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

Abstract

The invention relates to bacillus, a biocontrol inoculant prepared from the bacillus and application of the biocontrol inoculant, and belongs to the technical field of biological inoculants. The bacillus provided by the invention is bacillus subtilis and is named as ZZ1, the preservation unit is China General Microbiological Culture Collection Center, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the preservation center is numbered as CGMCC No.31374, and the preservation time is July 19, 2024. The bacillus ZZ1 disclosed by the invention has a certain bacteriostatic effect on different pathogenic bacteria regardless of living bacteria, supernate or cell lysis solution. The bacillus ZZ1 is high in living ability and wide in application range of nutrient sources, temperature, humidity and pH value. The compound has no pathogenicity to fruits and vegetables, has good tolerance to broad-spectrum bactericides, and has a great application prospect.

Description

Technical Field

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

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

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

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

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

[0006] However, although the currently developed biocontrol strains and biocontrol agents have shown certain effects, and some have broad-spectrum antibacterial properties, the inhibition rate of existing Bacillus biocontrol agents is generally low. Moreover, for fruit and vegetable pathogens such as Actinomucor elegans, Alternaria sp., and Aspergillus sp., there are currently few biocontrol strains found, and their inhibition rates are not high.

[0007] Therefore, there is a need to provide a Bacillus subtilis biocontrol strain having a broader spectrum of antibacterial effects and a very significant inhibition rate against fruit and vegetable pathogens such as Actinomucor elegans, Alternaria sp., and Aspergillus sp. Summary of the invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a biocontrol strain that can be used for disease control and bacteriostasis and preservation of fruits and vegetables. The present invention provides a Bacillus sp., which can be effectively used for disease control and bacteriostasis and preservation of fruits and vegetables, especially Actinomucor elegans, Alternaria sp., Aspergillus sp. and other pathogenic bacteria. The present invention also provides a biocontrol agent made from the Bacillus sp. and its application in antibacterial and bacteriostasis preservation of fruits and vegetables.

[0009] After a lot of experimental research and exploration, the inventors of the present invention obtained a strain of Bacillus subtilis, which is Bacillus subtilis, named ZZ1, 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.31374, and the preservation time is: July 31, 2024.

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

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

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

[0013] Specifically, the fruit and vegetable pathogens include Acremonium sclerotigenum, Actinomucorelegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthephoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichumcandidum, Moniliniafructicola, Periconia byssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporiumtenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichum gloeosporioides, Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsis cucurbtacearum, Stemphyliumeturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme, Fusariumtricinctum, Penicillium polonicum, Fusarium proliferatum, Gibberella fujikuroi, Fusarium avenaceum and Cladosporium colombiae. .

[0014] The concentration of Bacillus ZZ1 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 living bacillus ZZ1 of the present invention has a certain antibacterial effect on different pathogens. Among them, the living bacillus ZZ1 has the most significant 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, Didymosphaeriavariabile, Galactomyces candidum, Geotrichum candidum, Moniliniafructicola, Periconia byssoides and Talaromyces verruculosu, with an antibacterial rate of 100%, and can completely inhibit the growth of pathogenic fungi.Among them, ZZ1 viable cells are effective against Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichum The antibacterial rate of gloeosporioides, Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp. and Fusarium oxysporum is greater than 90%, and has a good inhibitory effect; it has a good inhibitory effect on Stagonosporopsis cucurbtacearum, Stephyliumeturmiunum, Fusariumfujikuroi, Aspergillus japonicus, Lasiodiplodiapseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme, Fusariumtricinctum and Penicillium polonicum had average antibacterial effects, with an inhibition rate of more than 80%.

[0017] (2) The supernatant of Bacillus ZZ1 of the present invention has a certain antibacterial effect on different pathogenic fungi. Among them, Bacillus ZZ1 supernatant was effective against Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeriavariabile, 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. had the best antibacterial effects, with inhibition rates all greater than 90%.The supernatant of Bacillus ZZ1 had a general antibacterial effect on Alternariaalstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsiscucurbtacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillusjaponicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusariumfalciforme and Fusarium tricinctum, with the inhibition rates all greater than 80%.

[0018] (3) The Bacillus ZZ1 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 ZZ1 strain supernatant. Among them, Bacillus ZZ1 cell lysate is effective against Acremoniumsclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporiumcladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichumfioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides, Talaromycesverruculosus, Penicillium sclerotiorum, Penicilliummallochil, Penicilliumchrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii and Colletotrichum gloeosporioides had better antibacterial effects, with an inhibition rate of more than 90%.The antibacterial effect of ZZ1 cell lysate on Penicillium rubens, Pestalotiasubcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp., Fusariumoxysporum, Stagonosporopsis cucurbtacearum, Stemphylium eturmiunum, Fusariumfujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae and Talaromycess p. was general, and the inhibition rate was greater than 80%.

[0019] (4) The Bacillus ZZ1 provided by the present invention has a wide antibacterial spectrum, covering 45 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 covers 32 fruit and vegetable pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the inhibition rate of ZZ1 live bacteria against different pathogens;

[0021] Figure 2 The turbidity of the suspension of Bacillus ZZ1 cultured at different temperatures;

[0022] Figure 3 The turbidity of suspension cultured at different pH values ​​for Bacillus ZZ1; DETAILED DESCRIPTION

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

[0024] Example

[0025] 1. Source of strains

[0026] In 2002, soil was collected from a fir tree planting base in Hongya County, Leshan 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 for isolation and purification of antagonistic bacteria.

[0027] 2. Identification of strains

[0028] The DNA of the bacteria was extracted, and the 16S rDNA sequence was analyzed by PCR. The gene sequence is shown in the sequence table SEQ NO.1. The obtained sequence was compared on the NCBI website and determined to be Bacillus subtilis, named ZZ1, and the deposited 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. 31374, and the deposit time is: July 19, 2024.

[0029] 3. Antibacterial test of strains

[0030] (I) Antibacterial test of live bacteria of Bacillus ZZ1

[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, rice, eggplants, garlic, and loofahs.

[0032] 1. Experimental Design

[0033] A single colony of the ZZ1 strain was inoculated into LB broth and cultured at 28°C with constant shaking at 150 rpm for 24 h. The cell suspension density was determined as 1 × 10 8 cfu / mL. Add the cell suspension of strain ZZ1 to the PDA medium, shake and mix to make a plate. The cell density of strain ZZ1 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 ZZ1 strain. Use the PDA plate containing 100mg / L carbendazim as the pesticide control, and the PDA plate medium without ZZ1 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 ZZ1 live bacteria have certain antibacterial effects on different pathogens. ZZ1 live bacteria have the most significant antibacterial effect on Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporiumoxysporum, Clathrospora diplospora, Colletotrichum fioriniae, Colletotrichumspaethianum, Diaporthe amygdali, Diaporthefusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Moniliniafructicola, Periconia byssoides and Talaromyces verruculosus, with an inhibition rate of 100%, which can completely inhibit the growth of pathogenic fungi.The ZZ1 live bacteria had an inhibition rate of more than 90% against Penicillium sclerotiorum, Penicillium mallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozymaguilliermondii, Colletotrichum gloeosporioides, Penicillium rubens, Pestalotia subcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp. and Fusarium oxysporum, and had a good inhibitory effect; it also had a good inhibitory effect on Stagonosporopsis cucurbtacearum, Stemphylium eturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme, Fusarium tricinctum and Penicilliumpolonicum had certain antibacterial effects, with the inhibition rate being greater than 80%.

[0039] Compared with the antibacterial rate of 100mg / L carbendazim, ZZ1 viable bacteria were effective against Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporiumcladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Diaportheamygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconiabyssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicilliummallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporiumxanthochromaticum, Cladosporium tenuissimum, Penicllium astrolabium, Penicilliumsolitum, Meyerozyma guilliermondii, Penicillium rubens, Pestalotiasubcuticularis, Acremonium sp., Alternaria alstroemeriae, Fusarium oxysporum, Stagonosporopsis cucurbtacearum, Stemphylium eturmiunum, Aspergillus japonicus, Lasiodiplodia pseudotheobromae, Talaromyces sp., Penicillium polonicum, Gibberella fujikuroi, Fusarium avenaceum and Cladosporium colombiae had more prominent antibacterial effects, and the antibacterial rates were significantly greater than those of the 100 mg / L carbendazim treatment.After treatment with 100 mg / L carbendazim, the antibacterial effect on Botrytis sp., Fusarium solani, Fusarium falciforme and Fusarium tricinctum was better, and the antibacterial rate was greater than that of Bacillus ZZ1 live bacteria treatment. After treatment with ZZ1 live bacteria and 100 mg / L carbendazim on Colletotrichum fioriniae, Colletotrichumspaethianum, Fusariumincarnatum, Colletotrichum gloeosporioides, Fusariumfujikuroi and Fusarium proliferatum, the antibacterial effect was similar.

[0040] Table 1 Antibacterial effects of live Bacillus ZZ1 and carbendazim on different pathogens

[0041]

[0042]

[0043] (II) Antibacterial test of supernatant of Bacillus ZZ1

[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 Bacillus ZZ1 for 24 hours, the live cells were removed by repeated centrifugation filtration to prepare ZZ1 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 ZZ1 supernatant on different pathogenic fungi are shown in Table 2. Among them, ZZ1 supernatant was effective against Acremonium sclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillus sp., Cladosporium cladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichumfioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Moniliniafructicola, Periconiabyssoides, Talaromyces verruculosus, Penicillium sclerotiorum, Penicilliummallochil, Penicillium chrysogenum, Colletotrichum siamense, Cladosporiumxanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Peniclliumastrolabium, Penicillium solitum, Meyerozyma guilliermondii, Colletotrichumgloeosporioides, Penicillium rubens, Pestalotia subcuticularis and Acremonium sp. had the best antibacterial effects, with inhibition rates all greater than 90%.The supernatant of Bacillus ZZ1 had a certain antibacterial effect on Alternaria alstroemeriae, Botrytis sp., Fusarium oxysporum, Stagonosporopsis cucurbtacearum, Stemphyliumeturmiunum, Fusarium fujikuroi, Aspergillus japonicus, Lasiodiplodiapseudotheobromae, Talaromyces sp., Fusarium solani, Fusarium falciforme and Fusarium tricinctum, and the antibacterial rate was greater than 80%. The supernatant of Bacillus ZZ1 had a general antibacterial effect on Penicillium polonicum, Fusarium proliferatum, Gibberella fujikuroi, Fusariumavenaceum and Cladosporium colombiae, and the antibacterial rate was greater than 70%.

[0052] Table 2 Antibacterial effect of Bacillus ZZ1 supernatant on different pathogenic fungi

[0053]

[0054]

[0055] (III) Antibacterial test of Bacillus ZZ1 cell lysate

[0056] 1. Experimental Design

[0057] Add 10 ml of sterile saline to the live cells of Bacillus ZZ1 and shake well. Put the shaken solution into a 50 ml centrifuge tube and crush the ZZ1 strain cells in an ultrasonic cell crusher. Filter the cell crushed solution once with a microporous membrane filter with a pore size of 0.22 μm to obtain a liquid, which is the Bacillus ZZ1 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 cell lysate of Bacillus ZZ1 has a certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the supernatant of ZZ1 strain. Among them, Bacillus ZZ1 cell lysate is effective against Acremoniumsclerotigenum, Actinomucor elegans, Alternaria sp., Aspergillussp., Cladosporiumcladosporioides, Cladosporium oxysporum, Clathrospora diplospora, Colletotrichumfioriniae, Colletotrichum spaethianum, Diaporthe amygdali, Diaporthe fusicola, Diaporthe phoenicicola, Didymosphaeria variabile, Galactomyces candidum, Geotrichum candidum, Monilinia fructicola, Periconia byssoides, Talaromycesverruculosus, Penicillium sclerotiorum, Penicilliummallochil, Penicilliumchrysogenum, Colletotrichum siamense, Cladosporium xanthochromaticum, Cladosporium tenuissimum, Fusarium incarnatum, Penicllium astrolabium, Penicillium solitum, Meyerozyma guilliermondii and Colletotrichum gloeosporioides had better antibacterial effects, with an inhibition rate of more than 90%.ZZ1 cell lysate had a certain antibacterial effect on Penicillium rubens, Pestalotiasubcuticularis, Acremonium sp., Alternaria alstroemeriae, Botrytis sp., Fusariumoxysporum, Stagonosporopsis cucurbtacearum, Stemphylium eturmiunum, Fusariumfujikuroi, Aspergillus japonicus, Lasiodiplodia pseudotheobromae and Talaromycess sp., and the inhibition rate was greater than 80%. ZZ1 cell lysate had a general antibacterial effect on Fusarium solani, Fusarium falciforme, Fusarium tricinctum, Penicillium polonicum, Fusariumproliferatum, Gibberella fujikuroi and Fusarium avenaceum, and the inhibition rate was greater than 70%.

[0061] Table 3 Antibacterial effect of Bacillus ZZ1 cell lysate on different pathogenic fungi

[0062]

[0063]

[0064] 4. Life characteristics of biocontrol bacteria

[0065] Bacillus ZZ1 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] The ZZ1 strain was inoculated into LB broth and cultured in a shaker at 4°C, 16°C, 28°C, 37°C, and 60°C at 150 r / min. Each treatment was repeated 3 times. After 12 hours, the absorbance and transmittance were measured at a wavelength of 600 nm using a UV-visible spectrophotometer. The LB liquid culture medium without bacteria was used as a blank control, and each treatment was repeated 3 times. Suspension turbidity = (100-transmittance) × 100%.

[0069] (2) Test results

[0070] The test results are shown in Table 4 and Figure 2As shown in the figure, Bacillus ZZ1 showed good survival ability at 16-37℃, and the turbidity of the culture solution was greater than 50% after 12h. Bacillus ZZ1 could grow at 45℃, but the growth was slow, and the turbidity of the culture solution was 32.97% after 12h. It was difficult for Bacillus ZZ1 to reproduce alive at 4℃ or 60℃, and the turbidity of Bacillus ZZ1 was 0 after 12h of culture.

[0071] Table 4 Determination of adaptability of Bacillus ZZ1 culture at different temperatures

[0072]

[0073]

[0074] 2. pH adaptability

[0075] (1) Experimental design

[0076] Use 1mol / L HCl or 1mol / L NaOH to adjust the pH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively. After sterilization, inoculate 1mL of Bacillus ZZ1 seed culture solution into 100mL LB liquid medium adjusted to different pH values, and culture at 37℃, 150r / min in a shaker. After 12h, measure the absorbance and transmittance at 600nm wavelength with a UV-visible spectrophotometer. Use the LB liquid medium without bacteria as the blank control, and repeat 3 times for each treatment. Suspension turbidity = (100-transmittance) × 100%.

[0077] (2) Test results

[0078] The test results are shown in Table 5 and Figure 3 From Table 5 and Figure 2 It can be seen that the turbidity of the culture solution of Bacillus ZZ1 is greater than 45% after 12 hours under the condition of pH = 6-8, indicating that Bacillus ZZ1 has good survival ability under the condition of pH = 6-8. Under the conditions of pH = 5 and pH = 9, the turbidity of the culture solution after 12 hours is 16.05% and 22.23% respectively, indicating that Bacillus ZZ1 can grow under acidic and alkaline conditions, but the growth is relatively slow. When pH <5 or >9, the turbidity of the culture solution is less than 10% after 12 hours, indicating that Bacillus ZZ1 is basically difficult to grow in strong acid or strong alkaline environments.

[0079] Table 5 Determination of the adaptability of Bacillus ZZ1 culture at different pH values

[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] It can be seen from Table 7 that Bacillus ZZ1 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 ZZ1 can survive well for more than 60 days. The ZZ1 strain can survive when the nitrogen, phosphorus, and potassium content is low. In the absence of nitrogen and potassium, respectively, Bacillus ZZ1 can survive in the soil for more than 60 days, but in the absence of phosphorus, Bacillus ZZ1 cannot survive in the soil for more than 60 days.

[0086] Table 6 Single factor level table of field nutrient sources of Bacillus ZZ1

[0087] Table 6 Single factor level table of nutrient sources for BacillusZZ1 in the field

[0088]

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

[0090] Table 760d Bacillus ZZ1 nutritional source adaptability

[0091] Table 7 The adaptability of the nutritional source of Bacillus ZZ1 at60days

[0092]

[0093] Note: “++” means the number of viable cells is ≥10 8 cfu / mL, "+" means the number of viable bacteria is ≥10 6 cfu / mL, “-” means no viable bacteria.

[0094] 4. Field humidity adaptability

[0095] (1) Experimental design

[0096] In a sterile sand tube, add a sterile nutrient solution with nutrient conditions of 23.59% organic carbon, 0.98% total nitrogen, 0.85% total phosphorus, and 2.75% total potassium, and control the water content at 10%, 30%, 50%, and 70%. Inoculate 1mL of ZZ1 seed culture solution respectively, 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.

[0097] (2) Test results

[0098] Table 8 shows the survival of Bacillus ZZ1 after 60 days under different field moisture content conditions. The results show that Bacillus ZZ1 can show good survival ability after 60 days under the condition of soil moisture content ≥30%. When the moisture content is 10%, Bacillus ZZ1 cannot survive for more than 60 days.

[0099] This strain has a strong tolerance to high humidity environments and is not adapted to extremely low temperature and humidity environments. However, soil moisture content below 10% does not occur in normal crop production areas, so the vitality of this strain is suitable for most crop soils. When the air humidity is high, this strain can be mixed with inorganic nutrient solution and sprayed on the surface of crops as foliar fertilizer and protective agent.

[0100] Table 8 Adaptability of 60d Bacillus ZZ1 to different field water contents

[0101]

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

[0103] 6. Tolerance to broad-spectrum fungicides

[0104] 1. Experimental Design

[0105] A total of 17 chemical fungicides, including pyraclostrobin, tebuconazole, epoxiconazole, carbendazim, azoxystrobin, propiconazole, oxazolidinone, myclobutanil, mancozeb, difenoconazole, boscalid, fluopyram, iprodione, 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 ZZ1 strain as a control. The culture was cultured in a shaking incubator at 37℃ 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 ZZ1 strain, with 3 replicates per treatment.

[0106] 2. Test results

[0107] The test results are shown in Table 9, and Bacillus ZZ1 has different resistance to different fungicides. Among them, after the normal dose and 10 times the dose of pyraclostrobin, tebuconazole, fluopyram, carbendazim, azoxystrobin, propiconazole, oxazolidinone, myclobutanil, difenoconazole, boscalid, fluopyram, isoproterenol and myclobutanil were co-cultured with Bacillus ZZ1 for 24 hours, Bacillus ZZ1 could grow on the LB plate, indicating that Bacillus ZZ1 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 ZZ1 can grow live bacteria after co-cultured with normal doses of mancozeb, pyrimidine nucleotide antibiotics and flusilazole for 24 hours, but cannot survive after co-cultured with 10 times the dose for 24 hours. Bacillus ZZ1 cannot survive after being treated with normal doses and 10 times the dose of prochloraz.

[0108] Table 9 Tolerance of Bacillus ZZ1 to common fungicides

[0109]

[0110]

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

[0112]

[0113]

Claims

1. A Bacillus strain, characterized in that: The Bacillus is Bacillus subtilis, named ZZ1, and the preservation unit is: General Microbiology Center of China Culture Collection Administration, the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation center number is: CGMCC No. 31374, 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 Acremoniumsclerotigenum, Actinomucor elegans, Alternaria alternata, Alternaria brassicae, Alternaria tenuissima, Apiospora mari, Aspergillus aculeatus, Aspergillus flavus, Aspergillus niger, Aspergillus sp., Aspergillus tubingensis, Aspergillustubingensis, Aspergilus niger, Aspergilus tamarii, Bjerkandera adusta, Botryosphaeria dothidea, Botrytis sp., Cladosporium anthropophilum, Cladosporium colombiae, Cladosporium tenuissimum, Colletotrichum acutatum, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Colletotrichumsiamense, Curvularia lunata, Diaporthephoenicicola, 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 cucurbtacearum, Talaromyces sp., Talaromyces verruculosus.

7. The use according to claim 4, characterized in that: The concentration of Bacillus ZZ1 in the biocontrol agent is 1×10 6-8 cfu / L.

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