, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, 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.The bacteriostatic rate of ZZ-1 active bacteria 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 is greater than 90%, and has a good inhibitory effect; the bacteriostatic effect on Stagonosporopsis cucurbitacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme, Fusarium tricinctum and Penicillium polonicum is general, and the bacteriostatic rate is greater than 80%.

[0017] (2) The supernatant of the Bacillus ZZ-1 has certain bacteriostatic effects on different pathogenic fungi. The bacteriostatic effects 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. are the best, and the bacteriostatic rates are all greater than 90%.The supernatant of Bacillus ZZ-1 had general antibacterial 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 was 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 inhibition 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] Fig. 1 is the inhibition rate of ZZ-1 live bacteria on different pathogens;

[0021] Fig. 2 is the turbidity of bacillus ZZ-1 cultured at different temperatures;

[0022] Fig. 3 is the turbidity of 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 description 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 the sequence table SEQ 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] (I) Bacteriostatic test of live Bacillus ZZ-1

[0031] The strain has rich antibacterial types, 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.

[0032] 1. Test design

[0033] ZZ-1 strain single colony 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 to be 1×10 8 cfu / mL using 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 PDA plate containing 100mg / L carbendazim as pesticide control, and PDA plate without ZZ-1 strain as blank control, with 3 replicates for each treatment. The plate with the fungus cake is sealed with a self-sealing bag and placed in a 26°C constant temperature incubator for culture. When the mycelium of the control group is full, the width of the inhibition zone is measured.

[0034] The inhibition rate R R(%) of mycelial growth is calculated as follows: R R(%)=(R1-R2) / R1×100%

[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 results are shown in Table 1 and Figure 1. As shown in Figure 1 and Table 1, the live Bacillus ZZ-1 has certain inhibitory effect on different pathogenic fungi. The live Bacillus ZZ-1 has the most significant 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 and Talaromyces verruculosus, and the inhibitory rate is 100%, which can completely inhibit the growth of pathogenic fungi.Among them, the viable cell of ZZ-1 has a bacteriostatic 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; has 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 has 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 byssoides, 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 inhibition effect of 100 mg / L carbendazim treatment on Botrytis sp., Fusarium solani, Fusarium falciforme and F. tricinctum was better than that of Bacillus ZZ-1 live bacteria treatment, and the inhibition rates were all greater than that of Bacillus ZZ-1 live bacteria treatment. The inhibition effect of ZZ-1 live bacteria treatment and 100 mg / L carbendazim treatment on Colletotrichum fioriniae, Colletotrichum spaethianum, Fusarium incarnatum, Colletotrichum gloeosporioides, Fusarium fujikuroi and Fusarium proliferatum was similar.

[0039] Table 1 Inhibition effect of Bacillus ZZ-1 live bacteria and carbendazim on different pathogenic fungi

[0040] (ii) Inhibition test of supernatant of Bacillus ZZ-1

[0041] The supernatant inhibition test mainly tests the inhibition effect of extracellular inhibition substance. This method mainly excludes the nutritional competition factor and is an important indicator for evaluating the biocontrol effect of biocontrol bacteria.

[0042] 1. Test design

[0043] After 24 h of culture, the live bacteria of Bacillus ZZ-1 were removed by repeated centrifuge filtration to prepare ZZ-1 supernatant. 200 μL of the supernatant was uniformly coated on the PDA plate, and 6 mm sterile puncher was used to take different pathogenic fungus cakes and place them on the surface center of the PDA plate. 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℃ constant temperature incubator for culture, and the inhibition band width was measured when the mycelium of the control group was full.

[0044] The inhibition rate R R(%) of mycelial growth was calculated as follows: R R(%)=(R1-R2) / R1x100%

[0045] Wherein 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.

[0046] 2. Test results

[0047] 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, 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 inhibition rate is more than 90%.The supernatant of Bacillus ZZ-1 had certain antibacterial 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 was greater than 80%. The antibacterial effect of the supernatant of Bacillus ZZ-1 on Penicillium polonicum, Fusarium proliferatum, Gibberella fujikuroi, Fusarium avenaceum and Cladosporium colombiae was general, and the inhibition rate was greater than 70%.

[0048] Table 2 Antibacterial effect of the supernatant of Bacillus ZZ-1 on different pathogenic fungi

[0049] (III) Bacillus ZZ-1 cell lysate antibacterial test

[0050] 1. Test design

[0051] 10 ml of sterile normal saline was added to the live Bacillus ZZ-1 cells, and the solution was shaken well. 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 0.22 μm pore size microporous filter to obtain the Bacillus ZZ-1 cell lysate, which was stored at 4°C for use.

[0052] 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. 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 inhibition zone width 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.

[0053] 2. Test results

[0054] As can be seen from Table 3, the cell lysate of Bacillus ZZ-1 had certain antibacterial effect on different pathogenic fungi, but the antibacterial effect was slightly lower than that of the supernatant of ZZ-1 strain. Among them, the cell lysate of Bacillus ZZ-1 had better antibacterial 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 inhibition 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 more 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 more than 70%.

[0055] Table 3 Inhibition effect of Bacillus ZZ-1 cell lysate on different pathogenic fungi

[0056] Four, life characteristics of biocontrol bacteria

[0057] The living ability of Bacillus ZZ-1 is strong, which is mainly reflected in the wide adaptability of temperature, pH, field nutrient source and humidity environment.

[0058] 1. Temperature adaptability

[0059] (1) Test design

[0060] ZZ-1 strain was inoculated into LB broth, and was cultured in a shaking bed at 4℃, 16℃, 28℃, 37℃ and 60℃, respectively, at 150 r / min. 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 had 3 repeats. Suspensibility = (100-transmittance) x 100%.

[0061] (2) Test results

[0062] The results are shown in Table 4 and Fig. 2. The bacillus ZZ-1 has good survival ability at 16-37°C, and the suspension density of the culture solution is more than 50% after 12 hours. The bacillus ZZ-1 can grow at 45°C, but the growth is slow, and the suspension density of the culture solution is 32.97% after 12 hours. The bacillus ZZ-1 is difficult to propagate in vivo at 4°C or 60°C, and the suspension density of the culture solution is 0 after 12 hours.

[0063] Table 4: Adaptability of the bacillus ZZ-1 at different temperatures

[0064] 2. Acid-base adaptability

[0065] (1) Test design

[0066] The LB liquid medium was adjusted to pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 by 1 mol / L HCl or 1 mol / L NaOH respectively, and then inoculated with 1 mL of the seed culture of the bacillus ZZ-1. The culture was incubated at 37°C and 150 r / min, and the absorbance and transmittance were measured at 600 nm after 12 hours. 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%.

[0067] (2) Test results

[0068] The results are shown in Table 5 and Fig. 3. As shown in Table 5 and Fig. 2, the suspension density of the culture solution is more than 45% after 12 hours at pH 6-8, indicating that the bacillus ZZ-1 has good survival ability at pH 6-8. The suspension density of the culture solution is 16.05% and 22.23% after 12 hours at pH 5 and pH 9 respectively, indicating that the bacillus ZZ-1 can grow at acidic and alkaline conditions, but the growth is slow. The suspension density of the culture solution is less than 10% after 12 hours at pH < 5 or > 9, indicating that the bacillus ZZ-1 is difficult to grow in strong acid or strong alkali environment.

[0069] Table 5: Adaptability of the bacillus ZZ-1 at different pH

[0070] 3. Field nutrient adaptability

[0071] (1) Test design

[0072] 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:

[0073] (2) Test results

[0074] 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 can survive for more than 60 days. ZZ-1 strain can 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 can survive in soil for more than 60 days, but in the case of lack of phosphorus element, Bacillus ZZ-1 cannot survive in soil for more than 60 days.

[0075] Table 6 Single factor level table of Bacillus ZZ-1 field nutrient source Note: In each single factor, the non-variable factor maintains the highest soil nutrient condition formula.

[0076] Table 760d Bacillus ZZ-1 nutrient source adaptability 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.

[0077] 4. Field humidity adaptability

[0078] (1) Test design

[0079] 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.

[0080] (2) Test results

[0081] 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.

[0082] 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.

[0083] Table 8 60d Bacillus ZZ-1 adaptability under different field water content 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.

[0084] Six, tolerance to broad-spectrum fungicides

[0085] 1. Test design

[0086] Pyraclostrobin, tebuconazole, epoxiconazole, carbendazim, azoxystrobin, propiconazole, azoxystrobin, thiophanate-methyl, mancozeb, difenoconazole, boscalid, fluopyram, iprodione, mepanipyrim, pyrimidine nucleotide antibiotic, pyraclostrobin 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 the same concentration of ZZ-1 strain. 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.

[0087] 2. Test results

[0088] The test results are shown in Table 9. The resistance of Bacillus ZZ-1 to different fungicides is different. Among them, after co-culturing Bacillus ZZ-1 with pyraclostrobin, tebuconazole, epoxiconazole, carbendazim, azoxystrobin, propiconazole, zoxam, thiram, myclobutanil, boscalid, fluopyram, dimethirimol and metsulfun for 24 hours at normal dose and 10 times dose, Bacillus ZZ-1 can grow on LB plate, indicating that Bacillus ZZ-1 has good resistance to the 10 commonly used fungicides at low concentration and can survive in the environment sprayed with the 10 fungicides. Bacillus ZZ-1 can grow after co-culturing with maneb-zinc, pyrimidine nucleotide antibiotics and flusilazole at normal dose for 24 hours, but cannot survive after co-culturing at 10 times dose for 24 hours. Bacillus ZZ-1 cannot survive after being treated with propanil at normal dose and 10 times dose.

[0089] Table 9 Tolerance of Bacillus ZZ-1 to common fungicides Note: "+" indicates viable bacteria, and "-" indicates no viable bacteria.

Claims

1. A Bacillus strain, characterized in that, The Bacillus is Bacillus subtilis, named ZZ-1, and is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Xibei Road, Beichen, Beijing, China, with a preservation center number of CGMCC No. 31374, and a preservation time of July 19, 2024.

2. The Bacillus of claim 1, characterized by, 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 according to claim 1 or 2.

4. Application of the biocontrol agent according to claim 3 in the antibacterial aspect of fruit and vegetable pathogens.

5. Application of the Bacillus according to claim 1 or 2 in the preparation of fruit and vegetable preservatives.

6. Use according to claim 4, characterized in that, The fruit and vegetable pathogens include Acremonium sclerotigenum, Actinomucor elegans, Alternaria alternata, Alternaria brassicae, Alternaria tenuissima, Apiospora mari, Aspergillus aculeatus, Aspergillus flavus, Aspergillus niger, Aspergillus sp., Aspergillus tubingensis, Aspergillus tubingensis, Aspergilus niger, Aspergilus tamarii, Bjerkandera adusta, Botryosphaeria dothidea, Botrytis sp., Cladosporium anthropophilum, Cladosporium colombiae, Cladosporium tenuissimum, Colletotrichum acutatum, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Colletotrichum siamense, Curvularia lunata, Diaporthe phoenicicola, Didymosphaeria variabile, Fusarium circinatum, Fusarium concentricum, Fusarium falciforme, Fusarium graminearum, Fusarium incarnatum, Fusarium metavorans, Fusarium oxysporum, Fusarium proliferatum, Fusarium proliferatum, Fusarium solani, Fusarium sp., Galactomyces candidum, Ganoderma sessile, Gibberella fujikuroi, Monilinia fructicola, Monilinia sp.Nectria rigidiuscula, Penicillium chrysogenum, Penicillium commune, Penicillium crustosum, Penicillium decumbens, Penicillium polonicum, Penicillium rubens, Penicillium sclerotiorum, Penicillium sp., Stagonosporopsis cucurbitacearum, Talaromyces sp., Talaromyces verruculosus.

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

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