Saxafor-bacillus atrophaeus compound bacteria and application thereof

Abstract

The invention discloses a Bacillus saffors-Bacillus atrophaeus compound bacterium and application thereof, the compound bacterium comprises Bacillus saffors BM-7 and Bacillus atrophaeus QB-2-7, the Bacillus saffors BM-7 is preserved in Guangdong Microbial Culture Collection Center on January 13, 2025, the preservation number is GDMCC NO: 65770, the preservation number is CGMCC NO: 65770, the preservation number is CGMCC NO: 65770, and the preservation number is CGMCC NO: 65770. The bacillus atrophaeus QB-2-7 is preserved in Guangdong Microbial Culture Collection Center on January 13, 2025, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 65771. The compound bacterium does not have hemolytic activity, is high in safety, has a good inhibition effect on colletotrichum gloeosporioides, has stable physicochemical properties, relatively strong biofilm forming ability, temperature tolerance and illumination and ultraviolet tolerance, is relatively wide in pH value application range, and has the potential of being applied to field disease control.

Description

Technical Field

[0001] The present invention belongs to the field of biocontrol bacteria, and particularly relates to a composite bacterium of Serratia fonticola - Bacillus atrophaeus and its application. Background Art

[0002] Potato anthracnose is caused by Colletotrichum coccodes ( Colletotrichum coccodes ). This pathogen has strong stress resistance and can survive for a long time. Its pathogen overwinters in the form of microsclerotia on the surface of potato tubers or the residues of its plants and can survive in the soil for a long time. Colletotrichum coccodes has a wide range of infection sites and can infect the leaves, the base of the stem, and the stolons, tubers, and roots of the underground part, causing diseases. In severe cases, it can cause plant shrinkage and withering, resulting in large - scale death and serious yield reduction, causing huge economic losses. Therefore, effectively controlling potato diseases during cultivation and storage has become an urgent problem in production.

[0003] At present, the prevention and control of potato anthracnose mainly rely on chemical control. However, long - term use of chemical agents can cause problems such as environmental pollution, ecological environment damage, pesticide residues, and the generation of pathogen resistance. Biological control is sustainable. Compared with traditional chemical control methods, biological control can not only effectively control diseases but also play a continuous control role.

[0004] The application of biological control technology in crops can not only effectively reduce crop diseases but also protect the ecological environment. Compared with traditional chemical pesticides, biological control technology has less negative impact on the ecosystem and non - target organisms. Biological control methods combat diseases by simulating biological interactions in natural ecosystems, promoting the natural balance among organisms, reducing long - term ecological damage and the risk to human health. As an important measure for the integrated management of crop diseases, biological control using antagonistic microorganisms has obvious advantages. Microorganisms can effectively control diseases by producing antibacterial substances, competing for nutrients and space, and inducing resistance. As an efficient means of reducing economic losses, biocontrol agents have been proven to provide promising solutions for the protection of various crops. However, the application effect of a single biocontrol agent is often unstable or has a short duration, and there are many problems such as low environmental adaptability, weak stress resistance, and low control effect. Mixed biocontrol microorganisms can form a more stable and efficient composite microbial community, thus achieving the purpose of synergy.

[0005] Therefore, it is necessary to explore the synthesis of biocontrol microbial communities that are friendly to humans and the environment and have good control effects to solve the problems existing in the chemical control of potato anthracnose.

[0006] It should be noted that the information disclosed in the above background art section is only used to enhance the understanding of the background of the present disclosure. Therefore, it may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

[0007] The object of the present invention is to provide a composite bacterium of Bacillus safensis - Bacillus atrophaeus and its application, which solves the problems of environmental pollution, ecological environment damage, pesticide residues, etc. caused by chemical control of anthracnose. The composite bacterium of the present invention does not have hemolytic property, has high safety, has good inhibitory effect on Colletotrichum coccodes, has stable physical and chemical properties and strong biofilm formation ability, has certain temperature tolerance, light and ultraviolet tolerance, and a wide pH adaptation range.

[0008] In order to achieve the above object, the present invention provides a composite bacterium of Bacillus safensis - Bacillus atrophaeus, which comprises: Bacillus safensis ( Bacillus safensis ) BM - 7 and Bacillus atrophaeus ( Bacillus atrophaeus ) QB - 2 - 7. Among them, the Bacillus safensis BM - 7 was deposited at the Guangdong Microbial Culture Collection Center on January 13, 2025, with the deposit number GDMCC NO: 65770, and the deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou; the Bacillus atrophaeus QB - 2 - 7 was deposited at the Guangdong Microbial Culture Collection Center on January 13, 2025, with the deposit number GDMCC NO: 65771, and the deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0009] The second object of the present invention is to provide the application of the fermentation broth or its bacterial suspension or supernatant of the composite bacterium of Bacillus safensis - Bacillus atrophaeus in the prevention and control of potato anthracnose.

[0010] Preferably, the volume ratio of the fermentation broth of Bacillus atrophaeus QB - 2 - 7 to Bacillus safensis BM - 7 is (1 - 9):(9 - 1) Preferably, the absorbance OD 600 value of the fermentation broth of Bacillus atrophaeus QB - 2 - 7 and Bacillus safensis BM - 7 is the same.

[0011] Preferably, the absorbance OD 600 of the fermentation broth of Bacillus atrophaeus QB - 2 - 7 and Bacillus safensis BM - 7 is both 1.0.

[0012] Preferably, the volume ratio of the fermentation broth of Bacillus atrophaeus QB - 2 - 7 to Bacillus safensis BM - 7 is 7:3.

[0013] Preferably, the preparation of the fermentation broth of the composite bacterium of Bacillus safensis - Bacillus atrophaeus comprises: (1) Preparation of seed liquid of each strain Pick single colonies of the active strains QB - 2 - 7 and BM - 7 and inoculate them into a liquid medium respectively, and shake - culture at 35 °C until the OD 600 value of the bacterial liquid is 1.0; (2)Preparation of Fermentation Broth, Supernatant and Bacterial Suspension of the Composite Bacteria Inoculate the seed solutions of QB-2-7 and BM-7 into the liquid medium and culture them with shaking at 35 °C until the OD 600 of the bacterial solution reaches 1.0 to obtain the fermentation broth of the composite bacteria.

[0014] Preferably, for the preparation of the bacterial suspension of the Bacillus safensis - Bacillus atrophaeus composite bacteria, centrifuge the fermentation broth of the Bacillus safensis - Bacillus atrophaeus composite bacteria, collect the bacterial cells, wash them with the buffer solution, and then add the buffer solution to the original volume to obtain the bacterial suspension.

[0015] Preferably, for the preparation of the supernatant of the Bacillus safensis - Bacillus atrophaeus composite bacteria, it includes: centrifuge the fermentation broth of the Bacillus safensis - Bacillus atrophaeus composite bacteria and collect the supernatant.

[0016] Preferably, the rotation speed of the shaking culture is 180 r / min; and / or, the liquid medium is selected as LB liquid medium.

[0017] Preferably, the pathogen of potato anthracnose includes: Colletotrichum coccodes ( Colletotrichum coccodes ).

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

[0019] The Bacillus safensis - Bacillus atrophaeus composite bacteria and its application of the present invention solve the problems such as environmental pollution, ecological environment damage, and pesticide residues caused by chemical control of anthracnose, and have the following advantages: (1) The present invention uses two strains of endophytic bacteria isolated from potato tubers in the early stage to construct a synthetic bacterial community, and finds that QB-2-7 and BM-7 can grow together. The synthetic bacterial community has an antibacterial effect on anthracnose pathogens. Mix different strains and ferment them together to promote their proliferation and coexistence with each other, and finally generate a microbial community with a complex composition and multiple functions. Different parts of it have antibacterial activity, and the antibacterial rates of the fermentation broth, bacterial suspension and supernatant of the synthetic bacterial community are all above 55.00%, and the antibacterial effect is obvious; (2) The present invention uses Bacillus safensis Bacillussafensis BM-7 and Bacillus atrophaeus Bacillus atrophaeusQB-2-7 is a biocontrol strain that forms a synthetic community. It has a control effect on potato anthracnose. This synthetic community does not have hemolytic activity, has high safety, has a good inhibitory effect on Colletotrichum gloeosporioides, and can also induce the activity of enzymes in potato tubers to resist pathogen infection. At the same time, the fermentation broth of the synthetic community has stable physical and chemical properties and strong biofilm formation ability, has a certain temperature tolerance, light and ultraviolet tolerance, and a wide pH adaptation range. Therefore, the synthetic community of the present invention has the potential to be applied to field disease control. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a photograph of the colony morphology and microscopic morphology of strains QB-2-7 and BM-7 on an LB plate in Experimental Example 1 of the present invention; a and c are the colony morphology and microscopic morphology of strain QB-2-7 respectively; b and d are the colony morphology and microscopic morphology of strain BM-7 respectively.

[0021] Figure 2 It is a phylogenetic tree constructed based on 16S rDNA and gyrA, gyrB multi-gene sequences of strains BM-7 and QB-2-7 in Experimental Example 1 of the present invention; A: phylogenetic tree of strain BM-7; B: phylogenetic tree of strain QB-2-7.

[0022] Figure 3 It is the hemolytic safety evaluation result of strains QB-2-7 and BM-7 in Experimental Example 2 of the present invention; a: strain QB-2-7; b: strain BM-7; c: Staphylococcus aureus.

[0023] Figure 4 It is the result of strain compatibility in Experimental Example 3 of the present invention; a: the horizontal line is strain QB-2-7, and the broken line is strain BM-7; b: the horizontal line is strain BM-7, and the broken line is strain QB-2-7.

[0024] Figure 5 It is the OD 600 value and antibacterial rate of single strains and mixed fermentation broths of strains QB-2-7 and BM-7 in Experimental Example 3 of the present invention.

[0025] Figure 6 It is the antibacterial activity of the synthetic community against Colletotrichum gloeosporioides in Experimental Example 4 of the present invention; a: colony morphology of the control plate; b: colony morphology of the plate confrontation.

[0026] Figure 7 It is the antibacterial activity of different parts of the synthetic community culture broth against Colletotrichum gloeosporioides in Experimental Example 4 of the present invention; a: control; b: synthetic community fermentation broth; c: synthetic community bacterial suspension; d: synthetic community supernatant.

[0027] Figure 8For the control effect of the synthetic microbial community on potato tuber anthracnose in Experimental Example 5 of the present invention; A1: Lesion conditions of potato tubers at different times; A2: Change in lesion diameter of potato tubers at different times.

[0028] Figure 9 For the effect of the synthetic microbial community on potato tuber-related defense enzymes in Experimental Example 6 of the present invention; A: SOD; B: MDA; C: POD; D: PPO.

[0029] Figure 10 For the antibacterial activity of the fermentation broth of the synthetic microbial community under different conditions in Experimental Example 7 of the present invention; A: Temperature; B: pH; C: Light exposure time; D: UV irradiation time.

[0030] Figure 11 For the formation of the biofilm of the synthetic microbial community in Experimental Example 8 of the present invention; A: Biofilm formation of the synthetic microbial community at 24 h and 48 h; B: Biofilm formation ability of the synthetic microbial community.

[0031] Note: The error bars in the figure represent standard deviation; CK: Blank control group; F: Group treated with only pathogen inoculation; B: Group treated with only synthetic microbial community inoculation; P: Prevention mode treatment group; T: Treatment mode treatment group; G: Difenoconazole treatment group. Detailed implementation manners

[0032] 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 of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts shall fall within the protection scope of the present invention.

[0033] It should be noted that: for those not specified with specific conditions in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the instruments not specified with the manufacturer, they are all conventional products that can be obtained through commercial purchase. For the raw materials and reagents not specified with the manufacturer, they are all commercially available products or can be prepared by known methods.

[0034] 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 the numerical range or percentage range should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0035] 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.

[0036] The experimental materials used in the following experimental examples are as follows: 1. Test strains Strains QB-2-7 and BM-7 are two endophytic bacteria with strong inhibitory effects on Colletotrichum coccodes. Potato disease samples were collected from the storage cellar of the Qinghai Potato Alpine Experiment Station (101°04′12″E, 36°73′31″N). After isolating and purifying the above two strains from the disease samples, they were stored in the Microbiology Laboratory of the Qinghai Academy of Agriculture and Forestry Sciences. The test pathogen is Colletotrichum coccodes Colletotrichum coccodes which was isolated and purified from the diseased potato samples of the potato variety Qingshu 10.

[0037] 2. Test potato varieties The potato variety Qingshu 10 was provided by the Institute of Biotechnology, Academy of Agriculture and Forestry Sciences, Qinghai University.

[0038] 3. Test media PDA medium: 200 g of potato, 20 g of glucose, 20 g of agar and 1000 mL of distilled water, natural pH; without adding agar is PDB liquid medium. Autoclaved at 121 °C for 20 min.

[0039] LA medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 20 g of agar and 1000 mL of distilled water, pH 7.4; without adding agar is LB liquid medium. Autoclaved at 121 °C for 20 min.

[0040] Blood agar medium: 10 g of trypticase peptone, 5 g of sodium chloride, 5 g of meat peptone, 1 g of corn starch, 3 g of heart peptone, 5 g of yeast extract powder, 15 g of agar and 1000 mL of distilled water, pH 7.1 - 7.5. Autoclaved at 121 °C for 15 min, cooled to about 55 °C and added with 7% - 10% sterile defibrinated sheep blood, mixed well and made into plates. Sterile defibrinated sheep blood (product number TX0020 - 100 ml) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0041] Experimental Example 1 Identification of Strains QB-2-7 and BM-7 1. Morphological identification The morphological characteristics of biocontrol strains QB-2-7 and BM-7 were observed with reference to the Manual for Systematic Identification of Common Bacteria. The strains were streaked on LB plates and cultured at 35 °C for 48 h. The colony morphology, color, and transparency were observed and recorded. The strains QB-2-7 and BM-7 were Gram-stained and observed under a microscope.

[0042] As Figure 1 shown in a of [reference], the results showed that the colonies of strain QB-2-7 had irregular edges and were opaque. When cultured at 35 °C for 48 h, its colonies were light brown on LB medium, with a rough surface, convex folds, and a dry texture. As Figure 1 shown in c of [reference], the results of staining and microscopic examination showed that the endophyte QB-2-7 was rod-shaped and Gram-positive.

[0043] As Figure 1 shown in b of [reference], the results showed that when cultured at 35 °C for 48 h, the colonies of strain BM-7 had uneven edges and were opaque, with a milky white front, a moist texture, convex folds in the middle, and were easy to pick up. As Figure 1 shown in d of [reference], the results of staining and microscopic examination showed that the endophyte BM-7 was rod-shaped and Gram-positive.

[0044] 2. Physiological and biochemical identification The physiological and biochemical characteristics of biocontrol strains QB-2-7 and BM-7, such as starch hydrolysis, indole production, gelatin liquefaction, catalase activity, citrate utilization, methyl red test, V-P test, and nitrate reduction, were identified with reference to the method in the Manual for Systematic Identification of Common Bacteria.

[0045] The results are shown in Table 1. Strain QB-2-7 was negative in the methyl red test, positive in the V-P test, could reduce nitrate, produce IAA and catalase, had the ability to hydrolyze starch and gelatin, and could utilize citrate. Strain BM-7 was negative in the methyl red test, positive in the V-P test, could reduce nitrate, produce IAA and catalase, could not hydrolyze starch, could hydrolyze gelatin, and could utilize citrate.

[0046] Table 1 Physiological and biochemical characteristics of strains QB-2-7 and BM-7 Note: "+": positive reaction; "-": negative reaction.

[0047] 3. Molecular biological identification The genomic DNA of strains QB-2-7 and BM-7 was extracted using a bacterial genomic DNA extraction kit. Genes 16S rDNA and DNA gyrase subunit A ( gyrA), DNA gyrase subunit B, namely gyrB ), were subjected to PCR amplification and sequencing (see Table 2). After the PCR amplification products were detected by 1% agarose gel electrophoresis and recovered, they were sent to Aiyouji Biotechnology (Xi'an) Co., Ltd. for sequencing. Using software MEGA 7.0 and Sequence Matrix, the gene sequences of 16S rDNA and gyrA, gyrB 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 QB-2-7 and BM-7.

[0048] Table 2 Primer information for molecular identification of Bacillus QB-2-7 and BM-7 The results are as shown in Figure 2 A, a multi-gene phylogenetic tree of BM-7 was constructed based on the gene sequences of 16S rDNA and gyrA, gyrB . The concatenated 16S rDNA and gyrA, gyrB gene sequences of strain BM-7 were found to be in the same clade as Bacillus.safensis strain NECC11095. Based on the comprehensive results of colony morphology, physiological and biochemical characteristics, and molecular biological identification, strain BM-7 was finally identified as Bacillus safensis Bacillus. safensis .

[0049] The results are as shown in Figure 2 B, a multi-gene phylogenetic tree of QB-2-7 was constructed based on the gene sequences of 16S rDNA and gyrA, gyrB . The concatenated 16S rDNA and gyrA, gyrB gene sequences of strain QB-2-7 were BLAST searched in NCBI and found to be in the same clade as Bacillus. atrophaeus CTD639-B3, Bacillus. atrophaeus SW , Bacillus. atrophaeus Z2, Bacillus. atrophaeus IHBB strains. Based on the comprehensive results of the unique colony morphology, physiological and biochemical characteristics, and molecular biological identification, strain QB-2-7 was further identified as Bacillus atrophaeus Bacillus. atrophaeus .

[0050] Experimental Example 2 Hemolytic safety evaluation of strains QB-2-7 and BM-7 Using a sterile inoculation loop, streak inoculate one loop of QB-2-7 and BM-7 bacteria on blood agar plates and incubate them in a 35 °C constant temperature incubator for 24 h. Using Staphylococcus aureus ( Staphylococcus aureus , GDMCC 1.221, purchased from the Guangdong Microbial Culture Collection Center) as a control, repeat 3 times, and observe the hemolytic phenomenon of the strains to judge their safety.

[0051] The results are as shown inFigure 3 As shown in Figure 3 , the hemolytic test results showed that the control Staphylococcus aureus formed a hemolytic ring on the blood agar plate, and as time prolonged, the hemolytic ring gradually expanded ( Figure 3 as shown in c) of Figure 3 . After culturing strains QB-2-7 and BM-7 on the blood agar plate for 24 h, no hemolytic zone was produced ( Figure 3 as shown in a) and b) of Figure 3 ), indicating no hemolytic property and high safety.

[0052] Experimental Example 3 Construction of Synthetic Bacterial Community 1. Compatibility Test of Strains Strains QB-2-7 and BM-7 were inoculated vertically on both sides of the LB solid medium. After culturing at 35 °C for 24 h, whether there was an antibacterial zone at the adjacent part of the strains was observed. If the two strains were compatible, they could grow with each other without the production of an antibacterial zone, while an antibacterial zone was produced between incompatible strains.

[0053] The results were as Figure 4 shown. No antibacterial zone was produced between the adjacent parts of the horizontal streak culture of strain QB-2-7 and the vertical streak culture of BM-7 ( Figure 4 as shown in a) of Figure 4 ), indicating that QB-2-7 and BM-7 could grow together without inhibition. No antibacterial zone was also produced between the adjacent parts of the horizontal streak culture of strain BM-7 and the vertical streak culture of QB-2-7, indicating no inhibitory effect between the two strains ( Figure 4 as shown in b) of Figure 4 ). Therefore, the two active endophytic bacteria were compatible with each other without mutual inhibition and could construct a synthetic bacterial community.

[0054] 2. Determination of the Optimal Inoculation Amount of the Synthetic Bacterial Community QB-2-7 and BM-7 were respectively inoculated into the LB liquid medium, and the absorbance of the bacterial solution was measured at a wavelength of 600 nm until the OD 600 value reached 1.0. A total of 11 inoculation amounts from K0 to K10 were set for each strain (see Table 3). The fermentation broths of QB-2-7 and BM-7 were proportioned according to Table 3 to make the total volume of the bacterial solution reach 1 mL, and then added to 100 mL of sterilized LB liquid medium according to the proportion. Each group was set with 3 parallel groups. After culturing at 35 °C and 180 r / min for 12 h, a Colletotrichum gloeosporioides mycelial disc with a diameter of 8 mm was inoculated in the center of the PDA plate, and filter paper was placed crosswise around it. The PDA without the fermentation broth of the synthetic bacterial community was used as the control. After culturing at 28 °C for 24 h, each treatment was repeated 3 times. The diameter of the control group and the diameter of the colony of the treatment group were measured by the cross method, and the antibacterial rate was calculated. Using the strain combination with the highest measured antibacterial rate as the object, a mixed bacterial solution was prepared.

[0055] Table 3 Proportion of the Fermentation Broths of Strains QB-2-7 and BM-7 The results were asFigure 5 As shown in the figure, according to the effects of different inoculation amounts, it can be obtained that after 24 hours of cultivation, the OD 600 values of the experimental combinations K0 to K10 are significantly different. K0 and K10 are single bacteria, and their OD 600 values are lower than those of the synthetic microbial community. The inhibitory effects of the fermentation broths of each synthetic microbial community on Colletotrichum gloesporioides are all above 50%. The antibacterial effects of K0 and K10 are not as good as those of the synthetic microbial community. Among the various combinations, the antibacterial rate of K7 against Colletotrichum gloesporioides is significantly different from that of other groups, and the antibacterial rate is the highest, reaching 64.68%. Considering comprehensively, the optimal combination ratio of the two bacteria is QB-2-7:BM-7 = 7:3.

[0056] Experimental Example 4 Antibacterial Activity Experiment 1. Antibacterial Activity of Synthetic Microbial Community against Colletotrichum gloesporioides Using the plate confrontation method, Colletotrichum gloesporioides was cultured in the dark at 28 °C on a PDA plate for 7 days. A mycelial disc with a diameter of 8 mm was punched from the edge of the colony and placed in the center of the PDA plate. At four relative points 25 mm away from the mycelial disc, sterile filter paper discs were placed. 2 μL of the synthetic microbial community fermentation broth (OD 600 = 1.0) was pipetted onto the filter paper discs (diameter 6 mm). The plate without inoculating the synthetic microbial community fermentation broth was used as the control group. There were 4 discs per plate. The plates were incubated upside down at 28 °C until the control colony covered the entire plate. Each treatment was repeated 3 times. The diameter of the inhibition zone was measured with a vernier caliper, and the inhibition rate was calculated.

[0057] Inhibition rate = (diameter of control colony - diameter of treated colony) / (diameter of control colony - diameter of mycelial disc) × 100%.

[0058] The results are as Figure 6 shown. The results of the plate confrontation test show that when the diameter of the control colony reached 79.99 mm, the diameter of the Colletotrichum gloesporioides colony inoculated with the synthetic microbial community fermentation broth was 34.52 mm, and the inhibition rate was 63.16%. It can be Figure 6 seen that the synthetic microbial community has an obvious inhibitory effect on Colletotrichum gloesporioides.

[0059] 2. Antibacterial Activity of Different Parts of Synthetic Microbial Community Culture Broth against Colletotrichum gloesporioides (1) Preparation of Seed Liquids of Each Strain Single colonies of the active strains QB-2-7 and BM-7 cultured for 1 day were picked and inoculated into LB liquid medium respectively. They were cultured with shaking at 35 °C and 180 r / min. When the OD 600 value of the bacterial liquid measured by a spectrophotometer reached 1.0, it was stored in the refrigerator for later use.

[0060] (2) Preparation of Synthetic Microbial Community Fermentation Broth The optimal ratio was obtained through the construction of synthetic microbiota. The seed solutions of QB-2-7 and BM-7 were inoculated into LB liquid medium at inoculation amounts of 0.7% and 0.3% respectively, with a filling volume of 100 mL / 250 mL (100 mL of culture solution in a 250 mL bottle), cultured at 35 °C and 180 r / min for 12 h until the OD 600 of the bacterial solution reached 1.0, which was the synthetic microbiota fermentation broth.

[0061] (3) Preparation of supernatant The synthetic microbiota fermentation broth was centrifuged at 4 °C and 12,000 r / min for 15 min, and the supernatant was collected, which was the supernatant.

[0062] (4) Preparation of bacterial suspension The synthetic microbiota fermentation broth was centrifuged at 4 °C and 12,000 r / min for 15 min, and the bacteria were collected. After washing 3 times with PBS buffer, PBS buffer was added to the original volume, which was the bacterial suspension.

[0063] Colletotrichum gloesporioides was inoculated in the center of a PDA plate, and filter papers were placed crosswise around it. 2 μL of the synthetic microbiota fermentation broth, bacterial suspension, and supernatant were respectively inoculated on sterile filter papers (diameter 6 mm). Without inoculating the synthetic microbiota fermentation broth, bacterial suspension, and supernatant was used as a blank control. When the control mycelium was almost full of the entire petri dish, the colony diameter was measured with a vernier caliper, and the inhibition rate of each part against Colletotrichum gloesporioides was calculated. Each treatment was repeated 3 times.

[0064] The results are as Figure 7 shown in and Table 4. Different components of the synthetic microbiota culture broth all had certain antibacterial activities against Colletotrichum gloesporioides ( Figure 7 b-d). The diameter of the control colony measured with a vernier caliper was 75.71 mm. Among them, the confrontation colony inoculated with the synthetic microbiota fermentation broth only grew to 32.35 mm. The antibacterial rate of the fermentation broth was calculated to be the highest, 64.13%, which was significantly higher than that of the synthetic microbiota supernatant. The confrontation colony of the synthetic microbiota bacterial suspension was 34.12 mm, and the antibacterial rate was lower than that of the fermentation broth, 61.43% (see Table 4). Therefore, subsequent research was carried out on the fermentation broth of the synthetic microbiota.

[0065] Table 4 Antibacterial activities of different parts of the synthetic microbiota culture broth against Colletotrichum gloesporioides Note: "-" indicates no inhibitory activity; the above values represent the mean ± standard deviation, and different letters indicate significant differences at the P < 0.05 level. The same applies to the following tables.

[0066] Experimental Example 5 Control effect of synthetic microbiota on potato tuber anthracnose Evaluate the control effect of the synthetic microbial community fermentation broth on potato anthracnose in Experimental Example 3 as follows: Wash the soil on the potatoes with tap water, air-dry them at room temperature, disinfect them with 1% sodium hypochlorite solution for 5 min, rinse them with sterile water 3 - 5 times, air-dry them on a clean bench, and punch a hole with a diameter of 8 mm and a depth of 10 mm in the middle of the potato tubers using a sterile borer. A total of 6 groups are set up, with 30 fruits in each group, and repeated 3 times. Each group is as follows:

[0067] Blank control group (CK): Sterile water, 40 μL per hole; Pathogen-only inoculation treatment group (F): Only inoculate 40 μL of pathogen spore suspension (1×10 8 cfu / mL) per hole; Synthetic microbial community-only inoculation treatment group (B): Only inoculate 40 μL of synthetic microbial community fermentation broth (OD 600 = 1.0) per hole; Prevention mode treatment group (P): Add 40 μL of synthetic microbial community fermentation broth (OD 600 = 1.0) per hole, culture at 28 °C for 24 h, and then inoculate 40 μL of pathogen spore suspension (1×10 8 cfu / mL); Treatment mode treatment group (T): Add 40 μL of pathogen spore suspension (1×10 8 cfu / mL) per hole, culture at 28 °C for 24 h, and then inoculate 40 μL of synthetic microbial community fermentation broth (OD 600 = 1.0).

[0068] Difenoconazole treatment group (G): Add 40 μL of pathogen spore suspension (1×10 8 cfu / mL) per hole, culture at 28 °C for 24 h, and then inoculate 40 μL of difenoconazole (active ingredient content 40%).

[0069] Seal and package the treated potatoes with sterile seal bags, and store them at 28 °C with humidity. Sample and observe the incidence of potato anthracnose on the 0th, 2nd, 4th, 8th, and 12th days respectively, and calculate the weight loss rate and control effect on the 12th day. The calculation formulas are as follows:

[0070] Weight loss rate (%) = (mass of potato tuber on the 0th day - mass of potato tuber on the 12th day) / mass of potato tuber on the 12th day × 100 Control effect (%) = (lesion diameter of pathogen-only inoculation - lesion diameter of treatment group) / (lesion diameter of pathogen-only inoculation - punching diameter) × 100 The results are as Figure 8 shown. From Figure 8As can be seen from A1, as the storage time extended, the lesions gradually enlarged. Among them, the lesions in the group treated only with the pathogen inoculation (F) enlarged rapidly on the 8th day, the interior of the potato rotted and became hollow, and the lesions were obvious; the same changes occurred in the treatment mode group (T) on the 8th d; while the lesions in the prevention mode group (P) and the difenoconazole treatment group (G) changed slowly, and there were no lesions in the group treated only with the synthetic community inoculation (B). This result was consistent with the lesion diameter ( Figure 8 in A2). On the 4th, 8th, and 12th d of storage, the lesion diameter of the group treated only with the pathogen inoculation (F) was higher than that of the prevention mode group (P), the treatment mode group (T), the difenoconazole treatment group (G), the group treated only with the synthetic community inoculation (B), and the blank control group (CK). The differences between the group treated only with the pathogen inoculation (F) and the three treatment mode groups indicated that the synthetic community composed of QB-2-7 and BM-7 could reduce the infection ability of Colletotrichum gloesporioides on potatoes, thereby delaying the decay of potatoes and extending the cellar storage time of potatoes.

[0071] In addition, the differences in the control effects of the three treatment mode groups (see Table 5) showed that the prevention mode group (P) had the highest control effect, which was 78.55%, significantly higher than the other two treatment mode groups, indicating that the synthetic community was significantly more effective in preventing potato anthracnose than in treatment. As shown in Table 5, the weight loss rate of potato tubers in the prevention mode group (P) was higher than that of the difenoconazole treatment group (G), the group treated only with the synthetic community inoculation (B), and the blank control group (CK), but lower than that of the treatment mode group (T) and the group treated only with the pathogen inoculation (F). On the 12th d of storage, the weight loss rate of the prevention mode group (P) was 5.16%, that of the treatment mode group (T) was 10.23%, that of the difenoconazole treatment group (G) was 5.05%, that of the blank control group (CK) was 3.87%, that of the group treated only with the synthetic community inoculation (B) was 3.88%, and that of the group treated only with the pathogen inoculation (F) was 14.50%. It can be seen that the weight loss rate of the group treated only with the pathogen inoculation (F) was significantly higher than that of other treatments, while inoculating the synthetic community had little effect on the weight loss rate of potatoes. Thus, it can be known that during storage, the synthetic community composed of QB-2-7 and BM-7 can inhibit the weight loss of potatoes caused by Colletotrichum gloesporioides infection during storage and maintain the weight of their tubers.

[0072] Table 5 Effects of synthetic community on control effect and quality of potato tuber anthracnose (12 d) Biocontrol bacteria and pathogenic bacteria compete for space and nutrients for survival and reproduction in a microenvironment. There are antagonistic substances in the secondary metabolites of microorganisms, which can inhibit the growth and reproduction of pathogenic bacteria. Bacillus is more likely to colonize in the rhizosphere than other strains. The preventive effect of the synthetic community of the present invention on potato anthracnose is much higher than the therapeutic effect. This may be because the synthetic community first occupies the infection sites of pathogenic bacteria in potatoes, thus improving the preventive effect on potato anthracnose.

[0073] Experimental Example 6 Effect of Synthetic Community on Defense Enzyme Activity of Potatoes In addition to the antagonism and competition for space and nutrients, biocontrol bacteria can also indirectly mediate biological control by stimulating the induced systemic resistance of crops. The defensive role of enzymes is one of the most basic and key reactions for plants to resist the invasion of pathogenic bacteria. This biological defense mechanism is achieved through a series of physiological mechanisms of disease-resistant substances in response to pathogens. Therefore, the defense enzyme activities of potatoes were measured as follows:

[0074] The potato tubers were treated as in Experimental Example 4. Samples were taken at 0, 2, 4, 8, and 12 d respectively. Each time, 3 g of the flesh of the damaged part was taken for the determination of the contents of superoxide dismutase (SOD), peroxidase (POD), polyphenoloxidase (PPO), and malondialdehyde (MDA). The extraction of crude enzyme solution and the detection of enzyme activity were carried out according to the steps of the enzyme activity kit (purchased from Suzhou Keming Biotechnology Co., Ltd., produced in Suzhou, Jiangsu).

[0075] The results are as Figure 9 shown in A. The SOD enzyme activity in each treatment showed a trend of first increasing and then decreasing, and all peaked at the 8th d. The SOD enzyme activity at the 8th d from high to low was the preventive mode treatment group > the group inoculated only with pathogenic bacteria > the therapeutic mode treatment group > the difenoconazole treatment group > the group inoculated only with the synthetic community > the blank control. Among them, the SOD enzyme activity of the preventive mode treatment group (P) was the highest, with an activity value of 604.53 U / g. The SOD enzyme activity of the group inoculated only with the synthetic community (B) was lower than that of the group inoculated only with pathogenic bacteria (F), the therapeutic mode treatment group (T), and the difenoconazole treatment group (G), with an activity value of 344.75 U / g. It can be seen that compared with other treatments, the preventive mode treatment significantly increased the SOD enzyme activity, and the treatment of inoculating only the synthetic community had a small impact on the change of SOD enzyme activity.

[0076] The results are as Figure 9As shown in B, through the analysis of the MDA content of potato tubers treated differently, it can be seen that the MDA content of the group treated only with the pathogen (F) showed a sharp change trend of first rising rapidly and then falling. The MDA content was significantly higher than that of other treatments at 8 d, reaching the peak value of 43.83 U / g. The change trend of the content of other treatments, which first increased and then decreased, was relatively gentle. The group treated only with the pathogen (F) was significantly higher than the blank control group (CK) by 238.92%, the group treated only with the synthetic flora (B) by 220.93%, the preventive mode treatment group (P) by 83.33%, the treatment mode treatment group (T) by 225.23%, and the difenoconazole treatment group (G) by 205.74% at 8 d. By 12 d, the MDA content of each treatment group decreased slightly, but the group treated only with the pathogen (F) was still significantly higher than other treatment groups. The results are as Figure 9 As shown in C, from 0 to 8 d, the POD enzyme activity of potato tubers in all treatments gradually increased. Especially from 2 to 4 d, the POD enzyme activity of each treatment increased sharply and reached the peak value at 8 d. Among them, the POD enzyme activity of the preventive mode treatment group (P) was significantly higher than that of other treatment groups at 8 d, which was 25033.33 U / g. At this time, the enzyme activity of each treatment inoculated with the synthetic flora was higher than that of the group treated only with the pathogen (F). At 12 d, the POD enzyme activity of each treatment decreased, but it was still significantly higher than the control treatment at the same time.

[0077] The results are as Figure 9 As shown in D, the change in the PPO enzyme activity of the group treated only with the pathogen (F) was the largest. The PPO enzyme activities of the four treatments inoculated with the synthetic flora were all relatively low, and showed a trend of first increasing and then decreasing from 0 to 12 d. From 4 to 8 d, the PPO enzyme activities of the treatment mode treatment group (T) and the difenoconazole treatment group (G) increased sharply and reached the peak values at 8 d, and the difference was the largest at this time. The increase in the PPO enzyme activities of the group treated only with the synthetic flora (B) and the preventive mode treatment group (P) was not significant. Thus, it can be seen that the preventive mode of inoculating the synthetic flora 24 h first and then inoculating Colletotrichum gloesporioides can reduce the PPO enzyme activity more significantly than the treatment mode.

[0078] From the above results, it can be seen that the activities of antioxidant enzymes SOD and POD in the potatoes in the prevention mode treatment group of the present invention are significantly higher than those in the control group (P<0.05). SOD and POD scavenge excessive reactive oxygen species, slow down and inhibit the occurrence of potato anthracnose during storage, and protect plant cells from oxidative damage. In addition, MDA is an important indicator of membrane lipid peroxidation, and its content can reflect the degree of cell damage to a certain extent. PPO is an important oxidase present in most plants, forming a physical barrier to protect plants against pathogen infection. In the present invention, the contents of MDA and PPO in the potatoes treated only with the pathogen (B) increased sharply, while after treatment with the synthetic bacterial community, the damage to potato cells was alleviated, maintaining its normal metabolic state to a certain extent and promoting the defense response of the potatoes.

[0079] Experimental Example 7 Determination of the Stability of the Fermentation Broth of the Synthetic Bacterial Community Using different treatment conditions, the stability of the antibacterial active substances in the fermentation broth of the synthetic bacterial community was explored. The treatment conditions included: thermal stability, acid-base stability, light stability, and ultraviolet tolerance time test. The antibacterial situation under different treatment conditions was determined by the plate confrontation method to determine the stability of the fermentation broth under different treatment conditions, as follows:

[0080] (1) Effect of Temperature on the Activity of the Fermentation Broth of the Synthetic Bacterial Community Take 35 mL of the fermentation broth of the synthetic bacterial community (OD 600 =1.0), and divide it equally into 7 10 mL centrifuge tubes, with 5 mL of the fermentation broth in each tube, numbered T1, T2, T3, T4, T5, T6, and T7 respectively. To clarify the effect of temperature on the antibacterial rate of the fermentation broth, a total of 7 temperature treatments were set. T1-T7 were placed at 4 °C, 20 °C, 40 °C, 60 °C, 80 °C, 100 °C, and 121 °C for 30 min respectively, and immediately cooled in an ice bath after taking out. The antibacterial activities of T1-T7 were measured, and each treatment was set with 3 replicates in total. The plate confrontation method was used to measure the antibacterial activities of the fermentation broth under different treatments.

[0081] The results are as Figure 10 shown in A. After the fermentation broth was treated at high temperature and high pressure of 100 °C and 121 °C for 30 min, it lost its antibacterial activity, indicating that the fermentation broth of the synthetic bacterial community has no tolerance to high temperature. After the fermentation broth of the synthetic bacterial community was incubated at temperatures in the range of 4-80 °C for 30 min, it still had antibacterial activity, and there were significant differences among the temperature treatment groups. After the fermentation broth was treated at 80 °C, the antibacterial rate was 49.42%. In agricultural use, environmental conditions above 80 °C rarely occur, but it is necessary to avoid the influence of too high temperature on the antibacterial activity of the fermentation broth of the synthetic bacterial community during storage.

[0082] (2) Effect of Acid and Base on the Activity of the Fermentation Broth of the Synthetic Bacterial Community Forty milliliters of synthetic microbial community fermentation broth (OD 600 = 1.0) was taken in 8 portions and aliquoted into 10 mL sterile centrifuge tubes. To understand the acid-base tolerance of the fermentation broth, a total of 8 pH treatments were set. The pH of the fermentation broth was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10 respectively with 1 mol / L HCl and NaOH. After incubation at 35 °C for 2 h, the pH was adjusted back to neutral, and the antibacterial activities of samples labeled 3 - 10 were measured. The fermentation broth without pH adjustment was set as the control group. Each treatment was replicated 3 times, and the antibacterial activities of the fermentation broth under different treatments were measured by the plate confrontation method.

[0083] The results are shown in Figure 10 Figure B. When the pH of the synthetic microbial community fermentation broth exceeds 9, the antibacterial rate of the fermentation broth decreases significantly, indicating low tolerance to a slightly alkaline environment; when the pH of the synthetic microbial community fermentation broth is below 5, the antibacterial rate of the fermentation broth continues to decrease, indicating low tolerance to a slightly acidic environment. The antibacterial ability of the fermentation broth remains at a relatively high level when the pH is in the range of 5 - 8. The synthetic microbial community grows well in a neutral environment and has weak tolerance to overly acidic or alkaline environments. Therefore, the antibacterial ability of the synthetic microbial community fermentation broth will be significantly weakened in soils with a pH greater than 9 or less than 5, and the soil in this environment is not suitable for using the synthetic microbial community to control potato anthracnose.

[0084] (3) Effect of light on the activity of synthetic microbial community fermentation broth Twenty-five milliliters of synthetic microbial community fermentation broth (OD 600 = 1.0) was aliquoted into 5 10 mL centrifuge tubes, with 5 mL of fermentation broth (OD 600 = 1.0) in each tube, numbered L1, L2, L3, L4, and L5 respectively. They were placed in a light incubator for irradiation treatment and cultured at 25 °C. The antibacterial activities of L1 were measured at 2 d, L2 at 4 d, L3 at 6 d, L4 at 8 d, and L5 at 10 d. The plate confrontation method was used, and each treatment was replicated 3 times.

[0085] The results are shown in Figure 10 Figure C. The effect of light on the antibacterial rate of the synthetic microbial community composed of QB-2-7 and BM-7 is not significant. With the extension of light exposure time, the antibacterial rate of the synthetic microbial community fermentation broth does not change significantly and remains at a relatively high level, indicating that the antibacterial active substances in the synthetic microbial community fermentation broth are not sensitive to light, and the influence of light on its antibacterial rate can be excluded in subsequent experiments.

[0086] (4) Effect of ultraviolet irradiation time on the activity of synthetic microbial community fermentation broth Thirty-five milliliters of synthetic microbial community fermentation broth (OD 600It was aliquoted into 7 10-mL centrifuge tubes (5 mL of the fermentation supernatant in each tube), numbered UV1 - UV7 respectively. A total of 7 time periods of ultraviolet irradiation treatment were set, and they were irradiated at equal distances under an ultraviolet lamp for 0, 30, 60, 90, 120, 150, and 180 min respectively. Each treatment was replicated 3 times, and the antibacterial activity of the fermentation broth under different treatments was measured by the plate confrontation method.

[0087] The results are as Figure 10 shown in D. After ultraviolet irradiation for 0 - 180 min, there was no significant difference in the antibacterial rate of each group, indicating that the synthetic community was not sensitive to long-term ultraviolet irradiation. Therefore, it can be directly exposed to sunlight for a long time during use, which is beneficial to the application of the synthetic community in potato potted plants and fields.

[0088] From the above results, it can be seen that the antibacterial substances of Bacillus atrophaeus and Bacillus safensis of the present invention both have strong stability, have a certain tolerance to adverse environmental conditions, have a certain tolerance to temperature, light, and ultraviolet, and have a wide pH adaptation range. The stability of the antibacterial substances of the biocontrol bacteria plays a key role in the biocontrol effect during the application process. Therefore, it can be known that the synthetic community of the present invention has the potential for application in the prevention and control of field diseases.

[0089] Experimental Example 8 Detection of the biofilm formation ability of the synthetic community 5 mL of LB medium was added to a 6-well plate, and then 500 μL of the synthetic community fermentation broth (OD 600 = 1.0) cultured overnight freshly was inoculated. It was placed in an incubator at 35 °C and statically cultured for 24 h and 48 h respectively. The 6-well plate was carefully taken out, and the medium and free cells under the biofilm in the 6-well plate were carefully aspirated with a sterile syringe. After gently washing once with sterile PBS, 5 mL of methanol solution was added to fix for 15 min; the methanol solution was carefully discarded, and it was left to stand at room temperature until the biofilm was dry. 5 mL of 1% crystal violet solution was added to stain for 15 min. The staining agent was carefully discarded, and the stain was washed with sterile PBS. It was inverted and dried in an incubator at 35 °C. 5 mL of 33% glacial acetic acid solution was added and incubated at 35 °C for 30 min to completely dissolve the biofilm, and the OD 590 was measured with a spectrophotometer. Each treatment was replicated 6 times, with the blank medium as the control.

[0090] As Figure 11 shown, after culturing at 35 °C for 24 h and 48 h, the synthetic community could form a biofilm. When the synthetic community fermentation broth was at 24 h and 48 h, the OD 590 values were both higher than 3.0, and the biofilm formation ability of the synthetic community cultured for 48 h was stronger than that of the fermentation broth cultured for 24 h, and the OD 590 value reached 3.42 ( Figure 11B). It shows that the fermentation broth of the synthetic bacterial community can form a biofilm and has a strong forming ability.

[0091] The synthetic bacterial community of the present invention has a strong biofilm forming ability. As the culture time increases, the biofilm forming ability also enhances. The formation of biofilm by biocontrol bacteria plays a positive role in biological control, probably because the biofilm can protect the biocontrol strains from external environmental pressures and stably exert their biocontrol effects. At the same time, the extracellular matrix can protect cells from being damaged by the external environment and also hinders the diffusion of small molecules within the biofilm. Therefore, inoculating the synthetic bacterial community onto the surface of potatoes to form a biofilm has great potential for protecting potato tubers from being infected by other pathogenic bacteria.

[0092] Although the content of the present invention has been introduced 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 to 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 safensis - Bacillus atrophaeus, characterized in that, The composite bacterium comprises: Bacillus safensis ( Bacillus safensis ), BM-7 and Bacillus atrophaeus ( Bacillus atrophaeus ), QB-2-7, wherein the Bacillus safensis BM-7 was deposited at the Guangdong Microbial Culture Collection Center on January 13, 2025, with the deposit number GDMCC NO: 65770, and the deposit address is the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou; the Bacillus atrophaeus QB-2-7 was deposited at the Guangdong Microbial Culture Collection Center on January 13, 2025, with the deposit number GDMCC NO: 65771, and the deposit address is the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

2. Use of the fermentation broth, bacterial suspension or supernatant of the Bacillus safensis - Bacillus atrophaeus complex bacteria according to claim 1 in controlling potato anthracnose.

3. The application according to claim 2, characterized in that, The volume ratio of the fermentation broths of Bacillus atrophaeus QB - 2 - 7 and Bacillus safensis BM - 7 is (1 - 9) : (9 - 1).

4. The application according to claim 3, characterized in that The absorbance OD of the fermentation broth of Bacillus atrophaeus QB-2-7 and Bacillus safensis BM-7 600 is the same.

5. The application according to claim 4, wherein The absorbance OD of the fermentation broth of Bacillus atrophaeus QB-2-7 and Bacillus safensis BM-7 600 is 1.0 for both.

6. The application according to any one of claims 2 to 5, characterized in that, The volume ratio of the fermentation broths of Bacillus atrophaeus QB - 2 - 7 and Bacillus safensis BM - 7 is 7 :

3.

7. The application according to claim 2, wherein The preparation of the fermentation broth of the Bacillus safensis - Bacillus atrophaeus complex bacteria comprises: (1) Preparation of the seed broth of each strain Single colonies of the active strains QB-2-7 and BM-7 were picked and inoculated into a liquid medium, and cultured with shaking at 35 °C until the OD 600 value of the bacterial solution reached 1.0; (2) Preparation of the fermentation broth of the complex bacteria Inoculate the seed solutions of QB-2-7 and BM-7 into a liquid medium and shake culture at 35 °C until the OD of the bacterial solution 600 reaches 1.0 to obtain the fermentation broth of the composite bacteria; The preparation of the bacterial suspension of the Bacillus safensis - Bacillus atrophaeus complex bacteria comprises: Centrifuge the fermentation broth of the Bacillus safensis - Bacillus atrophaeus complex bacteria, collect the bacterial cells, wash them with a buffer solution, and then add buffer solution to the original volume to obtain the bacterial suspension; The preparation of the supernatant of the Bacillus safensis - Bacillus atrophaeus complex bacteria comprises: Centrifuge the fermentation broth of the Bacillus safensis - Bacillus atrophaeus complex bacteria and collect the supernatant.

8. The application according to claim 7, wherein The rotation speed of the shaking culture is 180 r / min; Or / and, the liquid medium is selected as LB liquid medium.

9. The application according to claim 2, characterized in that, The pathogen of the potato anthracnose includes: Colletotrichum coccodes ( Colletotrichum coccodes ).

10. Use of the fermentation broth, bacterial suspension or supernatant of the composite bacterium of *Sphagnum*-*Bacillus atrophaeus* according to claim 1 in preventing and treating plant anthracnose caused by Colletotrichum coccodes ).

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