Bacillus strain, biocontrol microbial agent prepared from bacillus strain, and use thereof

Abstract

The present invention provides a Bacillus strain, a biocontrol microbial agent prepared from the bacillus strain, and a use thereof. The Bacillus strain provided in the present invention is Bacillus subtilis, named QY2, for which the deposition institution is the China General Microbiological Culture Collection Center, the deposition address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposition center number is CGMCC No. 31373, and the deposition time is 19 July 2024. The Bacillus stain QY2 of the present invention, whether in the form of viable bacteria, a supernatant, or a cell lysate, exhibits inhibitory effects on various pathogens. The Bacillus stain QY2 has strong viability, wide applicability in terms of nutrient sources, temperature, humidity, and pH, no pathogenicity to fruits and vegetables, and relatively good tolerance to broad-spectrum microbicides.

Description

Bacillus, biocontrol agent prepared from the bacillus and application thereof 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 thereof 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 technology 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 Aspergillus pseudoglaucus, Colletotrichum siamense, Cladosporium tenuissimum 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 biocontrol strain with a wider spectrum of antibacterial effects and a very significant inhibition rate against Aspergillus pseudoglaucus, Colletotrichum siamense, Cladosporium tenuissimum 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 for Aspergillus pseudoglaucus, Colletotrichum siamense, Cladosporium tenuissimum and other fruit and vegetable pathogenic bacteria. 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 QY2, and is deposited at the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, China, with the accession number CGMCC No. 31373, and the deposit date is July 19, 2024.

[0010] The gene sequence of the 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 pathogenic bacteria 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 alternata, Alternaria tenuissima, Apiospora mari, Aspergillus flavus, Aspergillus pseudoglaucus, Aspergillus sp., Aspergillus tubingensis, Aspergilus niger, Aspergilus tamarii, Botryosphaeria dothidea, Botrytis sp., Cladosporium anthropophilum, Cladosporium colombiae, Cladosporium tenuissimum, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Colletotrichum siamense, Curvularia lunata, Diaporthe phoenicicola, Didymosphaeria variabile, Fusarium circinatum, Fusarium falciforme, Fusarium graminearum, Fusarium incarnatum, Fusarium metavorans, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Fusarium sp., Galactomyces candidum, Ganoderma sessile, Nectria rigidiuscula, Penicillium chrysogenum, Penicillium commune, Penicillium polonicum, Penicillium rubens, Penicillium sclerotiorum, Stagonosporopsis cucurbtacearum, Talaromyces sp., and Talaromyces verruculosus.

[0014] The concentration of the Bacillus QY2 in the biocontrol agent of the present application is (1 x 10 6-8 ) cfu / mL.

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

[0016] (1) The Bacillus QY2 live bacteria of the present application has certain bacteriostatic effect on different pathogenic bacteria. Among them, the bacteriostatic effect of Bacillus QY2 live bacteria on Botrytis sp., Aspergillus pseudoglaucus, Colletotrichum siamense and Cladosporium tenuissimum is the most significant, and the inhibition rate is 100%, which can completely inhibit the growth of pathogenic fungi. Among them, the inhibition rate of QY2 live bacteria on Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp., Diaporthe phoenicicola, Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Alternaria tenuissima and Acremonium sclerotigenum is greater than 90%, which has good inhibition effect; the bacteriostatic effect on Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans, Fusarium solani and Fusarium graminearum is general, and the inhibition rate is greater than 80%.

[0017] (2) The supernatant of Bacillus QY2 of the present application has certain bacteriostatic effect on different pathogenic fungi. The bacteriostatic effect of the supernatant of Bacillus QY2 on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp. and Diaporthe phoenicicola is the best, and the inhibition rate is greater than 90%. The supernatant of Bacillus QY2 has certain bacteriostatic effect on Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Alternaria tenuissima, Acremonium sclerotigenum, Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans and Fusarium solani, and the inhibition rate is greater than 80%.

[0018] (3) The Bacillus QY2 cell lysate of the present application has certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the QY2 supernatant. Among them, the Bacillus QY2 cell lysate has good antibacterial effect on Acremonium Sclerotigenum, Aureobasidium melanogenum, Botryosphaeria laricina, Botrytis cinerea, Botrytis fabae, Byssochlamys spectabilis, Cladosporium cladosporioides, Diaporthe eres, Fusarium decemcellulare, Fusarium proliferatum, Galactomyces geotrichum, Glomerella acutata, Lasiodiplodia theobromae, Monilinia polystroma, Mucor circinelloides, Penicillium camemberti and Penicillium verruculosum, and the inhibition rate is greater than 90%. The QY2 cell lysate has good antibacterial effect on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum and Fusarium incarnatum, and the inhibition rate is greater than 90%. The QY2 cell lysate has certain antibacterial effect on Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp., Diaporthe phoenicicola, Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Alternaria tenuissima, Acremonium sclerotigenum, Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile and Talaromyces verruculosus, and the inhibition rate is greater than 80%.

[0019] (4) The bacillus QY-2 provided by the present application has a wide antibacterial spectrum, involving 42 kinds of fruit and vegetable pathogenic bacteria, 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 it involves only 32 kinds of fruit and vegetable pathogenic bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0020] Fig. 1 is the inhibition rate of QY2 live bacteria on different pathogenic bacteria;

[0021] Fig. 2 is the turbidity of bacillus culture at different pH values;

[0022] Fig. 3 is the turbidity of bacillus culture at different pH values. DETAILED DESCRIPTION

[0023] The present application will be specifically described below by 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] In 2023, the leaves of Bletilla striata were collected in a greenhouse of Chengdu Agricultural and Forestry Academy in Wenjiang District of Chengdu City, packed in a sterile sealed bag, and taken back to the Institute of Agricultural Products of Chengdu Agricultural and Forestry Academy 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, which is named as bacillus QY2. The preservation unit is: China General Microbiological Culture Collection Center, the preservation address is: No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the preservation center number is: CGMCC No. 31373, and the preservation time is: July 19, 2024.

[0029] III. Inhibition test of the strain

[0030] (I) Inhibition test of live bacteria of bacillus QY2

[0031] The strain has rich antibacterial types, and has good control effect on pathogenic bacteria on strawberry, blueberry, grape, apple, mango, cherry, kiwi fruit, winter jujube, pepper, lotus, eggplant, garlic and sponge gourd.

[0032] 1. Test design

[0033] The QY2 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 to be 1×10 8 cfu / mL using a hemocytometer. The QY2 strain cell suspension was added to PDA medium and mixed by shaking to make plates, with a QY2 strain cell density of 1×10 6 cfu / mL per plate. A 6mm sterile punch was used to take a gray botrytis cake and place it in the center of the PDA plate containing the QY2 strain. A PDA plate containing 100mg / L carbendazim was used as a pesticide control, and a PDA plate medium without QY2 strain was used as a blank control, with 3 replicates per treatment. The plates with the fungus cake were sealed with a self-sealing bag and placed in a 26°C constant temperature incubator for culture. When the mycelium of the control group was full, the width of the inhibition zone was measured.

[0034] The inhibition rate R R(%) of mycelial growth was 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 test results are shown in Table 1 and Figure 1. As can be seen from Figure 1 and Table 1, the live Bacillus QY2 has certain inhibition effect on different pathogenic bacteria. The live Bacillus QY2 has the most significant inhibition effect on Botrytis sp., Aspergillus pseudoglaucus, Colletotrichum siamense and Cladosporium tenuissimum, and the inhibition rate is 100%, which can completely inhibit the growth of pathogenic fungi. Among them, the live Bacillus QY2 has an inhibition rate of more than 90% on Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp., Diaporthe phoenicicola, Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Alternaria tenuissima and Acremonium sclerotigenum, and has a good inhibition effect; the live Bacillus QY2 has a certain inhibition effect on Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans, Fusarium solani and Fusarium graminearum, and the inhibition rate is more than 80%.

[0038] Compared with the inhibition rate of 100 mg / L carbendazim, QY2 live bacteria had more prominent inhibition effect on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum, Penicillium chrysogenum, Aspergillus sp., Talaromyces sp., Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Penicillium sclerotiorum, Ganoderma sessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillus flavus, Didymosphaeria variabile, Fusarium metavorans, Aspergilus niger, Aspergilus tamarii, Aspergillus tubingensis, Galactomyces candidum and Stagonosporopsis cucurbtacearum, and the inhibition rate was significantly higher than that of 100 mg / L carbendazim treatment. The inhibition effect of 100 mg / L carbendazim treatment on Botryosphaeria dothidea, Diaporthe phoenicicola, Fusarium falciforme, Alternaria alternata, Fusarium solani, Fusarium graminearum, Colletotrichum fioriniae, Fusarium sp., Colletotrichum gloeosporioides, Penicillium commune, Fusarium circinatum, Fusarium proliferatum and Fusarium oxysporum was better, and the inhibition rate was higher than that of Bacillus QY2 live bacteria treatment.The inhibition effect of QY2 living bacteria and 100mg / L carbendazim on Fusarium incarnatum, Alternaria tenuissima, Acremonium sclerotigenum, Penicillium polonicum and Penicillium rubens was similar.

[0039] Table 1 Inhibition effect of Bacillus QY2 living bacteria and carbendazim on different pathogenic fungi

[0040] (II) Inhibition test of supernatant of Bacillus QY2

[0041] The main purpose of the supernatant inhibition test is to test 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 24h of culture, Bacillus QY2 was removed by repeated centrifuge filtration to prepare QY2 supernatant. 200μL of supernatant was evenly coated on PDA plates, and different pathogenic fungi were placed on the surface of PDA plates with 6mm sterile puncher. The same amount of sterile water was used as 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.

[0044] The inhibition rate R R(%) of mycelial growth was calculated as follows: R R(%)=(R1-R2) / R1×100%

[0045] 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.

[0046] 2. Test results

[0047] The antibacterial effects of the supernatant of Bacillus QY2 on different pathogenic fungi are shown in Table 2. Among them, the antibacterial effect of the supernatant of QY2 on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp. and Diaporthe phoenicicola is the best, and the inhibition rate is more than 90%. The supernatant of Bacillus QY2 has certain antibacterial effect on Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Alternaria tenuissima, Acremonium sclerotigenum, Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans and Fusarium solani, and the inhibition rate is more than 80%. The supernatant of Bacillus QY2 has general antibacterial effect on Fusarium graminearum, Aspergilus niger, Colletotrichum fioriniae, Fusarium sp., Aspergilus tamarii, Colletotrichum gloeosporioides, Penicillium polonicum, Penicillium commune and Fusarium circinatum, and the inhibition rate is more than 70%.

[0048] Table 2 Antibacterial effect of the supernatant of Bacillus QY2 on different pathogenic fungi

[0049] (III) Antibacterial test of Bacillus QY2 cell lysate

[0050] 1. Test design

[0051] Add 10 ml sterile normal saline to Bacillus QY2 live bacteria, shake well, and put the shaken solution into a 50 ml centrifuge tube. Crush the QY2 strain cells in an ultrasonic cell crusher. Filter the crushed cell solution once with a 0.22 μm pore size micropore filter to obtain a liquid, which is the Bacillus QY2 cell lysate, and store at 4°C for later use.

[0052] Take 200 μL of the cell lysate and evenly spread it on a PDA plate. Place different pathogenic fungal mold cakes on the surface of the PDA plate in the center with a 6 mm sterile puncher, and use an equal amount of sterile water as a control. Repeat each treatment three times. Place the prepared plates in a 26°C constant temperature incubator for culture, and measure the inhibition zone width when the control plate mycelium is full. The mycelial growth inhibition rate R formula is the same as above.

[0053] 2. Test results

[0054] As can be seen from Table 3, the cell lysate of Bacillus QY2 has certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the supernatant of QY2 strain. Among them, the cell lysate of Bacillus QY2 has good antibacterial effect on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum and Fusarium incarnatum, and the inhibition rate is greater than 90%. The cell lysate of QY2 has certain antibacterial effect on Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp., Diaporthe phoenicicola, Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Alternaria tenuissima, Acremonium sclerotigenum, Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile and Talaromyces verruculosus, and the inhibition rate is greater than 80%. The cell lysate of QY2 has general antibacterial effect on Curvularia lunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans, Fusarium solani, Fusarium graminearum, Aspergilus niger and Colletotrichum fioriniae, and the inhibition rate is greater than 70%.

[0055] Table 3 Antibacterial effect of Bacillus QY2 cell lysate on different pathogenic fungi

[0056] Four, life characteristics of biocontrol bacteria

[0057] The living ability of Bacillus QY2 is strong, mainly in temperature, pH, field nutrient source and humidity environment.

[0058] 1. Temperature adaptability

[0059] (1) Test design

[0060] The QY2 strain was inoculated into LB broth and cultured at 4°C, 16°C, 28°C, 37°C, 60°C, and 150 r / min in a shaker, with 3 replicates for each treatment. After 12 h, 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, with 3 replicates for each treatment. The suspension density = (100-transmittance) x 100%.

[0061] (2) Test results

[0062] The test results are shown in Table 4 and Figure 2. Bacillus QY2 showed good survival ability at 28-37°C, with a suspension density of the culture solution of more than 50% after 12 h. At 16°C and 45°C, Bacillus QY2 could grow, but the growth was slow, with a suspension density of the culture solution of 45.55% and 36.22% after 12 h, respectively. At 4°C or 60°C, it was difficult for Bacillus QY2 to reproduce in vivo under these environmental conditions, and the suspension density of Bacillus QY2 was 0 after 12 h of culture.

[0063] Table 4 Adaptability of Bacillus QY2 to different temperatures

[0064] 2. Acid-base adaptability

[0065] (1) Test design

[0066] 1 mol / L HCl or 1 mol / L NaOH was used to adjust the pH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. After sterilization, 1 mL of Bacillus QY2 seed culture was inoculated into 100 mL of LB liquid medium adjusted to different pH values, and cultured at 37°C and 150 r / min in a shaker. After 12 h, 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, with 3 replicates for each treatment. The suspension density = (100-transmittance) x 100%.

[0067] (2) Test results

[0068] The test results are shown in Table 5 and Figure 3. As shown in Table 5 and Figure 2, the turbidity of the culture solution of Bacillus QY2 is greater than 50% after 12 hours under the condition of pH = 5-6, indicating that Bacillus QY2 has good survival ability under the condition of pH = 5-6. Under the conditions of pH = 5 and pH = 9, the turbidity of the culture solution is 44.45% and 30.20% respectively after 12 hours, indicating that Bacillus QY2 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 QY2 is basically difficult to grow in strong acid or strong alkali environment.

[0069] Table 5 Bacillus QY2 different pH culture adaptability test

[0070] 3. Field nutrient source adaptability

[0071] (1) Test design

[0072] According to the nutrient classification standard of the second national soil survey 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 water content 70%. According to the above conditions, the N, P, K element nutrients are designed as follows:

[0073] (2) Test results

[0074] As shown in Table 7, Bacillus QY2 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 QY2 can survive for more than 60 days. QY2 strain can survive in the case of low content of carbon, nitrogen, phosphorus, and potassium elements. In the case of lack of carbon, nitrogen, phosphorus, and potassium elements respectively, Bacillus QY2 can survive in soil for more than 60 days, indicating that QY2 strain does not depend on a certain specific nutrient element for survival, and can provide nutrients for itself through other elements.

[0075] Table 6 Bacillus QY2 field nutrient single factor level table Note: In each single factor, the non-variable factor maintains the highest soil nutrient condition formula.

[0076] Table 7 60d Bacillus QY2 nutrient 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 moisture adaptability

[0078] (1) Test design

[0079] In sterile sand tubes, add sterile nutrient solution with total nitrogen 2 g / kg, total phosphorus 1 g / kg, total potassium 20 g / kg, and control the moisture at 10%, 30%, 50%, and 70%. Inoculate 1 mL of QY2 seed culture solution, cover with sealing film, and incubate at 37°C. Repeat 3 times for each treatment. After 60 days, dissolve the soil sample in saline, take a small amount of sand and spread on LB plates, and observe the presence or absence of live bacteria and their number.

[0080] (2) Test results

[0081] As shown in Table 8, the survival of Bacillus QY2 after 60 days under different field moisture conditions. The results showed that Bacillus QY2 could show good survival ability after 60 days under the condition of soil moisture ≥ 30%. When the moisture content was 10%, Bacillus QY2 could not survive for more than 60 days.

[0082] The strain has strong tolerance to high temperature and high humidity environment, and is not adapted to very low temperature and humidity environment, but soil moisture content below 10% will not occur in normal crop production areas, so the viability of this 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 QY2 adaptability to different field moisture Note: “++” indicates that the number of live bacteria ≥ 10 8 cfu / mL, “+” indicates that the number of live bacteria ≥ 10 6 cfu / mL, “-” indicates no live bacteria.

[0084] Six, tolerance to broad-spectrum fungicides

[0085] 1. Test design

[0086] Pyraclostrobin, tebuconazole, epoxiconazole, carbendazim, azoxystrobin, propiconazole, zoxamide, mancozeb, myclobutanil, metconazole, boscalid, fluopimide, iprodione, mepanipyrim, 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; the LB liquid medium without fungicides and biocontrol bacteria was used as a control to inoculate the same concentration of QY2 strain. After 24h of culture at 37℃ and 120r / min in a shaking bed, the LB liquid medium was taken with an inoculation loop and streaked on LB plates, and the growth of QY2 strain was observed, with 3 repeats for each treatment.

[0087] 2. Test results

[0088] The test results are shown in Table 9. Bacillus QY2 has different resistance to different fungicides. Among them, normal dose and 10 times dose of pyraclostrobin, epoxiconazole, carbendazim, azoxystrobin, zoxamide, mancozeb, myclobutanil, metconazole, boscalid and fluopimide were co-cultured with Bacillus QY2 for 24h, and Bacillus QY2 could grow on LB plates, indicating that Bacillus QY2 had good resistance to the 10 commonly used fungicides at low concentrations and could survive in the environment sprayed with the 10 fungicides. Bacillus QY2 could grow after co-culturing with normal dose of tebuconazole, propiconazole, iprodione, mepanipyrim, pyrimidine nucleotide antibiotic and flusilazole for 24h, but could not survive after co-culturing with 10 times dose for 24h. Bacillus QY2 could not survive after treatment with normal dose and 10 times dose of prochloraz.

[0089] Table 9 Tolerance of Bacillus QY2 to common fungicides Note: "+" indicates viable bacteria, "-" indicates no viable bacteria.

Claims

1. A Bacillus strain, characterized in that, The Bacillus is Bacillus subtilis, named QY2, and is preserved in the China General Microbiological Culture Collection Center, located at No.3, Institute of Microbiology, Chinese Academy of Sciences, 1st North Chenxi Road, Chaoyang District, Beijing, with a preservation center number of CGMCC No.31373, and a preservation time of July 19, 2024.

2. The Bacillus of claim 1, characterized by, The gene sequence of the Bacillus is shown as 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 tenuissima, Apiospora mari, Aspergillus flavus, Aspergillus pseudoglaucus, Aspergillus sp., Aspergillus tubingensis, Aspergilus niger, Aspergilus tamarii, Botryosphaeria dothidea, Botrytis sp., Cladosporium anthropophilum, Cladosporium colombiae, Cladosporium tenuissimum, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Colletotrichum siamense, Curvularia lunata, Diaporthe phoenicicola, Didymosphaeria variabile, Fusarium circinatum, Fusarium falciforme, Fusarium graminearum, Fusarium incarnatum, Fusarium metavorans, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Fusarium sp., Galactomyces candidum, Ganoderma sessile, Nectria rigidiuscula, Penicillium chrysogenum, Penicillium commune, Penicillium polonicum, Penicillium rubens, Penicillium sclerotiorum, Stagonosporopsis cucurbtacearum, Talaromyces sp., and Talaromyces verruculosus.

7. Use according to claim 4, characterized in that, The concentration of Bacillus QY2 in the biocontrol agent is 1 x 10 6-8 cfu / L.

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