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 QY2, 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 number of the preservation center is CGMCC No.31373, and the preservation time is July 19, 2024. The bacillus QY2 disclosed by the invention has a certain bacteriostatic effect on different pathogenic bacteria regardless of living bacteria, supernate or cell lysis solution. The bacillus QY2 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 Aspergillus pseudoglaucus, Colletotrichum siamense, and Cladosporium tenuissimum, there are currently few biocontrol strains found, and their inhibition rates are not high.

[0007] Therefore, there is a need to provide a Bacillus biocontrol strain with a broader spectrum of antibacterial effects and a very significant inhibition rate against fruit and vegetable pathogens such as Aspergillus pseudoglaucus, Colletotrichum siamense, and Cladosporium tenuissimum. 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 efficiently used for disease control and bacteriostasis and preservation of fruits and vegetables, especially for Aspergillus pseudoglaucus, Colletotrichum siamense, Cladosporium tenuissimum and other fruit and vegetable pathogens. 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 QY2, 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.31373, and the preservation time 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 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 pathogenic bacteria include Acremonium sclerotigenum, Actinomucorelegans, Alternaria alternata, Alternaria tenuissima, Apiospora mari, Aspergillusflavus, Aspergillus pseudoglaucus, Aspergillus sp., Aspergillus tubingensis, Aspergilus niger, Aspergillus tamarii, Botryosphaeria dothidea, Botrytis sp., Cladosporium anthropophilum, Cladosporium colombiae, Cladosporium tenuissimum, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Colletotrichum siamense, Curvularia lunata,, Diaporthephoenicicola, Didymosphaeria variabile, Fusariumcircinatum, Fusarium falciforme, Fusarium graminearum, Fusarium incarnatum, Fusarium metavorans, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Fusarium sp., Galactomyces candidum, Ganodermasessile, Nectria rigidiuscula, Penicillium chrysogenum, Penicillium commune, Penicillium polonicum, Penicillium rubens, Penicillium sclerotiorum, Stagonosporopsis cucurbtacearum, Talaromyces sp., and Talaromyces verruculosus.

[0014] The concentration of Bacillus QY2 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 QY2 of the present invention has a certain antibacterial effect on different pathogens. Among them, the living bacillus QY2 has the most significant antibacterial effect on Botrytis sp., Aspergillus pseudoglaucus, Colletotrichum siamense and Cladosporium tenuissimum, with an antibacterial rate of 100%, which can completely inhibit the growth of pathogenic fungi. The inhibition rate of QY2 viable bacteria was greater than 90% against Cladosporium anthropophilum, Fusariumincarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces p., Diaporthephoenicicola, Actinomucor elegans, Apiospora mari, Nectriarigidiuscula, Alternaria tenuissima and Acremonium sclerotigenum, and it had a good inhibitory effect on Fusariumfalciforme, Penicillium sclerotiorum, Ganodermasessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusariummetavorans, Fusarium solani and Fusarium graminearum had a general antibacterial effect, with an inhibition rate of more than 80%.

[0017] (2) The supernatant of Bacillus sp. QY2 of the present invention has a certain antibacterial effect on different pathogenic fungi. Among them, the supernatant of Bacillus sp. QY2 has the best antibacterial effect on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp. and Diaporthe phoenicicola, and the antibacterial rate is greater than 90%. The supernatant of Bacillus QY2 had a certain antibacterial effect on Actinomucor elegans, Apiospora mari, Nectriarigidiuscula, Alternaria tenuissima, Acremonium sclerotigenum, Fusariumfalciforme, Penicillium sclerotiorum, Ganoderma sessile, Talaromycesverruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillusflavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans and Fusariumsolani, and the inhibition rates were all greater than 80%.

[0018] (3) The Bacillus QY2 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 QY2 strain supernatant. Among them, the Bacillus QY2 cell lysate has a 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 antibacterial rate is greater than 90%. QY2 cell lysate had good antibacterial effect on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum and Fusarium incarnatum, with the inhibition rate greater than 90%. QY2 cell lysate had certain antibacterial effect on Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp., Diaporthephoenicicola, Actinomucor elegans, Apiosporamari, Nectria rigidiuscula, Alternaria tenuissima, Acremonium sclerotigenum, Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile and Talaromyces verruculosus, with the inhibition rate greater than 80%.

[0019] (4) The Bacillus QY-2 provided by the present invention has a wide antibacterial spectrum, involving 42 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 involves 32 fruit and vegetable pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 It is the turbidity of suspension cultured with different pH of Bacillus;

[0022] Figure 3 It is the turbidity of suspension of Bacillus cultured at different pH. 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 2023, Bletilla striata leaves were collected from the greenhouse of Chengdu Academy of Agriculture and Forestry Sciences in Wenjiang District, Chengdu, placed in sterile sealed bags, and brought back to the Institute of Agricultural Products of Chengdu Academy of Agriculture and Forestry Sciences to isolate and purify antagonistic strains.

[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, which was named Bacillus QY2. The depository 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. 31373, and the deposit time is: July 19, 2024.

[0029] III. Antibacterial test of strains (I) Antibacterial test of live bacteria of Bacillus QY2

[0030] 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, wax apples, eggplants, garlic, and loofahs.

[0031] 1. Experimental Design

[0032] A single colony of 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 as 1 × 10 8 cfu / mL. Add QY2 strain cell suspension to PDA medium, shake and mix to make plates. The cell density of QY2 strain 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 QY2 strain. Use the PDA plate containing 100mg / L carbendazim as the pesticide control, and the PDA plate medium without QY2 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.

[0033] Calculate the inhibition rate R of mycelial growth

[0034] R(%)=(R1-R2) / R1×100%

[0035] 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.

[0036] 2. Test results

[0037] The test results are shown in Table 1 and Figure 1 As shown. Figure 1As shown in Table 1, Bacillus QY2 live bacteria have certain antibacterial effects on different pathogens. The antibacterial effect of QY2 live bacteria on Botrytis sp., Aspergillus pseudoglaucus, Colletotrichum siamense and Cladosporium tenuissimum is the most significant, with an inhibition rate of 100%, which can completely inhibit the growth of pathogenic fungi. The inhibition rate of QY2 viable bacteria was greater than 90% against Cladosporium anthropophilum, Fusariumincarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeriadothidea, Talaromyces sp., Diaporthephoenicicola, Actinomucor elegans, Apiosporamari, Nectria rigidiuscula, Alternaria tenuissima and Acremonium sclerotigenum, and it had a good inhibitory effect on Fusariumfalciforme, Penicilliumsclerotiorum, Ganoderma sessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillusflavus, Alternaria alternata, Didymosphaeriavariabile, Fusarium metavorans, Fusarium solani and Fusarium graminearum has a certain antibacterial effect, and the antibacterial rate is greater than 80%.

[0038] Compared with the antibacterial rate of 100mg / L carbendazim, QY2 viable bacteria are effective against Botrytis sp., Aspergilluspseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporiumanthropophilum, Penicillium chrysogenum, Aspergillus sp., Talaromyces sp., Actinomucor elegans, Apiospora mari, Nectria rigidiuscula, Penicilliumsclerotiorum, Ganoderma sessile, Talaromyces verruculosus, Curvularia lunata, Cladosporium colombiae, Aspergillusflavus, Didymosphaeria variabile, Fusariummetavorans, Aspergilus niger, Aspergilus tamarii, Aspergillustubingensis, Galactomyces candidum and Stagonosporopsis cucurbtacearum had more prominent antibacterial effects, and the antibacterial rates were significantly higher than those treated with 100 mg / L carbendazim. After treatment with 100 mg / L carbendazim, the antibacterial effects on Botryosphaeria dothidea, Diaporthephoenicicola, Fusariumfalciforme, Alternariaalternata, Fusarium solani, Fusarium graminearum, Colletotrichumfioriniae, Fusarium sp., Colletotrichum gloeosporioides, Penicillium commune, Fusariumcircinatum, Fusariumproliferatum and Fusarium oxysporum were better, and the antibacterial rates were higher than those treated with live cells of Bacillus QY2.The antibacterial effects of QY2 live bacteria and 100 mg / L carbendazim on Fusariumincarnatum, Alternaria tenuissima, Acremonium sclerotigenum, Penicilliumpolonicum and Penicillium rubens were similar.

[0039] Table 1 Antibacterial effects of live Bacillus QY2 and carbendazim on different pathogens

[0040]

[0041]

[0042] (II) Antibacterial test of supernatant of Bacillus QY2

[0043] 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.

[0044] 1. Experimental Design

[0045] After culturing Bacillus QY2 for 24 hours, the live cells were removed by repeated centrifugation filtration to prepare QY2 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.

[0046] Calculate the inhibition rate R of mycelial growth

[0047] R(%)=(R1-R2) / R1×100%

[0048] 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.

[0049] 2. Test results

[0050] The antibacterial effects of Bacillus QY2 supernatant on different pathogenic fungi are shown in Table 2. Among them, QY2 supernatant had the best antibacterial effect on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichumsiamense, Cladosporium anthropophilum, Fusarium incarnatum, Penicillium chrysogenum, Aspergillus sp., Botryosphaeria dothidea, Talaromyces sp. and Diaporthephoenicicola, and the inhibition rates were all greater than 90%. The supernatant of Bacillus QY2 had certain antibacterial effects 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 rates were all greater than 80%. The supernatant of Bacillus QY2 had a moderate antibacterial effect on Fusarium graminearum, Aspergilus niger, Colletotrichum fioriniae, Fusarium sp., Aspergilus tamarii, Colletotrichum gloeosporioides, Penicillium polonicum, Penicillium commune and Fusarium circinatum, with an inhibition rate of more than 70%.

[0051] Table 2 Antibacterial effect of Bacillus QY2 supernatant on different pathogenic fungi

[0052]

[0053]

[0054] (III) Antibacterial test of Bacillus QY2 cell lysate

[0055] 1. Experimental Design

[0056] Add 10 ml of sterile physiological saline to the live cells of Bacillus QY2 and shake well. Put the shaken solution into a 50 ml centrifuge tube and crush the cells of QY2 strain 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 QY2 cell lysate, and store it at 4°C for later use.

[0057] 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.

[0058] 2. Test results

[0059] It can be seen from Table 3 that the Bacillus QY2 cell lysate has a certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the QY2 strain supernatant. Among them, the Bacillus QY2 cell lysate has a good antibacterial effect on Botrytis sp., Aspergillus pseudoglaucus, Cladosporium tenuissimum, Colletotrichum siamense, Cladosporium anthropophilum and Fusarium incarnatum, with an inhibition rate of more than 90%. QY2 cell lysate had a certain antibacterial effect on Penicillium chrysogenum, Aspergillus sp., Botryosphaeriadothidea, Talaromyces sp., Diaporthephoenicicola, Actinomucor elegans, Apiosporamari, Nectria rigidiuscula, Alternaria tenuissima, Acremonium sclerotigenum, Fusarium falciforme, Penicillium sclerotiorum, Ganoderma sessile and Talaromycesverruculosus, and the inhibition rates were all greater than 80%. The antibacterial effect of QY2 cell lysate on Curvularialunata, Cladosporium colombiae, Aspergillus flavus, Alternaria alternata, Didymosphaeria variabile, Fusarium metavorans, Fusarium solani, Fusariumgraminearum, Aspergilus niger and Colletotrichum fioriniae was moderate, with an inhibition rate of more than 70%.

[0060] Table 3 Antibacterial effect of Bacillus QY2 cell lysate on different pathogenic fungi

[0061]

[0062]

[0063] 4. Life characteristics of biocontrol bacteria

[0064] Bacillus QY2 has strong vitality, which is mainly reflected in its wide applicability in temperature, pH, field nutrient sources and humidity environments.

[0065] 1. Temperature adaptability

[0066] (1) Experimental design

[0067] The QY2 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 medium without bacteria was used as a blank control, and each treatment was repeated 3 times. Suspension turbidity = (100-transmittance) × 100%.

[0068] (2) Test results

[0069] The test results are shown in Table 4 and Figure 2 As shown in the figure, Bacillus QY2 showed good survival ability at 28-37℃, and the turbidity of the culture solution was greater than 50% after 12h. Bacillus QY2 could grow at 16℃ and 45℃, but the growth was slow, and the turbidity of the culture solution was 45.55% and 36.22% after 12h, respectively. It was difficult for Bacillus QY2 to reproduce in vivo at 4℃ or 60℃, and the turbidity of Bacillus QY2 was 0 after 12h of culture.

[0070] Table 4 Determination of adaptability of Bacillus QY2 to culture at different temperatures

[0071]

[0072] 2. pH adaptability

[0073] (1) Experimental design

[0074] Use 1mol / LHCl or 1mol / LNaOH 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 QY2 seed culture solution in 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%.

[0075] (2) Test results

[0076] 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 solution of Bacillus QY2 was greater than 50% after 12 hours at pH = 5-6, indicating that Bacillus QY2 showed 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 was 44.45% and 30.20% after 12 hours, respectively, 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 was less than 10% after 12 hours, indicating that Bacillus QY2 is basically difficult to grow in strong acid or strong alkaline environments.

[0077] Table 5 Determination of the adaptability of Bacillus QY2 culture at different pH values

[0078]

[0079] 3. Adaptability of field nutrient sources

[0080] (1) Experimental design

[0081] 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):

[0082] (2) Test results

[0083] It can be seen from Table 7 that Bacillus QY2 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 QY2 can survive well for more than 60 days. The QY2 strain can survive when the carbon, nitrogen, phosphorus, and potassium content is low. In the absence of carbon, nitrogen, phosphorus, and potassium, respectively, Bacillus QY2 can survive in the soil for more than 60 days, indicating that the QY2 strain does not rely on a specific nutrient element to survive, and can provide itself with nutrient sources through other elements.

[0084] Table 6 Single factor level table of field nutrient sources of Bacillus QY2

[0085]

[0086] Note: Among each single factor, the non-variable factor maintains the highest soil nutrient condition formula.

[0087] Table 760d Bacillus QY2 nutrient source adaptability

[0088]

[0089] 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.

[0090] 4. Field humidity adaptability

[0091] (1) Experimental design

[0092] In a sterile sand tube, add a sterile nutrient solution with 2g / kg total nitrogen, 1g / kg total phosphorus, and 20g / kg total potassium, and control the moisture at 10%, 30%, 50%, and 70%. Inoculate 1mL of QY2 seed culture solution, 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.

[0093] (2) Test results

[0094] Table 8 shows the survival of Bacillus QY2 after 60 days under different field moisture content conditions. The results show that Bacillus QY2 can show good survival ability after 60 days under the condition of soil moisture content ≥30%. When the moisture content is 10%, Bacillus QY2 cannot survive for more than 60 days.

[0095] This strain has a strong tolerance to high temperature and 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.

[0096] Table 8 Adaptability of 60d Bacillus sp. QY2 to different field water contents

[0097]

[0098] 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.

[0099] 6. Tolerance to broad-spectrum fungicides

[0100] 1. Experimental Design

[0101] 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 QY2 strain as a control. The culture was cultured in a shaking incubator at 37℃ and 120r / min. After 24h, the LB liquid culture medium was dipped with an inoculation loop and streaked on the LB plate to observe the growth of the QY2 strain, and 3 replicates were used for each treatment.

[0102] 2. Test results

[0103] The test results are shown in Table 9. Bacillus QY2 has different resistance to different fungicides. Among them, after the normal dose and 10 times the dose of pyraclostrobin, fluopicolide, carbendazim, myclobutanil, oxazolidinone, mancozeb, difenoconazole, boscalid and fluopyram were co-cultured with Bacillus QY2 for 24 hours, Bacillus QY2 could grow on the LB plate, indicating that Bacillus QY2 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 QY2 can grow live bacteria after co-cultured with normal doses of tebuconazole, propiconazole, isoprodinil, myclobutanil, pyrimidine nucleotide antibiotics and flusilazole for 24 hours, but it cannot survive after co-cultured with 10 times the dose for 24 hours. Bacillus QY2 cannot survive after being treated with normal doses and 10 times the dose of prochloraz.

[0104] Table 9 Tolerance of Bacillus QY2 to common fungicides

[0105]

[0106]

[0107] Note: “+” indicates the presence of live bacteria, “-” indicates the absence of live bacteria.

Claims

1. A Bacillus strain, characterized in that The Bacillus is Bacillus subtilis, named QY2, 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. 31373, 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 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, Diaporthephoenicicola, 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. The use according to claim 4, characterized in that: The concentration of Bacillus QY2 in the biocontrol agent is 1×10 6-8 cfu / L.

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