Atrophy-atrophy-bacillus velezensis compound bacteria and application thereof

Abstract

The invention discloses an atrophia-atrophia-bacillus velezensis compound bacterium and application thereof, and the compound bacterium is prepared from bacillus velezensis QS10-6, bacillus velezensis QS2-5 and bacillus velezensis QS2-13, wherein the bacillus atrophaeus QS10-6, the bacillus atrophaeus QS2-5 and the bacillus velezensis QS2-13 are preserved in Guangdong Province Microbial Culture Collection Center on January 13, 2025, the preservation numbers are respectively GDMCC NO: 65772, GDMCC NO: 65773 and GDMCC NO: 65774, and the preservation address is 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. According to the invention, the three strains are compounded together to have an obvious bacteriostatic effect on colletotrichum gloeosporioides, and a synergistic effect possibly exists among the complex flora, so that the antibacterial effect is mutually promoted, and the overall bacteriostatic rate is increased.

Description

Technical Field

[0001] The present invention belongs to the field of biocontrol bacteria, and particularly relates to a Bacillus velezensis complex bacterium of Atrophiibacter-Atrophiibacter and its application. Background Art

[0002] Potato anthracnose is one of the important diseases of potatoes, and its pathogen is Colletotrichum coccodes ( Colletotrichum coccodes ), which usually survives for a long time in the form of small sclerotia inside and on the surface of potato tubers or stems, crop debris, soil and air. The infection sites of this pathogen are extensive, and it can infect the leaves, the base of the stem and the stolons, daughter tubers and roots of the underground part and cause diseases; after infecting the tubers, the lesions are irregular or nearly circular, which will cause the tubers to gradually turn brown and rot; after infecting the rhizomes, it will turn black and rot, and a large number of black sclerotia will be produced in the lumen when the humidity is high; after infecting the stems, brown long stripes will be formed; there are usually no obvious symptoms on the leaves.

[0003] It is reported that C. coccodes can spread along the intercellular spaces in potato stem tissues and tubers, eventually leading to necrosis of stem tissues and tuber rot. With the changes in the environment and agricultural production conditions, the incidence of potato anthracnose and the economic losses caused by this disease are also continuously increasing, and the incidence of individual varieties can be as high as more than 50%.

[0004] In recent years, a large number of chemical agents have been used for the control of plant diseases. They have the characteristics of quick effect, high control efficiency and simple operation, and are widely used in the control of potato anthracnose. However, the long-term use of chemical agents will cause the pathogens to develop drug resistance and tolerance, reducing the control effect of chemical agents. Therefore, using biocontrol bacteria agents that are harmless to humans and the ecological environment to replace chemical agents has become the direction of plant disease control worldwide.

[0005] It should be noted that the information disclosed in the above background art section is only used to strengthen the understanding of the background of the present disclosure, and thus may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

[0006] The purpose of the present invention is to provide a Bacillus velezensis complex bacterium of Atrophiibacter-Atrophiibacter and its application, which solves the problem of environmental pollution to humans and the ecological environment in the existing chemical agent control. It has an obvious antibacterial effect on Colletotrichum coccodes of potatoes. There may be a synergistic effect between the complex bacterial populations, promoting each other's antibacterial effects and improving the overall antibacterial rate. Moreover, it does not have hemolytic property and is safe to use.

[0007] To achieve the above purpose, the present invention provides a Bacillus velezensis complex bacterium of Atrophiibacter-Atrophiibacter, which comprises: Atrophiibacter sp. ( Bacillus atrophaeus QS10-6, Atrophiibacter sp. (Bacillus atrophaeus ), QS2-5 and Bacillus velezensis ( Bacillus velezensis ), QS2-13; among them, the Bacillus atrophaeus QS10-6 was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on January 13, 2025, with the deposit number GDMCC NO: 65772 and the deposit address being the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou; the Bacillus atrophaeus QS2-5 was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on January 13, 2025, with the deposit number GDMCC NO: 65773 and the deposit address being the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou; the Bacillus velezensis QS2-13 was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on January 13, 2025, with the deposit number GDMCC NO: 65774 and the deposit address being the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0008] The second object of the present invention is to provide the application of the described Bacillus atrophaeus - Bacillus atrophaeus - Bacillus velezensis complex bacteria or their fermentation broth in the prevention and control of potato anthracnose.

[0009] Preferably, the volume ratio of the fermentation broths of Bacillus atrophaeus QS10-6, Bacillus atrophaeus QS2-5, and Bacillus velezensis QS2-13 is (1~3):(1~3):(1~3).

[0010] Preferably, the volume ratio of the fermentation broths of Bacillus atrophaeus QS10-6, Bacillus atrophaeus QS2-5, and Bacillus velezensis QS2-13 is 3:1:3.

[0011] Preferably, the absorbance OD 600 of the fermentation broths of Bacillus atrophaeus QS10-6, Bacillus atrophaeus QS2-5, and Bacillus velezensis QS2-13 is 2.0~2.1.

[0012] Preferably, in the preparation of the fermentation broth of the complex bacteria, the carbon source in the medium is selected from wheat bran; or / and, the nitrogen source in the medium is selected from yeast extract powder; or / and, the inorganic salt in the medium is selected from NaCl.

[0013] Preferably, the concentration of the wheat bran is 2.0~3.0%; or / and, the concentration of the yeast extract powder is 1.5~3.0%; or / and, the concentration of the NaCl is 0.5~1.5%.

[0014] Preferably, the concentration of the wheat bran is 2.5%, the concentration of the yeast extract powder is 2.6%, and the concentration of the NaCl is 1.0%.

[0015] Preferably, in the preparation of the fermentation broth of the composite bacteria, the culture conditions are: inoculum amount 1.0 - 2.0%, pH 5 - 10, culture temperature 27 - 37°C, and rotation speed 180 - 200 r / min.

[0016] More preferably, in the preparation of the fermentation broth of the composite bacteria, the culture conditions are: inoculum amount 1.0 - 2.0%, pH 5, culture temperature 27 - 37°C, and rotation speed 180 - 200 r / min.

[0017] Even more preferably, in the preparation of the fermentation broth of the composite bacteria, the culture conditions are: inoculum amount 2.0%, pH 5, culture temperature 32°C, and rotation speed 190 r / min.

[0018] The third object of the present invention is to provide the application of the Bacillus atrophaeus - Bacillus atrophaeus - Bacillus velezensis composite bacteria or its fermentation broth in preventing and controlling plant anthracnose caused by Colletotrichum gloeosporioides Colletotrichum coccodes ).

[0019] The Bacillus atrophaeus - Bacillus atrophaeus - Bacillus velezensis composite bacteria and its application of the present invention solve the problem of environmental pollution to humans and the ecosystem caused by chemical agent control, and have the following advantages: (1) The present invention uses Colletotrichum gloeosporioides as the pathogen, and screens endophytic bacteria with antagonistic effects against potato anthracnose from potato tubers. Among them, 3 strains of Bacillus with high antibacterial activity are Bacillus atrophaeus QS10 - 6, Bacillus atrophaeus QS2 - 5, and Bacillus velezensis QS2 - 13. Combining these 3 strains has an obvious antibacterial effect on Colletotrichum gloeosporioides of potatoes. There may be a synergistic effect among the composite bacterial populations, promoting each other's antibacterial effects and improving the overall antibacterial rate. The antibacterial activity of the composite bacterial population against potato anthracnose pathogens is stronger and more stable; (2) The present invention uses the response surface method to analyze and optimize, and obtains that the best fermentation medium for the composite bacterial population HQS6513 is wheat bran 2.5%, yeast extract powder 2.6%, NaCl 1.0%. The best fermentation conditions are culture temperature 32°C, pH 5, inoculum amount 2.0%, and shaker rotation speed 190 r / min. After optimizing the fermentation formula and conditions, the antibacterial rate of the composite bacterial population HQS6513 reaches 69.66%. After optimizing its fermentation formula and conditions, the spore production and antibacterial activity of the composite bacterial population HQS6513 increase, providing support for the research of biocontrol agents for potato anthracnose; (3) Bacillus atrophaeus QS10 - 6, Bacillus atrophaeus QS2 - 5, and Bacillus velezensis QS2 - 13 of the present invention do not show hemolysis and have safety, and are suitable for use as composite biocontrol agents. Description of the Drawings

[0020] Figure 1Antibacterial activity of bacteria against Colletotrichum coccodes in Experimental Example 1 of the present invention; a: CK; b: strain QS10-1; c: strain QS10-2; d: strain QS10-3; e: strain QS10-4; f: strain QS10-5; a1: strain QS10-6; b1: strain QS2-4; c1: strain QS2-5; d1: strain QS2-7; e1: strain QS2-11; f1: strain QS2-12; a2: strain QS2-13; b2: strain QS10-15; c2: strain QS10-16; d2: strain QS2-15; e2: strain QS2-17; f2: strain QS2-19; a3: strain QS10-18; b3: strain QS10-19; c3: strain QS10-20.

[0021] Figure 2 Colony morphology and phylogenetic tree of strains in Experimental Example 2 of the present invention; a, b, and c are the colony morphologies of QS10-6, QS2-5, and QS2-13 respectively; a1, b1, and c1 are the Gram staining morphologies of strains QS10-6, QS2-5, and QS2-13 respectively; a2, b2, and c2 are the phylogenetic trees of strains QS10-6, QS2-5, and QS2-13 respectively.

[0022] Figure 3 Blood safety evaluation results in Experimental Example 3 of the present invention; a: Staphylococcus aureus; b: QS10-6; c: QS2-5; d: QS2-13.

[0023] Figure 4 Determination of compatibility of 3 active bacteria in Experimental Example 4 of the present invention; a: QS10-6 vs QS2-5; b: QS10-6 vs QS2-13; c: QS2-5 vs QS2-13; d: QS2-5 vs QS10-6; e: QS2-13 vs QS10-6; f: QS2-13 vs QS2-5.

[0024] Figure 5 Antibacterial activity of strains QS10-6, QS2-5, QS2-13 and the composite bacterial community composed thereof in Experimental Example 5 of the present invention; A: Plate test, wherein, a: CK; b: QS10-6; c: QS2-5; d: QS2-13; e: HQS65; f: HQS613; g: HQS513; h: HQS6513; B: Results of the treatment group with biocontrol bacteria fermentation broth, C: Results of the treatment group with biocontrol bacteria suspension, D: Results of the prevention group with biocontrol bacteria fermentation broth, E: Results of the prevention group with biocontrol bacteria suspension, wherein, a: CK; b: H2O; c: Chlorothalonil; d: Carbendazim; e: HQS6513; f: QS10-6; g: QS2-5; h: QS2-13; i: HQS65; j: HQS613; k: HQS513.

[0025] Figure 6 This is the antibacterial effect of different compound ratios of the composite flora HQS6513 on potato anthracnose in Experimental Example 6 of the present invention.

[0026] Figure 7 This is the influence of different culture medium components and their concentrations on the growth and antibacterial activity of the composite flora HQS6513 in Experimental Example 7 of the present invention; bar chart: OD 600 ; broken line: antibacterial rate; A: treatment with different carbon sources; B: treatment with different concentrations of wheat bran; C: treatment with different nitrogen sources; D: treatment with different concentrations of yeast extract powder; E: treatment with different inorganic salts; F: treatment with different concentrations of NaCl.

[0027] Figure 8 This is the contour (left) and response surface diagram (right) of the interaction of different culture medium components on the antibacterial activity of the composite flora HQS6513 in Experimental Example 7 of the present invention; A1, B1: interaction between wheat bran and yeast extract powder; C1, D1: interaction between wheat bran and NaCl; E1, F1: interaction between yeast extract powder and NaCl.

[0028] Figure 9 This is the influence of fermentation conditions on the growth and antibacterial activity of the composite flora HQS6513 in Experimental Example 8 of the present invention; A: influence of temperature; B: influence of pH; C: influence of inoculum size; D: influence of rotation speed.

[0029] Note: Different lowercase letters on the bars or broken lines indicate significant differences ( P <0.05). Detailed implementation manners

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] It should be noted that: for those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the instruments not specified for the manufacturer, they are all conventional products that can be obtained by commercial purchase. For the raw materials and reagents not specified for the manufacturer, they are all commercially available products or can be prepared by known methods.

[0032] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0033] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features. All possible combinations should be considered as within the scope described in this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0034] The experimental materials used in the following experimental examples are as follows: 1. Strains and samples The pathogen of potato anthracnose, Colletotrichum coccodes Colletotrichum coccodes (Wallr.) Hughes, was isolated, identified and preserved by the Microbiology Laboratory of the Academy of Agriculture and Forestry Sciences, Qinghai University. The tested diseased potato samples were Qingshu 10 and Qingshu 2, which were collected from the storage cellars of the scientific research base of the Academy of Agriculture and Forestry Sciences, Qinghai University.

[0035] 2. Tested culture media PDA medium: 200.0 g of potato, 20.0 g of glucose, 20.0 g of agar and 1000 mL of distilled water, natural pH; without adding agar, it is PDB liquid medium.

[0036] LB medium: 10.0 g of NaCl, 10.0 g of tryptone, 5.0 g of yeast extract, 20.0 g of agar and 1000 mL of distilled water, natural pH; without adding agar, it is LB liquid medium.

[0037] Glucose peptone liquid medium: 2.0 g of K2HPO4, 5.0 g of glucose, 5.0 g of peptone and 1000 mL of distilled water, natural pH.

[0038] Glucose fermentation medium: 2.7 g of peptone, 5.0 g of NaCl, 0.3 g of dipotassium hydrogen phosphate, 3.0 mL of 1.0% bromothymol blue and 5.0 g of agar, sterilized at 121 °C for 15 min.

[0039] V-P medium: 5.0 g of tryptone, 5.0 g of glucose, 5.0 g of K2PO4, 1000 mL of distilled water, sterilized at 121 °C for 15 min.

[0040] Starch hydrolysis medium: 10.0 g of peptone, 5.0 g of NaCl, 2.0 g of soluble starch, 20.0 g of agar, 5.0 g of beef extract, 1000 mL of distilled water.

[0041] Nitrate reduction medium: 10.0 g of peptone, 2.0 g of potassium nitrate, 3.0 g of yeast extract powder and 1000 mL of distilled water, sterilized at 121 °C for 15 min.

[0042] Experimental Example 1 Isolation, Purification and Screening of Biocontrol Bacteria Potato disease samples were collected. The soil on the surface of the potato was rinsed with sterile water, then rinsed 2 - 3 times with 75% ethanol, and then rinsed 3 - 4 times with sterile water. Then, the diseased and healthy parts of the potato were cut into small pieces of 5 * 5 mm and placed into a sterilized mortar. The sample was ground until there were no obvious particles. 1.0 mL of the sample was taken out with a pipette, and 1.0 mL was aspirated for gradient dilution to make the concentration of the suspension 10 -1 to 10 −6 . 6 kinds of dilution solutions with concentration gradients (100 μL) were inoculated into PDA and LB media, and cultured at 28°C for 2 d until single colonies appeared. The plate streaking method was used to purify the strains with different morphologies, and the above operations were repeated until single strains with the same morphology were obtained.

[0043] Potato anthracnose pathogen discs (7 mm) were taken and inoculated in the center of the PDA medium. The above - purified bacteria were streaked at two points at the same distance of 3.5 cm from the potato anthracnose pathogen disc. The plate inoculated only with the pathogen was used as the control group (CK). The petri dishes were placed in an incubator at 28°C for 7 d, and the inhibition rate was measured. Each treatment had 3 replicates, and the experiment was repeated 3 times.

[0044] Inhibition rate (%) = (Colony diameter of the control group - Colony diameter of the treatment group) / Colony diameter of the control group × 100 The results are as Figure 1 shown in and Table 1. 20 strains with different morphologies were isolated and screened from the diseased tubers of potato No. 10 and No. 2. Among them, QS10 - 1, QS10 - 2, QS10 - 3, QS10 - 4, and QS2 - 13 had no antibacterial effect. The remaining 15 strains had inhibitory effects on potato anthracnose pathogen, and the inhibition rate was 42.07% - 59.97% (see Table 1). Among them, the inhibition rates of strains QS10 - 6, QS2 - 5, and QS2 - 13 were 42.25%, 56.75%, and 59.97% respectively. QS10 - 6, QS2 - 5, and QS2 - 13 were selected as active strains for subsequent experiments to further explore the antibacterial activity of these 3 strains as a complex flora.

[0045] Table 1 Activity of Bacteria in Inhibiting Potato Anthracnose Pathogen Note: The data are mean ± standard deviation. Different lowercase letters indicate significant differences at the 0.05 level tested by Duncan's new multiple range method.

[0046] Experimental Example 2 Identification of Active Strains 1. Morphological Observation The morphological characteristics of biocontrol strains QS10-6, QS2-5, and QS2-13 were observed with reference to the Manual for the Systematic Identification of Common Bacteria. The strains were streaked on LB plates and cultured at 28 °C for 36 h. The colony morphology, color, and transparency were observed and recorded, and strains QS10-6, QS2-5, and QS2-13 were Gram-stained and observed under a microscope.

[0047] The results are as Figure 2 shown in a–c and a1–c1 of

[0048] 2. Physiological and biochemical identification The physiological and biochemical characteristics of biocontrol strains QS10-6, QS2-5, and QS2-13, such as methyl red test, glucose fermentation test, V-P test, starch hydrolysis test, catalase test, nitrate reduction test, and acid-base determination test, were carried out with reference to the methods in the Manual for the Systematic Identification of Common Bacteria.

[0049] The results of the physiological and biochemical identification are shown in Table 2. The methyl red test and V-P test of strains QS10-6, QS2-5, and QS2-13 were both negative. Strains QS10-6, QS2-5, and QS2-13 could utilize starch. Strains QS2-5 and QS2-13 could reduce nitrate, while strain QS10-6 could not reduce nitrate. Strains QS10-6, QS2-5, and QS2-13 grew normally in an environment with pH 5, but could not grow normally in an environment with pH 10. Strain QS2-5 could utilize glucose, while strains QS10-6 and QS2-13 could not utilize glucose.

[0050] Table 2 Physiological and biochemical characteristics of strains QS10-6, QS2-5, and QS2-13 Note: “+”: positive reaction; “−”: negative reaction.

[0051] 3. Molecular biology identification The genomic DNA of strains QS10-6, QS2-5, and QS2-13 was extracted using a bacterial genomic DNA extraction kit. Conservative genes 16S rDNA, DNA gyrase subunit A (i.e., gyrA ), and RNA polymerase subunit β (i.e., rpoD ), which can be used to identify Bacillus species, were selected for PCR amplification and sequencing (see Table 3). The PCR amplification products were detected by 1.0% agarose gel electrophoresis, recovered, and sequenced. Using software MEGA 7.0 and Sequence Matrix, the gene sequences of 16S rDNA, gyrA , rpoD were concatenated and aligned, and a phylogenetic tree was constructed using the maximum likelihood method (bootstrap value set to 1000) to determine the taxonomic status of strains QS10-6, QS2-5, and QS2-13.

[0052] Table 3 Primer information for molecular identification of Bacillus QS10-6, QS2-5, and QS2-13 The results of molecular biological identification are shown in a2-c2 of Figure 2 . Based on the 16S rDNA, gyrA , , rpoD gene sequences of strains QS10-6, QS2-5, and QS2-13, a multi-gene phylogenetic tree of QS10-6, QS2-5, and QS2-13 was constructed. The results showed that strain QS10-6 clustered with Bacillus atrophaeus XT28, strain QS2-5 clustered with Bacillus atrophaeus C4-1-2, and strain QS2-13 clustered with Bacillus velezensis YW0041.

[0053] Based on the comprehensive results of colony morphology, physiological and biochemical, and molecular biological identification, strain QS10-6 was finally identified as Bacillus atrophaeus ( Bacillus atrophaeus ), strain QS2-5 was identified as Bacillus atrophaeus ( Bacillus atrophaeus ), and strain QS2-13 was identified as Bacillus velezensis ( Bacillus velezensis ).

[0054] Experimental Example 3 Safety evaluation of active strains Using the streak plate method, active strains QS10-6, QS2-5, and QS2-13 were streaked and inoculated on blood agar plates, respectively, and incubated upside down in a constant temperature incubator at 28 °C for 48 h. The hemolysis phenomenon of the strains on the blood plate medium was observed to judge their safety.

[0055] The results are shown in Figure 3As shown in the figure, a completely transparent and clear area appeared around Staphylococcus aureus on the blood agar plate medium, while no completely transparent and clear area appeared around the colonies of strains QS10-6, QS2-5, and QS2-13, indicating that the strain did not exhibit hemolysis and was suitable for the preparation of a compound biocontrol agent.

[0056] Experimental Example 4 Antagonistic Test of Strains Single colonies of QS10-6, QS2-5, and QS2-13 were picked from the plate with an inoculation loop respectively and inoculated onto the same plate by the "cross-cross method". After culturing in a constant temperature incubator for 24 h, the growth of the colonies on the plate was observed. The cross-cross method stipulates that if the bacteria at the intersection of the strains grow normally, then the strains have no antagonistic effect and can be co-cultured; otherwise, it indicates that there is an antagonistic effect between the strains and they cannot be co-cultured.

[0057] The results are as Figure 4 shown. Six rounds of tests were carried out on each of the three bacterial strains using the 2×2 antagonistic assay. The results of the six test groups showed that there was no inhibition of each other's growth at the intersections of the strains, indicating that these three strains were not antagonistic to each other, suggesting that they could coexist and thrive in the mixed culture and could therefore be used in combination as a composite bacterial population.

[0058] Experimental Example 5 Antibacterial Activity Test of Composite Bacterial Population 1. Preparation of Pathogen Seed Liquid and Fermentation Liquid of Biocontrol Bacteria Preparation of pathogen seed liquid: The Colletotrichum gloesporioides activated on the PDA solid plate was punched with a puncher and then transferred to the PDA liquid medium with forceps. It was cultured in a constant temperature shaker at 28°C and 180 r / min for 10 d. When used, it was filtered through four layers of sterile gauze into a 50 mL high-speed centrifuge tube and centrifuged at 4°C and 11000 r / min for 15 min. The supernatant was removed, and sterile water was added to the original volume for standby.

[0059] Preparation of biocontrol bacteria seed liquid: The strains QS10-6, QS2-5, and QS2-13, which were isolated and purified and had good antibacterial activity against potato anthracnose, were inoculated onto the LB solid medium by streaking with an inoculation loop and cultured at 28°C for 24 h for activation. Then, one loop of the activated colony was taken with an inoculation loop and transferred to the LB liquid medium, and cultured in a constant temperature shaker at 28°C and 180 r / min for 24 h for standby.

[0060] Preparation of biocontrol bacteria fermentation liquid: Each seed liquid with an inoculation amount of 1.0% was inoculated into the LB liquid medium respectively. The liquid volume in a 250 mL triangular flask was 100 mL, and it was cultured in a constant temperature shaker at 28°C at 180 r / min for 24 h to obtain the fermentation liquid.

[0061] Preparation of biocontrol bacteria suspension: Centrifuge the above-mentioned biocontrol bacteria fermentation broth at 4°C and 11,000 r / min for 15 min, discard the supernatant, and add sterile water to the original volume to obtain the bacteria suspension.

[0062] 2. Antibacterial activity test (1)In vitro plate antibacterial test Determined by the drug sensitivity paper method. Punch and inoculate the potato pathogen in the center of the PDA solid medium. Uniformly stick four drug sensitivity papers at 3.5 cm from the four corners of the pathogen, and place them in an incubator at 28°C for 24 h. Then, drop 4.0 μL of the fermentation broth or bacteria suspension (1×10 7 cfu / mL) of the biocontrol bacteria (single biocontrol bacteria or complex flora) on the drug sensitivity papers respectively. The treatment with only the pathogen inoculated is used as the positive control, and it is placed in an incubator at 28°C for 7 d. Measure the diameter of the lesion and calculate the inhibition rate. Each treatment group is repeated 3 times.

[0063] Inhibition rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100 The results show that the fermentation broth of the strain has better and more stable antibacterial effect than the bacteria suspension. The antibacterial results of the fermentation broth are shown in Table 4 and Figure 5 as shown in A. The antibacterial rate of strain QS10-6 on the in vitro plate is only 36.31%, while the antibacterial rates of the complex flora HQS65 (QS10-6 and QS2-5), HQS613 (QS10-6 and QS2-13), HQS513 (QS2-5 and QS2-13), and HQS6513 (QS10-6, QS2-5, and QS2-13) composed of strains QS10-6, QS2-5, and QS2-13 reach 50.29%, 54.99%, 53.64%, and 58.91% on the plate antibacterial rate. Therefore, the antibacterial rate of the complex flora HQS6513 is higher than that of the single bacteria QS10-6 and QS2-5. The reason for this phenomenon may be that the complex flora has more complex metabolic pathways and bioactive products, can produce more types of antibacterial substances, improve the antibacterial effect, and there may be a synergistic effect between the complex flora, promoting each other's antibacterial effect and increasing the overall antibacterial rate. Generally speaking, the complex flora has stronger and more stable antibacterial activity against potato anthracnose pathogens.

[0064] Table 4 Antibacterial activities of the fermentation broths of strains QS10-6, QS2-5, QS2-13 and the composed complex flora Note: The data are mean ± standard deviation, and different lowercase letters indicate significant differences at the 0.05 level by Duncan's new multiple range test.

[0065] (2)Potato antibacterial test Select healthy, fresh, and undamaged potatoes. After cleaning them thoroughly, soak them in 75% ethanol for 2 - 3 min, then wash them with pure water 2 - 3 times, dry them on a superclean bench, and use a sterile borer to make 1 hole of the same size in the middle of the potato tuber for the following experiments: Treatment group with biocontrol bacteria fermentation broth: Inoculate 100 μL of the pathogen seed solution (1×10 7 cfu / mL, the same below) into the hole, and after 1 day, inoculate 100 μL of the biocontrol bacteria fermentation broth (1×10 7 cfu / mL, the same below); Prevention group with biocontrol bacteria fermentation broth: Inoculate 100 μL of the biocontrol bacteria fermentation broth into the hole, and after 1 day, inoculate 100 μL of the pathogen seed solution; Treatment group with biocontrol bacteria suspension: Inoculate 100 μL of the pathogen seed solution (1×10 7 cfu / mL, the same below) into the hole, and after 1 day, inoculate 100 μL of the biocontrol bacteria suspension (1×10 7 cfu / mL, the same below); Prevention group with biocontrol bacteria suspension: Inoculate 100 μL of the biocontrol bacteria suspension into the hole, and after 1 day, inoculate 100 μL of the pathogen seed solution; Positive treatment control group: Inoculate 100 μL of the pathogen seed solution into the hole, and after 1 day, inoculate 100 μL of chlorothalonil or carbendazim; Positive prevention control group: Inoculate 100 μL of chlorothalonil or carbendazim into the hole, and after 1 day, inoculate 100 μL of the pathogen seed solution; Blank control group (CK): Inoculate 100 μL of the pathogen seed solution into the hole.

[0066] Place the above groups at 28°C and culture them in the dark for 7 days. Measure the diameter of the lesion and calculate the inhibition rate (the same as above). Each treatment group is repeated 3 times.

[0067] The results are shown in B - E of Figure 5 . B of Figure 5 is the inhibition experiment result of the fermentation broth of the treatment group against potato anthracnose, Figure 5 C of Figure 5 is the inhibition experiment result of the suspension of the treatment group against potato anthracnose, Figure 5Figure E shows the results of the antibacterial experiment of the bacterial suspension of the prevention group against potato anthracnose. It can be seen from the figure that the antibacterial activity of the strain fermentation broth is greater than that of the bacterial suspension. Moreover, it can be seen that the antibacterial effect of the bacterial suspension of QS-13 on potato anthracnose is significantly poor, and the potato tubers rot severely. However, the antibacterial effect of the composite bacterial community HQS6512 is obvious whether it is the fermentation broth or the bacterial suspension, indicating that the composite bacterial community has stronger stability than single bacteria. Among the fermentation broths, the antibacterial effect of HQS6513 is equivalent to that of the standard pesticides chlorothalonil and carbendazim. Therefore, the composite bacterial community HQS6513 composed of the strains QS10-6, QS2-5, and QS2-13 was selected as the research object for subsequent studies.

[0068] Experimental Example 6 Determination of the strain ratio The strains QS10-6, QS2-5, and QS2-13 were mixed and inoculated for fermentation, with a total inoculation amount of 1.0%. The inoculation ratios were set as shown in Table 5, and they were inoculated into LB medium in 100 / 250 mL (100 mL of culture solution in a 250 mL bottle), and cultured in a constant temperature shaking flask cabinet at 28 °C for 24 h, and the OD 600 value and antibacterial rate were measured to determine the optimal inoculation ratio.

[0069] Table 5 shows the ratio settings of the contents of the 3 strains The results are as Figure 6 shown. By measuring the OD 600 value and antibacterial rate, there are no particularly large differences in the OD 600 value among the combinations T1~T9. However, in terms of the antibacterial rate, the antibacterial rate of combination T7 is significantly higher than that of other ratios. At this ratio, the OD 600 and antibacterial rate of the composite bacterial community HQS6513 reached 2.055 and 56.10% respectively. Therefore, the optimal ratio of active bacteria was determined as T7 = 3:1:3, and all subsequent experiments were inoculated at this ratio.

[0070] Experimental Example 7 Optimization of the components of the optimal medium 1. Single-factor experiment on the components of the medium Six carbon sources (glucose, sucrose, fructose, lactose, soluble starch, wheat bran), six nitrogen sources (beef extract, tryptone, yeast extract powder, glutamic acid, sodium nitrate, urea) and five inorganic salts (NaCl, MgSO4, K2HPO4, CaCl2, (NH4)2SO4, NaCl) were respectively selected to equivalently replace the corresponding components in the basal medium (weigh 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride, add 1000 mL of distilled water, sterilize at 121 °C for 20 min; among them, tryptone is used as the nitrogen source, yeast extract is used as the carbon source, and sodium chloride is used as the inorganic salt). After shaking culture for 24 h under the conditions of a liquid loading of 100 mL, an inoculation amount of 1.0%, a shaker speed of 180 r / min, and a culture temperature of 28 °C, the absorbance at 600 nm (OD 600 ) was measured and the antibacterial rate was calculated to determine the best carbon source, nitrogen source and type of inorganic salt. The effects of the selected best carbon source, nitrogen source and inorganic salt at different concentrations (0.5%, 1.0%, 1.5%, 2.0%, 2.5% and 3.0%) on the growth and antibacterial activity of the complex microbial community HQS6513 were explored to determine the subsequent addition concentration.

[0071] As can be seen from Figure 7 A, among different carbon sources, the OD 600 values of the fermentation broth of the complex microbial community decreased in the order of wheat bran > soluble starch > lactose > sucrose > fructose > glucose, and the antibacterial rates decreased in the order of wheat bran > sucrose > glucose > lactose > soluble starch > fructose. When wheat bran was used as the carbon source, the OD 600 of the fermentation broth and the antibacterial rate were both the highest, reaching 2.093 and 58.01%. Therefore, wheat bran was selected as the optimal carbon source for the fermentation broth of the complex microbial community.

[0072] As can be seen from Figure 7 B, the OD 600 and antibacterial rates of the fermentation broth of the complex microbial community were different under different wheat bran treatments. The OD 600 and antibacterial rate of the fermentation broth both reached the maximum value when the wheat bran concentration was 2.5%. Therefore, wheat bran concentrations of 2.0%, 2.5% and 3.0% were selected as the subsequent carbon source concentrations for the preparation of the fermentation broth of the complex microbial community.

[0073] As can be seen from Figure 7 C, among different nitrogen sources, the OD 600 values of the fermentation broth of the complex microbial community decreased in the order of glutamic acid > yeast extract powder > sodium nitrate > tryptone > beef extract powder > urea, and the antibacterial rates decreased in the order of yeast extract powder > beef extract powder > sodium nitrate > tryptone > glutamic acid > urea. When yeast extract powder was used as the nitrogen source, the OD 600 of the fermentation broth and the antibacterial rate were both the highest, reaching 2.970 and 58.74%. Therefore, yeast extract powder was selected as the optimal nitrogen source for the fermentation broth of the complex microbial community.

[0074] As can be seen from Figure 7 D in Figure 7 , the OD of the fermentation broth of the complex microbial community under different concentrations of yeast extract powder treatment 600 and the antibacterial rate are different. The OD 600 of the fermentation broth and the antibacterial rate both reach the maximum value at a concentration of 2.5%. Therefore, the concentrations of 1.5%, 2.5% and 3.0% are selected as the subsequent nitrogen source concentrations for the preparation of the fermentation broth of the complex microbial community.

[0075] As can be seen from Figure 7 E in Figure 7 , among different inorganic salts, the OD 600 values of the fermentation broth of the complex microbial community from high to low are K2HPO4 > CaCl2 > NaCl > MgSO4 > NaCl > (NH4)2SO4, and the antibacterial rates from high to low are NaCl > NaCl > MgSO4 > K2HPO4 > (NH4)2SO4 > CaCl2. When NaCl is the inorganic salt, the OD 600 and the antibacterial rate of the fermentation broth are both the highest, reaching 2.779 and 62.02%. Therefore, NaCl is selected as the optimal inorganic salt for the fermentation broth of the complex microbial community.

[0076] As can be seen from Figure 7 F in Figure 7 , the OD 600 of the fermentation broth of the complex microbial community and the antibacterial rate are different under different concentrations of NaCl treatment. The OD 600 of the fermentation broth and the antibacterial rate both reach the maximum value at a concentration of 1.0%. Therefore, the concentrations of 0.5%, 1.0% and 1.5% are selected as the subsequent inorganic salt concentrations for the preparation of the fermentation broth of the complex microbial community.

[0077] 2. Response surface test for medium optimization On the basis of single-factor experiments, the optimal carbon source concentration (A), nitrogen source concentration (B) and inorganic salt concentration (C) are selected as three factors. Taking the inhibitory activity of the fermentation broth of the complex microbial community HQS6513 against Colletotrichum gloesporioides as the response value, the single factors selected are used as independent variables, and Design Expert 13 is used for response surface test design to determine the optimal composition of the fermentation medium and conduct 5 repeated verification tests on it.

[0078] The effects of the bran concentration (A), yeast extract powder concentration (B) and NaCl concentration (C) in the medium of the complex microbial community on the antibacterial activity of its fermentation broth were studied by the response surface method (Table 6), and its antibacterial activity was fitted with a quadratic multiple regression equation to obtain the quadratic multiple regression equation of the antibacterial rate (y) of the complex microbial community HQS6513: y = 63.61 - 0.2362A + 0.7200B - 0.3938C - 0.2500AB - 0.2475AC + 0.5500BC - 4.03A 2 - 3.63B 2-2.91°C 2 。

[0079] Table 6 Factors and Levels of the Optimization Test of the Fermentation Medium Components of the Composite Bacterial Community HQS6513 Table 7 Significance Test of the Regression Model for the Optimization of the Medium Components of the Composite Bacterial Community HQS6513 Note: * and ** indicate significant difference ( P <0.05) or extremely significant difference ( P <0.01), respectively.

[0080] As can be seen from Table 7, the fitting model P <0.0001, which is an extremely significant level ( P <0.01, the same below), and the lack-of-fit term is not significant ( P >0.05, the same below), indicating that the experimental data has a high degree of fitting with the model, and the established model is meaningful. In addition, except that the interaction terms AB and AC are not significant and the first-order term A shows a significant level ( P <0.05, the same below), other terms all show extremely significant levels, indicating that the influence of the three components on the antibacterial rate changes complexly and is not a common linear relationship. Among the correlation coefficients, R 2 >0.900; R 2 adj =0.9935; the signal-to-noise ratio (Adeq Precision) = 39.7638 all indicate that the correlation of the model is good, the credibility is high, and the prediction of the fermentation component ratio is relatively accurate. The F value is more important for the experimental factors. The larger the F value, the greater the influence of the factor on the antibacterial rate. Therefore, F(B) > F(C) > F(A), that is, the influence degree on the antibacterial rate of the composite bacterial community HQS6513 is yeast extract powder > NaCl > wheat bran.

[0081] As Figure 8 shown, the response surface and contour lines can more intuitively reflect the interaction of the three components and their influence on the antibacterial rate. As Figure 8 shown in A and B, the contour lines are elliptical, the response surface graph opens downward, there is a highest point, and it is found that when the concentration of yeast extract powder is constant, the antibacterial rate of the fermentation broth of the composite bacterial community HQS6513 first increases and then decreases with the increase of the wheat bran concentration, and the antibacterial rate reaches the maximum value when the wheat bran concentration reaches about 2.5%; when the wheat bran concentration is constant, the antibacterial rate of the fermentation broth of the composite bacterial community HQS6513 shows the same change trend with the increase of the yeast extract powder concentration, and the antibacterial rate reaches the maximum value when the yeast extract powder concentration is 2.6%.

[0082] As Figure 8As can be seen from C and D, the contour lines of NaCl and bran are elliptical. At the same time, the response surface plot bulges with the opening downward, indicating an obvious interaction between NaCl and bran. When the NaCl concentration is fixed, the antibacterial rate of the fermentation broth of the complex bacterial community HQS6513 first increases and then decreases with the increase of the bran concentration. When the NaCl content is about 1.0%, the antibacterial rate reaches the maximum value. When the bran concentration is fixed, the antibacterial rate first increases and then decreases with the increase of the NaCl concentration.

[0083] From Figure 8 As can be seen from E and F, the contour plot is elliptical, indicating a significant interaction between yeast extract powder and NaCl. When the yeast extract powder concentration is fixed, the antibacterial rate of the fermentation broth of the complex bacterial community HQS6513 first increases and then decreases with the increase of the NaCl concentration. When the NaCl concentration is fixed, the antibacterial rate of the fermentation broth of the complex bacterial community HQS6513 first increases and then decreases with the increase of the yeast extract powder concentration.

[0084] Figure 8 The response surfaces of B, D, and F all have the opening downward. The highest point of the model represents the optimal point of the response value, that is, the optimal concentrations of each culture medium component (A, B, C) corresponding to the highest antibacterial rate (y), which are bran concentration (A) = 2.5%, yeast extract powder concentration (B) = 2.6%, and NaCl concentration (C) = 1.0%, respectively. At this time, the maximum antibacterial rate of the fermentation broth of the complex bacterial community HQS6513 is 63.66%.

[0085] 3. Verification of the optimal culture medium Adjust the corresponding components in the basic culture medium to 2.5% bran, 2.6% yeast extract powder, and 1.0% NaCl. Five repeated experiments were carried out under this condition, and the antibacterial rates of the fermentation broth of the complex bacterial community HQS6513 were measured to be 64.35%, 64.24%, 63.78%, 64.84%, and 63.56%, with an average value of 64.16%, which is very close to the predicted 63.66% in the experiment. This further shows that the culture medium component parameters of the fermentation broth of the complex bacterial community HQS6513 predicted by the response surface optimization method are relatively accurate and can be used as a reference for subsequent industrial production.

[0086] The present invention discovers that both lower or higher carbon and inorganic salt concentrations can reduce the growth of the composite bacterial community HQS6513 and lower its antibacterial rate. Insufficient medium components are not conducive to the reproduction of strains, while high concentrations of carbon sources and inorganic salts not only cause material waste but also inhibit the growth of bacteria to a certain extent. Generally speaking, accurate component ratios have important reference value for the subsequent industrial fermentation of strains. Moreover, bran has a low cost and abundant raw materials, and can be applied to large-scale industrial production. The present invention adds bran as the optimal carbon source for the composite bacterial community HQS6513, which improves the yield of antibacterial active substances in the composite bacterial community and the resource utilization rate of bran.

[0087] Optimization of the Optimal Culture Conditions in Experimental Example 8 Using the optimized medium components in Experimental Example 7, with an inoculation amount of 1.0%, pH of 7, liquid filling amount of 100 / 250 mL, culture temperature of 28 °C, and rotation speed of 180 r / min as the initial culture conditions, single-factor experiments were successively conducted on different setting values of four factors: fermentation temperature (27, 32, 34, 37, and 42 °C), inoculation amount (1.0%, 2.0%, 3.0%, 4.0%, and 5.0%), pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), and shaker rotation speed (140, 150, 160, 170, 180, 190, and 200 r / min). After selecting the optimal value from the previous single-factor experiment, it was used as the condition for the next single-factor experiment, and the OD of the fermentation broth of the composite bacterial community HQS6513 was measured. 600 and antibacterial rate to optimize each fermentation condition parameter.

[0088] As can be seen from Figure 9 A in, except at 42 °C, the OD of the fermentation broth of the composite bacterial community HQS6513 under the other 4 temperature treatments 600 was 2.783 - 3.014, among which 32 °C was the most suitable, with an OD 600 of 3.014 and the highest antibacterial rate, reaching 60.32%. Excessive culture temperature will inhibit the growth of strain HQS6513. Perhaps most of the bacteria are inactive or enter a dormant state at high temperatures and cannot carry out normal growth and reproduction. For example, when the culture temperature is 42 °C, the OD of the fermentation broth 600 significantly decreases, only being 2.297; too high a temperature will also affect the bacterial mass in the fermentation broth and at the same time significantly reduce the inhibitory activity of the fermentation broth against pathogenic bacteria. Therefore, 32 °C was selected as the optimal culture temperature for the composite bacterial community HQS6513.

[0089] As can be seen from Figure 9 B in, the composite bacterial community HQS6513 can grow at pH 4 - 10, but its growth is significantly inhibited under strong acid and strong base conditions. For example, when the pH is 4, the OD of the fermentation broth 600It is only 0.037, and 0.051 at pH 10. Generally speaking, as the pH increases, the OD of the fermentation broth 600 shows a trend of first increasing and then decreasing; the antibacterial rate fluctuates accordingly with the change of OD 600 . When the pH is 5, the OD 600 is relatively large, being 2.874, and at this time the antibacterial rate also reaches the maximum value, which is 55.94%. Therefore, pH 5 is selected as the optimal pH for culturing the composite flora HQS6513.

[0090] As can be seen from Figure 9 C of 600 , the growth of the composite flora HQS6513 under different inoculum amounts is different, and its fermentation broth has a good inhibitory effect on pathogenic bacteria, and the antibacterial rates are all above 50.00%. Among them, when the inoculum amount is 2.0%, the OD

[0091] reaches the maximum value, being 3.217, and the antibacterial rate is the highest, being 62.43%. Therefore, 2.0% is selected as the optimal inoculum amount for culturing the composite flora HQS6513. Figure 9 As can be seen from 600 E of

[0092] , the growth and antibacterial activity of the composite flora HQS6513 are greatly affected by the shaker speed. When the speed is 140 - 200 r / min, the OD Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A composite bacterium of Bacillus velezensis with atrophy-atrophy, characterized in that, The composite bacterium includes: Bacillus atrophaeus ( Bacillus atrophaeus ), QS10-6, Bacillus atrophaeus ( Bacillus atrophaeus ), QS2-5, and Bacillus velezensis ( Bacillus velezensis ), QS2-13; Among them, the Bacillus atrophaeus QS10-6 was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 13, 2025, with the deposit number GDMCC NO: 65772, and the deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou; The Bacillus atrophaeus QS2-5 was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 13, 2025, with the deposit number GDMCC NO: 65773, and the deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou; The Bacillus velezensis QS2-13 was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 13, 2025, with the deposit number GDMCC NO: 65774, and the deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

2. Use of the Bacillus atrophaeus - Bacillus atrophaeus - Bacillus velezensis complex bacterium or its fermentation broth according to claim 1 in the prevention and control of potato anthracnose.

3. The application according to claim 2, wherein The volume ratio of the fermentation broths of the Bacillus atrophaeus QS10-6, Bacillus atrophaeus QS2-5 and Bacillus velezensis QS2-13 is (1~3):(1~3):(1~3).

4. The application according to claim 3, characterized in that, The volume ratio of the fermentation broths of the Bacillus atrophaeus QS10-6, Bacillus atrophaeus QS2-5 and Bacillus velezensis QS2-13 is 3:1:

3.

5. The application according to claim 3, wherein The absorbance OD of the fermentation broth of Bacillus atrophaeus QS10-6, Bacillus atrophaeus QS2-5, and Bacillus velezensis QS2-13 600 is 2.0 - 2.

1.

6. The application according to claim 2, characterized in that, In the preparation of the fermentation broth of the complex bacterium, the carbon source in the culture medium is selected from wheat bran; or / and, the nitrogen source in the culture medium is selected from yeast extract powder; or / and, the inorganic salt in the culture medium is selected from NaCl.

7. The application according to claim 6, wherein The concentration of the wheat bran is 2.0~3.0%; or / and, the concentration of the yeast extract powder is 1.5~3.0%; or / and, the concentration of the NaCl is 0.5~1.5%.

8. The application according to claim 7, wherein The concentration of the wheat bran is 2.5%, the concentration of the yeast extract powder is 2.6%, and the concentration of the NaCl is 1.0%.

9. The application according to any one of claims 6 to 8, characterized in that, In the preparation of the fermentation broth of the complex bacterium, the culture conditions are: inoculum amount 1.0~2.0%, pH 5~10, culture temperature 27~37°C and rotation speed 180~200 r / min.

10. Use of the Bacillus velezensis complex bacteria of atrophy-atrophy or its fermentation broth according to claim 1 in preventing and treating plant anthracnose caused by Colletotrichum coccodes ).

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