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

Abstract

The present application relates to a bacillus, a biocontrol agent made from the bacillus and an application thereof, and belongs to the technical field of biological bacterial agents. The bacillus provided by the present application is Bacillus subtilis, named QY2, and the preservation unit is the General Microbiological Center of China Microorganism Bacterial Strain Preservation Management Committee, the preservation address is the Institute of Microbiology of Chinese Academy of Sciences, No. 3, Xili, Beichen, Chaoyang District, Beijing, the preservation center number is CGMCC No. 31373, and the preservation time is July 19, 2024. The bacillus QY2 has certain inhibition effects on different pathogenic bacteria, whether it is a living bacterium, a supernatant or a cell lysate. The bacillus QY2 has strong living ability, and is suitable for a wide range of nutrition sources, temperature and humidity and pH. The bacillus QY2 has no pathogenicity to fruits and vegetables, and has good tolerance to a wide range of fungicides, and has great application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological agents technology, specifically relating to a strain of Bacillus, a biocontrol agent made from the Bacillus, and its application in antibacterial and antimicrobial preservation of fruits and vegetables. Background Technology

[0002] Fruits and vegetables are susceptible to soil-borne pathogens during cultivation, frequently threatened by bacterial and fungal infections, with fungal diseases being the most common. Common symptoms include necrosis (leaf spot, leaf blight), rot (root rot, fruit rot), and wilting (infection of roots, stem base, and vascular tissue). Traditionally, disease control methods include chemical fungicides, breeding disease-resistant varieties, intercropping, and grafting, but each of these measures has its advantages and disadvantages.

[0003] On the other hand, fresh fruits and vegetables undergo changes in their physiological and biochemical characteristics after harvest and during long-term storage, making them susceptible to varying degrees of infectious diseases (pathogenic microbial infection). Postharvest infectious diseases in fruits and vegetables are caused by pathogenic microorganisms infecting the host plants during their growth and development in the field. Cross-contamination during transportation and storage is also a contributing factor. The presence of pores in the skin of most fruits and vegetables, as well as wounds caused by improper transportation, facilitates the invasion of pathogenic microorganisms into the internal flesh, exacerbating the severity of postharvest infectious diseases. Currently, the main methods for controlling postharvest infectious diseases in fruits and vegetables are pre-harvest pesticide spraying and postharvest lowering of storage temperature. However, excessive pesticide use can lead to serious pesticide residue exceeding standards, endangering human health. Furthermore, long-term excessive pesticide use can cause pathogenic microorganisms to develop resistance, significantly reducing the effectiveness of disease control.

[0004] Biological control is a technique that uses live 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 inhibit and kill pathogenic fungi on the one hand, and induce plants to improve their disease resistance on the other.

[0005] Biological control methods have significant advantages over chemical control methods. Besides the ease of production and use of biological agents, mixed inoculants made from biocontrol bacteria and their metabolites are more environmentally friendly, producing no pollution or pesticide residues, thus aligning with my country's sustainable development strategy. Furthermore, biocontrol agents are less sensitive to environmental changes and exhibit stable efficacy. Additionally, the nutrients decomposed and transformed by biocontrol bacteria can be utilized by crops, thereby improving crop quality and increasing yield.

[0006] However, while existing biocontrol strains and agents have shown some effectiveness, and some possess broad-spectrum antibacterial properties, the inhibition rates of existing Bacillus biocontrol agents are generally low. Furthermore, regarding… Aspergillus pseudoglaucus , Colletotrichum siamense, Cladosporium tenuissimum For pathogenic bacteria affecting fruits and vegetables, there are currently few biocontrol strains that have been discovered, and their antibacterial rate is not high.

[0007] Therefore, there is a need to provide a drug with broader antibacterial effects and targeting specific pathogens. Aspergillus pseudo-glaucus , Colletotrichum siamense, Cladosporium tenuissimum Bacillus biocontrol strains with extremely high inhibition rates against pathogens in fruits and vegetables. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the purpose of this invention is to provide a biocontrol strain capable of being used for the prevention and control of fruit and vegetable diseases and for antibacterial preservation. This invention provides a Bacillus strain that is highly effective in the prevention and control of fruit and vegetable diseases and for antibacterial preservation, particularly effective against… Aspergillus pseudoglaucus , Colletotrichum siamense、Cladosporium very thin The inhibitory rate against pathogens in fruits and vegetables is extremely significant. This invention also provides a biocontrol agent made from this Bacillus and its application in antibacterial and antimicrobial preservation of fruits and vegetables.

[0009] Through extensive experimental research and exploration, the inventors of this invention obtained a strain of Bacillus subtilis. Bacillus subtilis It was named QY2, and the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The depositary number is CGMCC No. 31373, and the deposit date is July 19, 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 the application of the biocontrol agent in the antibacterial activity against pathogens in fruits and vegetables, and the application of the Bacillus in the preparation of fruit and vegetable preservatives.

[0013] Specifically, the fruit and vegetable pathogens include Acremonium sclerotigenum、Actinomucor elegans, Alternaria alternata, Alternaria tenuissima, Apiospora mari, Aspergillus yellow、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 variable、 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 cucurbitacearum, Talaromycessp. and Talaromyces verruculosus .

[0014] The concentration of Bacillus QY2 in the biocontrol agent of this invention is (1×10⁻⁶). 6-8 ) cfu / mL.

[0015] The beneficial effects of this invention are as follows:

[0016] The live Bacillus QY2 cells of this invention exhibit certain antibacterial effects against various pathogens. Among them, the live Bacillus QY2 cells... Botrytis sp.、 Aspergillus pseudoglaucus, Colletotrichum siamense and Cladosporium tenuissimum The antibacterial effect is most significant, with an inhibition rate of 100%, completely inhibiting the growth of pathogenic fungi. Among them, QY2 live bacteria exhibit the most significant antibacterial effect. 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 Its antibacterial rate is greater than 90%, exhibiting good inhibitory effect; [it has a] bacteriostatic rate of over 90%, demonstrating good inhibitory effect; Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile、Talaromyces verruculosus、Curvularia lunata、Cladosporium colombiae、 Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans、Fusarium solani and Fusarium gramineae Its antibacterial effect is average, with an antibacterial rate greater than 80%.

[0017] The supernatant of Bacillus QY2 of this invention has a certain inhibitory effect on different pathogenic fungi. Among them, the supernatant of Bacillus QY2... Botrytis sp. 、Aspergillus pseudoglaucus、Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp. 、Botryosphaeria dothidea、 Talaromyces sp. and Diaporthe phoenicicola The supernatant of Bacillus subtilis QY2 showed the best antibacterial effect, with an inhibition rate greater than 90%. 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 It has a certain antibacterial effect, with an antibacterial rate of over 80%.

[0018] The Bacillus QY2 cell lysate of this invention exhibits certain antibacterial effects against various pathogenic fungi, but the antibacterial effects are slightly lower than those of the QY2 strain supernatant. Specifically, the Bacillus QY2 cell lysate shows antibacterial activity against... 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、 Mucer circinelloides、Penicillium camemberti and Penicillium verruculosum It exhibits good antibacterial effect, with an inhibition rate greater than 90%. QY2 cell lysate has a good antibacterial effect. Botrytis sp. 、Aspergillus pseudoglaucus、 Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum and Fusarium incarnate It exhibits good antibacterial effect, with an inhibition rate greater than 90%. QY2 cell lysate has a good antibacterial effect. Penicillium chrysogenum、Aspergillus sp. 、Botryosphaeria dothidea、Talaromyces sp. 、Diaporthe phoenicicola、Actinomucor elegans、Apiospora mari、Nectria rigidiuscula、 Alternaria tenuissima, Acremonium sclerotigenum, Fusarium falciforme, Penicillium sclerotia, Ganoderma sessile and Talaromyces verruculosus It has a certain antibacterial effect, with an antibacterial rate of over 80%.

[0019] The Bacillus QY-2 provided by this invention has a broad antibacterial spectrum, covering 42 types of fruit and vegetable pathogens. In contrast, the existing technology (CN114369556B-A strain of Bacillus, a biocontrol agent made from the Bacillus and its application) also provides a strain of Bacillus biocontrol, but it only covers 32 types of fruit and vegetable pathogens. Attached Figure Description

[0020] Figure 1 The inhibition rate of QY2 live bacteria against different pathogens;

[0021] Figure 2 The turbidity of Bacillus cultured at different pH values;

[0022] Figure 3 The turbidity of Bacillus cultured at different pH values. Detailed Implementation

[0023] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention. Example

[0024] I. Source of the strain

[0025] In 2023, leaves of Bletilla striata were collected from a greenhouse of the Chengdu Academy of Agricultural and Forestry Sciences in Wenjiang District, Chengdu, and placed in sterile sealed bags. The antagonistic bacterial strains were then isolated and purified at the Institute of Agricultural Products of the Chengdu Academy of Agricultural and Forestry Sciences.

[0026] II. Identification of the strain

[0027] DNA was extracted from the bacteria, and 16S rDNA sequencing was performed using PCR. The gene sequence is shown in SEQ NO.1 of the sequence listing. The obtained sequence was compared with the NCBI website and identified as *Bacillus subtilis*. Bacillus subtilisIt was named Bacillus QY2, and the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit center number is CGMCC No. 31373, and the deposit date is July 19, 2024.

[0028] III. Antibacterial Tests of Strains

[0029] (a) Live cell inhibition test of Bacillus QY2

[0030] The strain has a wide range of antibacterial properties and is effective in preventing and controlling pathogens on strawberries, blueberries, grapes, apples, mangoes, cherries, kiwifruit, jujubes, peppers, wax apples, eggplants, garlic, and loofahs.

[0031] 1. Experimental Design

[0032] Single colonies of strain QY2 were 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 × 10⁻⁶ using a hemocytometer. 8 cfu / mL. Add the QY2 strain cell suspension to PDA medium, vortex to mix, and prepare plates. The cell density of QY2 strain on each plate is 1×10⁻⁶. 6 cfu / mL. Using a 6 mm aseptic punch, *Botrytis cinerea* mycelial cakes were placed in the center of a PDA plate containing strain QY2. A PDA plate containing 100 mg / L carbendazim served as a pesticide control, and a PDA plate without QY2 cells served as a blank control. Each treatment was repeated in triplicate. The plates containing the mycelial cakes were sealed in resealable bags and incubated at 26°C. The width of the inhibition zone was measured when the control plate had fully grown mycelium.

[0033] Calculate the inhibition rate R of mycelial growth.

[0034] R (%) = (R1 - R2) / R1 × 100%

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

[0036] 2. Test Results

[0037] The experimental results are shown in Table 1 and Figure 1 As shown. By Figure 1 As shown in Table 1, live Bacillus QY2 cells exhibit certain antibacterial effects against various pathogens. Live QY2 cells... Botrytis sp.、 Aspergillus pseudoglaucus, Colletotrichum siamense and Cladosporium tenuissimumThe antibacterial effect is most significant, with an inhibition rate of 100%, completely inhibiting the growth of pathogenic fungi. Among them, QY2 live bacteria exhibit the most significant antibacterial effect. 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 Its antibacterial rate is greater than 90%, exhibiting good inhibitory effect; [it has a] bacteriostatic rate of over 90%, demonstrating good inhibitory effect; Fusarium falciforme, Penicillium sclerotiorum、Ganoderma sessile、Talaromyces verruculosus、Curvularia lunata、 Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile、Fusarium metavorans、Fusarium solani and Fusarium gramineae It has a certain antibacterial effect, with an antibacterial rate of over 80%.

[0038] Compared with the antibacterial rate of 100 mg / L carbendazim, QY2 live bacteria showed a higher inhibition rate. 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、、Aspergillus niger、Aspergillus tamarii、Aspergillus tubingensis、 Galactomyces candidum and Stagonosporopsis cucurbtaceae The antibacterial effect was more pronounced, with inhibition rates significantly higher than those of the 100 mg / L carbendazim treatment. After treatment with 100 mg / L carbendazim, the antibacterial effect was significantly reduced. Botryosphaeria dothidea, Diaporthe phoenicicola, Fusarium falciforme, Alternaria alternata, Fusarium solani, Fusarium graminearum, Colletotrichum fioriniae, Fusarium sp. 、 Colletotrichum gloeosporioides、Penicillium common、Fusarium circinatum、 Fusarium proliferatum and Fusarium oxysporum The antibacterial effect was better, with antibacterial rates exceeding those of live Bacillus QY2 cells. In the treatment of... Fusarium incarnatum, Alternaria tenuissima, Acremonium sclerotigenum, Penicillium polonicum and Penicillium rubens After treatment with live QY2 bacteria and 100 mg / L carbendazim, the antibacterial effects were similar.

[0039] Table 1. Antibacterial effects of Bacillus QY2 live cells and carbendazim against different pathogens.

[0040] disease <![CDATA[10 6 QY2 antibacterial rate / % 100mg / L sp. 100.00 96.38 100.00 35.42 100.00 86.78 100.00 96.56 96.49 49.63 96.15 95.13 96.15 77.78 sp. 95.73 38.78 95.73 100.00 sp. 95.30 76.89 94.44 98.67 93.94 65.78 93.22 58.89 92.51 58.12 91.30 90.89 90.48 90.12 89.74 93.56 89.74 77.67 89.74 77.19 89.32 74.54 88.89 60.50 88.48 62.44 87.98 37.16 87.94 97.34 87.33 79.33 86.06 79.56 84.27 90.05 81.29 98.42 79.95 40.56 79.49 100.00 sp. 79.49 88.89 78.41 40.89 76.64 95.12 75.00 76.34 75.00 80.67 74.07 90.12 73.98 78.89 73.89 39.44 73.21 74.38 69.37 86.58 68.53 47.38 61.90 56.33

[0041] (II) Antibacterial test of Bacillus QY2 supernatant

[0042] The supernatant antibacterial test mainly tests the antibacterial effect of extracellular antibacterial substances. This method mainly eliminates nutrient competition factors and is an important indicator for evaluating the biocontrol effect of biocontrol bacteria.

[0043] 1. Experimental Design

[0044] After 24 hours of culture, Bacillus strain QY2 was repeatedly centrifuged and filtered to remove viable cells, resulting in QY2 supernatant. 200 μL of the supernatant was evenly spread onto a PDA plate. Different pathogenic bacterial pellets were placed in the center of the PDA plate using a 6 mm sterile punch, with an equal volume of sterile water as a control. Each treatment was repeated three times. The prepared plates were incubated at 26°C. The width of the inhibition zone was measured when the control plate reached full mycelial growth.

[0045] Calculate the inhibition rate R of mycelial growth.

[0046] R (%) = (R1 - R2) / R1 × 100%

[0047] Where R is the percentage of radial hyphal growth inhibited, R1 is the hyphal growth of the blank control, and R2 is the hyphal growth of the treatment group.

[0048] 2. Test Results

[0049] The antibacterial effects of Bacillus subtilis QY2 supernatant against different pathogenic fungi are shown in Table 2. Among them, the antibacterial effect of QY2 supernatant against... Botrytis sp. Aspergillus pseudoglaucus Cladosporium tenuissimum Colletotrichum siamense, Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp. 、Botryosphaeria dothidea、Talaromyces sp. and Diaporthe phoenicicola The supernatant of Bacillus subtilis QY2 showed the best antibacterial effect, with an inhibition rate greater than 90%. 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 variable、Fusarium metavorans and Fusarium solani It exhibits a certain antibacterial effect, with an inhibition rate greater than 80%. The supernatant of Bacillus subtilis QY2 has a certain antibacterial effect. Fusarium graminearum, Aspergillus niger, Colletotrichum fioriniae、Fusarium sp. 、Aspergilus tamarii、Colletotrichum gloeosporioides、 Penicillium polonicum, Penicillium commune and Fusarium circinatum Its antibacterial effect is average, with an antibacterial rate greater than 70%.

[0050] Table 2. Antibacterial effects of Bacillus QY2 supernatant against different pathogenic fungi.

[0051] disease QY2 sp. 99.12 98.89 98.73 98.04 94.46 94.65 92.75 sp. 90.33 91.44 sp. 91.80 90.47 89.74 88.92 86.91 87.34 86.78 85.94 86.14 85.73 85.47 84.09 84.48 83.98 82.91 83.73 82.56 80.97 74.11 73.95 74.09 sp. 73.89 73.11 72.84 72.03 71.78 70.97 69.98 69.67 69.58 66.07 64.83 59.20

[0052] (III) Antibacterial test of Bacillus QY2 cell lysate

[0053] 1. Experimental Design

[0054] Add 10 ml of sterile physiological saline to live Bacillus QY2 cells and shake well. Transfer the solution to a 50 ml centrifuge tube and lyse the QY2 cells using an ultrasonic cell disruptor. Filter the lysate once through a 0.22 μm microporous membrane filter to obtain the Bacillus QY2 cell lysis buffer, and store at 4 °C for later use.

[0055] 200 μL of cell lysis buffer was evenly spread onto a PDA plate. Using a 6 mm sterile punch, different pathogenic fungal mold cakes were placed in the center of the PDA plate surface. An equal volume of sterile water was used as a control. Each treatment was repeated three times. The prepared plates were incubated at 26°C. The width of the inhibition zone was measured when the control plate reached full mycelial growth. The inhibition rate R of mycelial growth was calculated using the same formula as above.

[0056] 2. Test Results

[0057] Table 3 shows that Bacillus QY2 cell lysate exhibits certain antibacterial effects against different pathogenic fungi, but the antibacterial effects are slightly lower than those of the QY2 strain supernatant. Specifically, Bacillus QY2 cell lysate shows... Botrytis sp. 、 Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum and Fusarium incarnate It exhibits good antibacterial effect, with an inhibition rate greater than 90%. QY2 cell lysate has a good antibacterial effect. 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 warty It has a certain antibacterial effect, with an antibacterial rate greater than 80%. QY2 cell lysate has a certain antibacterial effect. Curved lunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile、Fusarium metavorans、Fusarium solani、Fusarium grasses, Aspergillus niger and Colletotrichum fioriniae Its antibacterial effect is average, with an antibacterial rate greater than 70%.

[0058] Table 3. Antibacterial effects of Bacillus QY2 cell lysate against different pathogenic fungi.

[0059] Pathogenic species QY2 cell lysate antibacterial rate / % sp. 96.92 95.93 94.73 94.94 90.86 90.75 89.45 sp. 87.03 86.49 sp. 86.89 85.97 85.45 84.12 82.97 82.67 81.98 81.64 81.94 80.98 80.77 79.69 79.98 78.92 77.95 77.93 76.86 75.97 70.21 70.05 70.49 sp. 69.78 69.81 68.94 68.43 67.28 66.17 65.18 65.27 64.18 62.27 60.43 54.63

[0060] IV. Life Characteristics of Biocontrol Bacteria

[0061] Bacillus QY2 has a strong survival ability, mainly due to its wide adaptability to temperature, pH, field nutrient sources and humidity.

[0062] 1. Temperature adaptability

[0063] (1) Experimental design

[0064] The QY2 strain was inoculated into LB broth and cultured at 4°C, 16°C, 28°C, 37°C, and 60°C in shakers at 150 r / min, with each treatment repeated three times. After 12 h, the absorbance and transmittance were measured at 600 nm using a UV-Vis spectrophotometer. Uninoculated LB broth served as a blank control, and each treatment was repeated three times. Turbidity = (100 - transmittance) × 100%.

[0065] (2) Test results

[0066] The test results are shown in Table 4 and Figure 2 As shown, Bacillus QY2 exhibited good survival ability at 28-37℃, with the turbidity of the culture medium exceeding 50% after 12 hours. Bacillus QY2 could grow at 16℃ and 45℃, but growth was slow, with turbidity of the culture medium at 45.55% and 36.22% after 12 hours, respectively. At 4℃ or 60℃, Bacillus QY2 struggled to reproduce viable cells, with the turbidity reaching 0 after 12 hours.

[0067] Table 4. Determination of the adaptability of Bacillus QY2 to different temperatures during culture.

[0068] Processing temperature (°C) Suspension turbidity (%) 4 0 16 45.55 28 89 37 54.7 45 36.22 60 0

[0069] 2. pH adaptability

[0070] (1) Experimental design

[0071] The pH of the culture medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using 1 mol / L HCl or 1 mol / L NaOH, respectively. After sterilization, 1 mL of Bacillus QY2 seed culture was inoculated into 100 mL of LB liquid medium adjusted to different pH values. The medium was incubated 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-Vis spectrophotometer. Uninoculated LB liquid medium served as a blank control. Each treatment was repeated in triplicate. Turbidity = (100 - transmittance) × 100%.

[0072] (2) Test results

[0073] The test results are shown in Table 5 and Figure 3 From Table 5 and Figure 2It can be seen that the turbidity of the culture medium of Bacillus QY2 was greater than 50% after 12 hours under pH 5-6 conditions, indicating that Bacillus QY2 exhibited good survival ability under these conditions. Under pH 5 and pH 9 conditions, the turbidity of the culture medium after 12 hours was 44.45% and 30.20%, respectively, indicating that Bacillus QY2 can grow under slightly acidic and slightly alkaline conditions, but the growth is relatively slow. When pH < 5 or > 9, the turbidity of the culture medium after 12 hours was less than 10%, indicating that Bacillus QY2 has very little chance of growing in strongly acidic or strongly alkaline environments.

[0074] Table 5. Determination of Bacillus QY2's adaptability to different pH cultures.

[0075] Processing Number Suspension turbidity (%) pH=4 5.13 pH=5 44.45 pH=6 50.07 pH=7 54.53 pH=8 55.33 pH=9 30.20 pH=10 1.02

[0076] 3. Adaptability to field nutrient sources

[0077] (1) Experimental design

[0078] Based on the nutrient classification standards of the Second National Soil Survey and the existing soil conditions in Sichuan Province, the following soil nutrient conditions were proposed: total nitrogen 2 g / kg, total phosphorus 1 g / kg, total potassium 20 g / kg, and field capacity 70%. Based on these conditions, the following single-factor variable design was conducted for N, P, and K nutrients, with five different gradients for each factor (see Table 6):

[0079] (2) Test results

[0080] Table 7 shows that Bacillus QY2 exhibited strong survival ability after 60 days of cultivation under different nutrient source conditions. Under the nutrient source conditions of 2 g / kg total nitrogen, 1 g / kg total phosphorus, and 20 g / kg total potassium, Bacillus QY2 could survive well for more than 60 days. The QY2 strain can survive under low levels of carbon, nitrogen, phosphorus, and potassium. Even when carbon, nitrogen, phosphorus, and potassium were deficient respectively, Bacillus QY2 could survive in soil for more than 60 days, indicating that the QY2 strain does not depend on any specific nutrient element for survival and can provide itself with nutrients from other elements.

[0081] Table 6. Single-factor levels of field nutrient sources for Bacillus QY2

[0082]

[0083] Note: Among the individual factors, the non-variable factors maintain the formula for the highest soil nutrient conditions.

[0084] Table 7. Nutrient source adaptability of Bacillus QY2 at 60 days

[0085] Processing Number Total nitrogen (g / kg) Total phosphorus (g / kg) Total potassium (g / kg) Survival status 1 0 1 20 ＋ 2 0.5 1 20 ＋ 3 1 1 20 ＋＋ 4 1.5 1 20 ＋＋ 5 2 0 20 ＋ 6 2 0.25 20 ＋ 7 2 0.5 20 ＋ 8 2 0.75 20 ＋ 9 2 1 0 ＋ 10 2 1 5 ＋ 11 2 1 10 ＋ 12 2 1 15 ＋ 13 2 1 20 ＋＋

[0086] Note: "++" indicates a viable count ≥10 8 cfu / mL, "+" indicates a viable count ≥10 6 cfu / mL, "-" indicates no viable bacteria.

[0087] 4. Field humidity adaptability

[0088] (1) Experimental design

[0089] In sterile sand tubes, a sterile nutrient solution containing 2 g / kg total nitrogen, 1 g / kg total phosphorus, and 20 g / kg total potassium was added, with moisture content controlled at 10%, 30%, 50%, and 70%. 1 mL of QY2 seed culture medium was inoculated into each tube, and the tubes were sealed with a sealing film and incubated at 37°C. Each treatment was repeated three times. After 60 days, soil samples were taken and dissolved in physiological saline. A small amount of sand was spread on LB agar plates to observe the presence and quantity of viable bacteria.

[0090] (2) Test results

[0091] Table 8 shows the survival of Bacillus QY2 after 60 days under different field moisture contents. The results indicate that Bacillus QY2 exhibits good survival ability after 60 days under soil moisture contents ≥30%. However, when the moisture content is 10%, Bacillus QY2 cannot survive for more than 60 days.

[0092] This strain exhibits strong tolerance to high temperature and humidity environments, but is not adapted to environments with extremely low temperature and humidity. However, soil moisture content below 10% is not observed in normal crop production areas. Therefore, the viability of this strain is suitable for most crop soils. When air humidity is high, this strain can be mixed with inorganic nutrient solution and sprayed on the crop surface as a foliar fertilizer and protectant.

[0093] Table 8. Adaptability of 60-day Bacillus thuringiensis QY2 to different field water contents

[0094] Moisture content (%) Survival status 10 - 30 + 50 ++ 70 ++

[0095] Note: "++" indicates a viable count ≥10 8 cfu / mL, "+" indicates a viable count ≥10 6 cfu / mL, "-" indicates no viable bacteria.

[0096] VI. Tolerance to broad-spectrum fungicides

[0097] 1. Experimental Design

[0098] Seventeen chemical fungicides, including pyraclostrobin, tebuconazole, flutriafol, carbendazim, azoxystrobin, propiconazole, pyraclostrobin-mancozeb, cyazofamid, mancozeb, difenoconazole, boscalid, fluopyram, iprodione, pyraclostrobin, pyrimidine nucleotide antibiotics, prochloraz, and flusilazole, were added to LB liquid medium to achieve pesticide concentrations equal to or 10 times the normal field application doses of the aforementioned fungicides. LB liquid medium without fungicides or biocontrol bacteria served as a control, inoculated with the same concentration of strain QY2. The cultures were incubated at 37°C and 120 r / min in a shaker. After 24 hours, LB liquid medium was streaked onto LB plates using an inoculation loop, and the growth of strain QY2 was observed. Each treatment was replicated three times.

[0099] 2. Test Results

[0100] The experimental results are shown in Table 9. Bacillus QY2 exhibited varying resistance to different fungicides. After co-culturing Bacillus QY2 with normal doses and 10 times the concentration of pyraclostrobin, flutriafol, carbendazim, azoxystrobin, pyraclostrobin-mancozeb, cyproconazole, mancozeb, difenoconazole, cyazofamid, and fluopyram for 24 hours, Bacillus QY2 successfully grew on LB agar plates, indicating that Bacillus QY2 showed good resistance to these 10 commonly used fungicides at low concentrations and could survive under these conditions. Bacillus QY2 could grow viable cells after co-culturing with normal doses of tebuconazole, propiconazole, iprodione, azoxystrobin, pyrimidine nucleotides, and flusilazole for 24 hours, but could not survive after co-culturing with 10 times the concentration. Bacillus QY2 could not survive treatment with normal doses and 10 times the concentration of prochloraz.

[0101] Table 9. Tolerance of Bacillus QY2 to common fungicides

[0102]

[0103] Note: "+" indicates the presence of live bacteria, and "-" indicates the absence of live bacteria.

Claims

1. A strain of Bacillus, characterized in that, The Bacillus species described is Bacillus subtilis, named QY2. The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No. 31373, and the deposit date is July 19, 2024.

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

3. The application of the biocontrol agent according to claim 2 in the antibacterial treatment of fruit and vegetable pathogens, characterized in that, The fruit and vegetable pathogenic bacteria are selected from Acremonium sclerotigenum, Actinomucor elegans, Alternaria alternata, Alternaria tenuissima, Apiospora mari, Aspergillus flavus, Aspergillus pseudoglaucus, Aspergillus tubingensis, Aspergilus niger, Aspergillus tamarii, Botryosphaeria dothidea, 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, Galactomyces candidum, Ganoderma sessile, Nectria rigidiuscula, Penicillium chrysogenum, Penicillium commune, Penicillium polonicum, Penicillium rubens, Penicillium sclerotiorum, Stagonosporopsis cucurbtacearum, and Talaromyces verruculosus.

4. Use of the Bacillus as claimed in claim 1 in the preparation of a fruit and vegetable freshness-keeping agent.

5. The application according to claim 3, characterized in that, The concentration of Bacillus QY2 in the biocontrol agent is 1×10⁻⁶. 6-8 cfu / L.

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