Compound microbial inoculum containing bacillus velezensis TA-5 strain and application of compound microbial inoculum

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound fungicide containing bacillus velezensis TA-5 strains and application of the compound fungicide. The microbial agent disclosed by the invention comprises the following components: (1) a viable bacterium, a freeze-dried bacterium, an inactivated bacterium body or a strain culture of a bacillus velezensis TA-5 strain; (2) bacillus subtilis and / or chitosan oligosaccharide, and / or pharmaceutically acceptable pharmaceutic adjuvants; wherein the preservation number of the bacillus velezensis TA-5 strain in the China General Microbiological Culture Collection Center is CGMCC (China General Microbiological Culture Collection Center) NO. 33034. The bactericidal composition has an excellent prevention and treatment effect on fruits and vegetables with diseases, and especially has an excellent effect on field tests; meanwhile, the dosage of chemical agents is reduced, safety and environmental protection are achieved, and a brand new thought is provided for agricultural control.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound bacterial agent containing Bacillus belye TA-5 strain and its application. Background Technology

[0002] Black rot fungus ( Valsa mali Apple tree rot, caused by [unspecified disease], has caused serious damage in all major apple-producing areas of my country and is a major limiting factor restricting the sustainable development of the apple industry. The rot mainly occurs on the trunk and main branches of the fruit tree. The surface of the diseased area is moist and slightly raised, appearing as a reddish-brown rot, later drying and collapsing, eventually causing localized ulceration. In severe cases, it can lead to dead branches, dead trees, and even the destruction of the orchard.

[0003] Staphylococcus aureus ( Botryosphaeria dothidea *Staphyllum spp.* is an important plant pathogenic fungus belonging to the genus *Staphyllum* in the phylum Ascomycota. It is a parasitic fungus with latent infection characteristics. It can widely infect various fruit trees and forest trees such as apples, pears, and grapes. In the early stages, it causes dark brown, spindle-shaped or irregular lesions on the trunks of fruit trees. The bark at the lesions rots and cracks, and in severe cases, it causes fruit rot. Later, it does not produce small black spots, seriously harming the health of fruit trees and causing irreparable economic losses.

[0004] Current chemical control methods have significant drawbacks, including severe environmental pollution, high residues in fruit, and increasing pathogen resistance. (Bacillus belyceae) Bacillus velezensis As a practical and effective biocontrol agent, *Bacillus vesiliflorus* has broad application prospects in the biological control of plant diseases. First isolated from the soil of the Belém River estuary in Spain in 1999, this bacterium possesses advantages such as a broad antibacterial spectrum, rapid growth, strong resistance, and high biocompatibility. Its mechanism of action mainly involves the secretion of various antibacterial metabolites, such as cell wall degrading enzymes (glucanase and protease), lipopeptide antibiotics (surfactants, iturobrine, and fentanyl), and polyketide antibiotics. These substances can hydrolyze the cell wall of pathogens, inhibit conidial germination and germ tube growth, alter the structure and permeability of the cell membrane, and ultimately lead to the death of pathogen cells. However, the colonization, reproduction, and effectiveness of single-agent biocontrol are heavily dependent on environmental conditions, especially temperature, humidity, and light.

[0005] Therefore, using a combination of multiple biocontrol agents, or combining biocontrol agents with other biological agents, to synergistically regulate plant diseases is an effective technical means to solve the drawbacks of single microbial agents, and also an important measure to reduce the amount of chemical pesticides and increase their efficiency, and promote the green development of the fruit industry. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the prior art by providing a compound microbial agent containing Bacillus belye TA-5 strain and its application. This compound microbial agent possesses unique advantages such as functional synergy, environmental adaptability, and application stability, exhibiting significant control effects against various important fruit and vegetable diseases; simultaneously, it reduces the amount of chemical agents used, providing a completely new approach to agricultural pest control.

[0007] To address the above problems, the present invention provides the following solutions.

[0008] In a first aspect, the present invention provides a compound bacterial agent containing Bacillus belyss TA-5 strain, wherein the active ingredient of the bacterial agent includes (1) Bacillus belyss TA-5 strain. Bacillus velezensis (2) Bacillus subtilis and / or chitosan oligosaccharides; The Bacillus vesiculosus TA-5 strain has the accession number CGMCC NO.33034 at the China General Microbiological Culture Collection Center.

[0009] In this invention, the Bacillus belye ( Bacillus velezensis The TA-5 strain was isolated from the surface of healthy apple branches in Qingdao, Shandong. Through comparative experiments, the inventors discovered that this *Bacillus belyceae* strain TA-5 inhibits the growth of mycelia of various pathogens, including apple rot fungus and ring rot fungus, leading to morphological deformities of the pathogenic mycelia and significantly reducing sporulation and spore germination rates. Furthermore, through compound studies, it was found that the microbial agent *Bacillus subtilis* wettable powder and / or the biological agent agricultural chitosan oligosaccharide can be used as a component of this *Bacillus belyceae* strain. Bacillus velezensis TA-5 is an synergist for inoculants, enhancing their biological control effects, especially showing significant advantages in the control of fruit tree diseases.

[0010] Optionally, in the compound bacterial agent, the Bacillus berberis ( Bacillus velezensis The TA-5 strain culture is the aforementioned Bacillus belesii ( Bacillus velezensis Fermentation broth, fermentation supernatant and / or the Bacillus belesiensis strain TA-5 Bacillus velezensis Fermentation broth, concentrated fermentation supernatant, dried product, or volatile product of TA-5 strain.

[0011] In a preferred embodiment, the Bacillus belye ( Bacillus velezensis TA-5 strain culture is Bacillus belesiensis ( Bacillus velezensis Fermentation broth of strain TA-5.

[0012] Optionally, the degree of polymerization of the chitosan oligosaccharide is between 2 and 20, and the molecular weight is ≤3200 Da.

[0013] Optionally, the compound microbial agent may be in the form of a liquid or solid preparation.

[0014] The solid formulation may be a conventional solid formulation used in pesticides in the art, preferably a powder, wettable powder or dry suspension.

[0015] The liquid formulation may be a conventional liquid formulation used for pesticides in the art, preferably a water dispersible agent or a bacterial suspension.

[0016] Optionally, when the active ingredient of the compound bacterial agent includes the Bacillus belyssus (… Bacillus velezensis When using TA-5 strain and the aforementioned Bacillus subtilis, the compound bacterial agent is 1×10⁻⁶. 3 ~1×10 8 CFU / mL of Bacillus belesi ( Bacillus velezensis TA-5 strain and 1×10 3 1×10 8 The Bacillus subtilis CFU / mL was prepared at a volume ratio of (0.4~3):1.

[0017] Preferably, when the active ingredient of the compound bacterial agent includes the Bacillus belyssus (… Bacillus velezensis When using TA-5 strain and the aforementioned Bacillus subtilis, the compound bacterial agent is 1×10⁻⁶. 5 ~1×10 6 CFU / mL of Bacillus belesi ( Bacillus velezensis TA-5 strain and 1×10 5 ~1×10 6 The Bacillus subtilis CFU / mL was prepared at a volume ratio of (0.5~2):1.

[0018] In a preferred embodiment, when the active ingredient of the compound bacterial agent includes the Bacillus belyssus (… Bacillus velezensis When using TA-5 strain and the aforementioned Bacillus subtilis, the compound bacterial agent is 1×10⁻⁶. 5 ~1×10 6 CFU / mL of Bacillus belesi ( Bacillus velezensis TA-5 strain and 1×10 6 The Bacillus subtilis CFU / mL is compounded at a volume ratio of (0.5~2):1, preferably at a volume ratio of 0.5:1, 1:1, or 2:1.

[0019] Preferably, the volume ratio of Bacillus subtilis to the compound bacterial agent is (0.2~1):1.

[0020] Optionally, when the active ingredient of the compound bacterial agent includes the Bacillus belyssus (… Bacillus velezensis When using TA-5 strain and the chitosan, the compound bacterial agent is 1×10⁻⁶. 3 ~1×10 8 The Bacillus belesiensis (CFU / mL) Bacillus velezensis The TA-5 strain was mixed with chitosan oligosaccharide at a concentration of (5~10) µg / mL at a volume ratio of (1~3):1.

[0021] Preferably, when the active ingredients of the compound microbial agent include the Bacillus vesiculus TA-5 strain and the chitosan, the compound microbial agent is 1×10⁻⁶. 5 ~1×10 6 The Bacillus belesiensis (CFU / mL) Bacillus velezensis The TA-5 strain was mixed with the chitosan oligosaccharide at a concentration of (8~10) µg / mL at a volume ratio of 1:1.

[0022] Preferably, the volume ratio of the chitosan oligosaccharide to the compound bacterial agent is (0.2~1):1.

[0023] Optionally, when the compound bacterial agent is a liquid preparation, the Bacillus belyssus (…) Bacillus velezensis The effective viable cell concentration of strain TA-5 is greater than 1×10⁻⁶. 3 CFU / mL, preferably an effective viable bacteria concentration of 1×10⁻⁶. 5 CFU / mL, for example 1×10 5 CFU / mL ~ 1×10 8 CFU / mL.

[0024] Optionally, in the compound bacterial agent, the Bacillus subtilis and the chitosan can enhance the performance of Bacillus belye (… Bacillus velezensis The control effect of TA-5 strain.

[0025] Preferably, in the compound bacterial agent, the Bacillus subtilis contains Bacillus subtilis that can enhance the antagonistic effect of the Bacillus belye TA-5 strain against Valsa mali (black rot fungus) and / or Staphylococcus aureus. Botryosphaeria dothidea Its function.

[0026] Preferably, in the compound bacterial agent, the chitosan can promote the growth of Bacillus belye (B. belye). Bacillus velezensis The TA-5 strain colonized the surface of the crop to be applied.

[0027] In a preferred embodiment, the active ingredient of the compound bacterial agent is derived from the Bacillus berberis (B. berberis). Bacillus velezensis The TA-5 strain consists of live, freeze-dried, inactivated cells or strain cultures, and Bacillus subtilis or chitosan oligosaccharides; preferably Bacillus belye ( Bacillus velezensis The TA-5 strain consists of live bacteria, freeze-dried bacteria, inactivated bacteria, or fermentation broth, along with Bacillus subtilis or chitosan oligosaccharides.

[0028] Optionally, the compound microbial agent further includes excipients. Preferably, the excipients are acceptable excipients for pesticide-based microbial agents, such as one or more of high-mesh diatomaceous earth, glucose, starch, amino acid powder, sodium dodecyl sulfonate, sodium dodecyl sulfate, and kaolin (e.g., 6000 mesh kaolin).

[0029] Secondly, the present invention provides an application of the compound microbial agent as described in the first aspect in the preparation of biological pesticides.

[0030] Optionally, the biopesticide is used for any one or more of the following purposes: (1) Used to antagonize black rot fungus Valsa mali ; (2) Used to prevent and control diseases caused by black rot fungi. Valsa mali This causes apple tree rot disease; (3) Used to antagonize Staphylococcus aureus Botryosphaeria dothidea ; (4) Used to prevent and treat Staphylococcus aureus Botryosphaeria dothidea The resulting ring-shaped marks.

[0031] Optionally, the ring rot is apple ring rot or pear ring rot.

[0032] Thirdly, the present invention provides a solution containing Bacillus belysinus ( Bacillus velezensis Live, lyophilized, inactivated, or chitosan oligosaccharide-based bacterial agents of strain TA-5 are used in the preparation of agents for antagonizing black rot crustaceans. Valsa mali and / or prevention of black rot caused by scabies Valsa mali Its use in pesticides that treat apple tree rot disease.

[0033] Fourthly, the present invention provides a solution containing Bacillus belye ( Bacillus velezensis A compound bacterial agent consisting of live, lyophilized, inactivated cells or cultures of TA-5 strain and Bacillus subtilis was prepared for use against Staphylococcus aureus. Botryosphaeria dothidea And / or, prevention and control of Staphylococcus aureus Botryosphaeria dothidea Uses of pesticides in treating ring spot disease.

[0034] In some preferred embodiments, the ring rot is apple ring rot or pear ring rot.

[0035] Fifthly, the present invention provides an antagonistic fungus against black rot. Valsa mali and / or Staphylococcus aureus Botryosphaeria dothidea The method, wherein a compound microbial agent as described in the first aspect is sprayed onto apple or pear branches or fruits (e.g., to control black rot caused by *Heteropoda spp.*). Valsa mali Apple tree rot caused by and / or caused by Staphylococcus aureus Botryosphaeria dothidea To prevent and control apple and / or pear ring rot caused by this disease.

[0036] Beneficial effects The compound fungal agent described in this embodiment of the invention is effective against black rot fungi. Valsa mali With Staphylococcus aureus Botryosphaeria dothidea It possesses excellent antagonistic activity, effectively controlling pathogenic fungal mycelial growth, reducing sporulation and spore germination rates, and effectively preventing and controlling diseases caused by *Heteropoda spp.* due to its high colonization ability on fruit trees. Valsa mali Caused by apple tree rot and grape seed rot Botryosphaeria dothidea The resulting ring rot (specifically apple ring rot and pear ring rot) plays a key role in the biological control of fruit tree diseases.

[0037] The compound microbial agent described in this invention has the unique advantages of being green and environmentally friendly, highly efficient in disease control, and safe and reliable. It can significantly reduce the total amount of chemical agents used, which is highly in line with the current needs and long-term direction of green and high-quality agricultural development. Attached Figure Description

[0038] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the implementation. The term “illustrative” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “illustrative” is not necessarily to be construed as superior to or better than other embodiments.

[0039] Figure 1 Colony morphology of Bacillus belyss TA-5 strain on NB medium; Figure 2 Phylogenetic tree of Bacillus belyss TA-5 strain; Figure 3 The effects of Bacillus vesicularis TA-5 on the mycelial growth of the pathogen causing apple tree canker are shown in the figures. Figure A shows a plate experiment illustrating the effect of Bacillus vesicularis TA-5 on the mycelial growth of the pathogen causing apple tree canker, and Figure B shows a bar chart illustrating the effect of Bacillus vesicularis TA-5 on the mycelial growth of the pathogen causing apple tree canker. This indicates a significant difference between the treatment group and the control group. p <0.01); Figure 4The image shows the effect of Bacillus belyceta TA-5 on the spore germination of the pathogen causing apple tree canker. Figure A shows a microscopic morphological image of the effect of Bacillus belyceta TA-5 on the spore germination of the pathogen causing apple tree canker, and Figure B shows a bar chart of the effect of Bacillus belyceta TA-5 on the spore germination of the pathogen causing apple tree canker. (See attached figure.) This indicates a significant difference between the treatment group and the control group. p <0.01); Figure 5 The colonization of Bacillus vesiculosus TA-5 strain on apple branches and buds; Figure 6 The control efficacy of the compound Bacillus vesicularis TA-5 and chitosan oligosaccharide against apple tree canker is shown in Figure A. Figure B is a graph illustrating the control efficacy of the compound Bacillus vesicularis TA-5 and chitosan oligosaccharide against apple tree canker, and Figure B is a bar chart showing the control efficacy of the compound Bacillus vesicularis TA-5 and chitosan oligosaccharide against apple tree canker. In the figures, different letters represent significant differences between different treatment groups. p <0.01); Figure 7 The control efficacy of the compound agent of Bacillus vesicularis TA-5 and Bacillus subtilis is shown in Figure A, which shows the control efficacy against apple ring rot, and Figure B shows the control efficacy against pear ring rot. In the figures, different letters represent different treatment groups, and the differences are significant. p <0.01); Figure 8 The effect of chitosan oligosaccharide on the colonization of Bacillus vesicle TA-5 strain on apple branch surface; Figure 9 The effects of the compound agent of Bacillus vesiculosus TA-5 and Bacillus subtilis on the expression of disease resistance-related genes in apple fruit are shown in the figures. A represents the effect on the expression of the MdPR1 gene, B represents the effect on the expression of the MdPR5 gene, and C represents the effect on the expression of the MdPR8 gene. In the figures, different letters represent significant differences between different treatment groups. p <0.01). Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0041] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0042] Unless otherwise specified, all reagents used in the experiments of this invention are commercially available.

[0043] The Bacillus subtilis used in this invention was purchased from Yantai Lvyun Biotechnology Co., Ltd., and is a 10 billion CFU / g Bacillus subtilis wettable powder.

[0044] The chitosan used in this invention was purchased from Weifang Dongxing Chitosan Products Factory and is 99% agricultural chitosan oligosaccharide.

[0045] Source of tested biocontrol strains: Bacillus belye ( Bacillus velezensis Strain TA5 was isolated from the surface of healthy apple branches in Qingdao, Shandong Province, and deposited on December 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33034. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0046] Source of the tested pathogen: Strain LXS080901, the pathogen of apple tree canker, was isolated from diseased branches of apple trees in our laboratory. Based on morphological observation, conserved gene sequence analysis, and verification using Koch's postulates, it was identified as *Heterocarpus spp.* Valsa mali ; The pathogenic fungus strain LXS030101, which causes apple and pear ring rot, was isolated from diseased branches of apple and pear trees in our laboratory. Based on morphological observation, conserved gene sequence analysis, and verification using Koch's postulates, it was identified as *Botrytis cinerea*. Botryosphaeria dothidea .

[0047] Example 1: Isolation, screening and identification of Bacillus belyss TA-5 strain (1) Isolation of strains The experiment was conducted in Qingdao, Shandong Province. Healthy branches and fruits of the "Fuji" apple variety were randomly selected, and 1 cm² samples of the branch and fruit bark tissue were prepared. The samples were homogenized in 0.1 mmol / L PBS buffer, serially diluted 1000-fold with sterile water, and 100 μL of the diluted solution was spread onto NA medium. After incubation at 28°C, single colonies were picked and streaked onto fresh NA medium plates for further incubation. The streaking was repeated 1-2 times until the colonies on the plates exhibited uniform morphology, thus obtaining a pure culture. Ten single bacterial strains were ultimately isolated in this experiment.

[0048] (2) Screening of strains The control efficacy of various bacterial strains against apple ring rot was determined using the puncture inoculation method. The pathogen of apple ring rot used in this experiment was *Botrytis cinerea* (*Botrytis cinerea*). Botryosphaeria dothidea This strain can be obtained by the inventors from diseased fruit using conventional methods in the field, or it can be a commercially available strain. The results are shown in Table 1, among which the branch-isolated biocontrol strain TA-5 showed the best control efficacy against apple ring rot.

[0049] Table 1. Efficacy determination of the isolated biocontrol bacteria

[0050] Note: "++" indicates a control efficacy of ≥80% against apple ring rot; "++" indicates 50% < control efficacy < 80%; "+" indicates 30% < control efficacy < 50%.

[0051] (3) Identification of strain TA-5 a. Morphological characteristics like Figure 1 As shown, after streaking on LB plates at 25°C for 24 hours, the bacteria showed good growth, forming opaque, milky-white colonies with irregular edges. The colony surface was rough, with a raised center. Microscopic examination revealed rod-shaped cells. The characteristics exhibited by this strain are consistent with the description of Bacillus in the "Handbook of Systematic Identification of Common Bacteria".

[0052] The LB medium consisted of: 10g tryptone, 5g yeast extract, and 5g NaCl, diluted to 1000mL with distilled water, and the pH was adjusted to 7.0.

[0053] b. Physiological and biochemical characteristics Physiological and biochemical tests revealed that strain TA-5 is a Gram-positive bacterium. It can utilize glucose, sucrose, lactose, mannose, sorbitol, arabinose, maltose, and cellobiose as carbon sources. This strain can liquefy gelatin, hydrolyze starch, and form a biofilm. It is positive for catalase and VP tests, but negative for methyl red, urease, and indole production tests (Table 2). The physiological and biochemical reactions exhibited by this strain are consistent with the description of Bacillus in the *Handbook of Systematic Identification of Common Bacteria*.

[0054] Table 2. Physiological and biochemical characteristics of biocontrol strain TA-5

[0055] Note: "+" indicates positive; "-" indicates negative.

[0056] c. Genetic identification A. Experimental Methods: Using DNA from strain TA-5 as a template, PCR amplification was performed using 16S rDNA primers. The amplified PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequences were analyzed for BLAST homology using NCBI, and multiple sequence comparisons were performed using MEGA11 software to construct a phylogenetic tree.

[0057] B. Results and Analysis: The obtained sequences were compared with nucleic acid sequences in GenBank using BLAST software in the NCBI database (http: / / www.ncbi.nlm.nih.gov). Strain TA-5 was found to be similar to *Bacillus belye*. Bacillus velezensis The homology of (accession number: OP435757.1) is up to 100%. Figure 2 ) Based on the above-mentioned morphological characteristics, physiological and biochemical properties, and sequence analysis results, strain TA-5 was identified as Bacillus belye (B. belye). Bacillus velezensis ).

[0058] Example 2: Study on the control effect of Bacillus belyssus TA-5 (1) Effects of Bacillus belyss TA-5 on the mycelial morphology of the pathogen causing apple tree canker Bacillus berberis Bacillus velezensisTo counteract the apple tree rot pathogen, streaks of TA-5 bacterial suspension were applied to both sides of a PDA plate. The plates were incubated at 25°C in the dark for 24 hours. Activated pathogens were then inoculated into the center of the plate. (This activation method is a standard procedure in the field: the strain was removed from a 4°C refrigerator slant, incubated on a PDA plate at 25°C for 3 days, and then mycelial cakes were collected using a 5mm diameter sterile punch and re-inoculated onto another PDA plate. After 3 days, the plates were ready for use.) A control was prepared by inoculating the pathogen onto a PDA plate without streaks of TA-5. After incubation at 25°C in the dark for 48 hours, the size of the pathogen colonies was recorded.

[0059] The solvent in the TA-5 bacterial culture was sterile water, and the concentration of the prepared mother liquor was 10. 9 CFU / mL.

[0060] The composition of PDA medium is as follows: 200g of peeled potatoes are weighed, cut into small pieces, boiled in water for 15-20 minutes, filtered through eight layers of gauze, and then 20g of glucose and 15g of agar powder are added. The volume is adjusted to 1000mL with distilled water, the pH value is natural, and the medium is sterilized by autoclaving at 121℃ for 20 minutes.

[0061] In this invention, the pathogenic fungus strain LXS080901, which causes apple tree rot, was preserved in the laboratory of Qingdao Agricultural University. Through morphological observation, conserved gene sequence analysis, and verification using Koch's postulates, it was identified as *Heterophyton rubrum*. Valsa mali .

[0062] See results Figure 3 And Table 3.

[0063] Table 3. Effects of Bacillus belyss TA-5 on mycelial growth of the pathogen causing apple tree canker.

[0064] Results and Analysis: In the confrontation culture with putrefactive bacteria ( Figure 3 The TA-5 strain colonies formed a distinct inhibition zone. The canker pathogen colonies located in the TA-5 inhibition zone were not connected to the TA-5 colonies. The colony diameter in the control group was 7.77 cm, while that in the TA-5 confrontation group was only 3.90 cm, indicating that Bacillus belye TA-5 strain has excellent inhibitory effect on the pathogen of apple tree canker.

[0065] (2) Effect of Bacillus belyss TA-5 strain on spore germination of apple tree rot pathogen Preparation of barley grain culture medium: Wash the barley grains with husks, soak them in distilled water for 1 hour, weigh 70g of soaked barley grains, place them in a 250mL Erlenmeyer flask, add 20mL of 12% honey and 20mL of 2% peptone solution, and autoclave at 121℃ for 1 hour.

[0066] Preparation of conidial suspension of *Pseudomonas aubergina*: 20 activated *Pseudomonas aubergina* mycelial cakes (5 mm) were inoculated into each bottle of barley grain culture medium and cultured in a 25℃ incubator in the dark for 15 days. The barley grains, now fully colonized with mycelium, were transferred to petri dishes (25-30 g per dish) and induced under a black light (365 nm) for 15 days. The barley grains were then kept moist on 1% water agar. Once orange conidial horns appeared, the horns were collected and placed in sterile water. The concentration was determined using a hemocytometer, and the conidial suspension was adjusted to a concentration of 1×10⁻⁶ using sterile water. 6 Use one spore per mL.

[0067] Preparation of fermentation broth for strain TA-5: 100 μL of glycerol bacteria was added to a 50 mL Erlenmeyer flask containing 10 mL of LB liquid medium. The flask was incubated at 25°C with shaking at 180 rpm for 12 h. The culture was then streaked onto a solid LB agar plate and incubated at 25°C in the dark for 48 h. A single colony was picked and added to 15 mL of LB liquid medium and incubated at 25°C with shaking at 180 rpm for 12 h. 1 mL of this culture was then added to 100 mL of LB medium and incubated at 25°C with shaking at 180 rpm for 48 h to obtain the fermentation broth for strain TA-5 (5 × 10⁻⁶). 9 Centrifuge the TA-5 fermentation broth at 5000 rpm for 10 min, collect the supernatant and filter it through a 0.22 μm bacterial filter to obtain the TA-5 fermentation filtrate for later use.

[0068] Experimental Methods: The inhibitory effect of the fermentation filtrate of strain TA-5 on the germination of apple tree rot fungus spores was determined by the slide method. The fermentation filtrate of TA-5 and the conidial suspension of rot fungus were thoroughly mixed at a volume ratio of 1:1. The control group was replaced with sterile water instead of fermentation filtrate. 20 μL of the mixture (TA-5 fermentation filtrate and conidial suspension of rot fungus) was dropped into the center of a glass slide, placed in a petri dish and kept moist. The slide was incubated in a 25℃ incubator in the dark for 12 h, and the spore germination was observed under a microscope.

[0069] See results Figure 4 And Table 4.

[0070] Table 4. Effects of Bacillus belyss TA-5 on spore germination of apple rot pathogen.

[0071] Results and analysis: Using the slide method, the germination rate of *Pseudomonas aeruginosa* spores in the control group was 87.55%, while that in the TA-5 fermentation filtrate treatment group was 55.81%. Compared with the control group, the TA-5 fermentation filtrate treatment significantly inhibited the germination of *Pseudomonas aeruginosa* conidia. Figure 4 (and Table 4).

[0072] (3) Determination of colonization of Bacillus vesiculosus TA-5 strain on apple branches and buds Preparation of TA-5 bacterial suspension: TA-5 fermentation broth (5 × 10⁻⁶) was obtained according to the above preparation method. 9 Centrifuge the TA-5 fermentation broth at 5000 rpm for 10 min, collect the precipitate, and resuspend it in sterile water to obtain the TA-5 bacterial suspension. Adjust the concentration of the bacterial suspension to 1×10⁻⁶ CFU / mL with sterile water. 7 CFU / mL available for use.

[0073] Experimental method: Healthy Fuji apple branches were randomly cut, disinfected by wiping with 75% ethanol, and then dried before being exposed to a 1×10⁻⁶ molasses solution. 7 Soak the branches in a CFU / mL TA-5 bacterial suspension for 0.5 h, air dry, and then incubate at 25°C in a humidifier. Periodically and randomly sample the branch bark and axillary bud tissue. Cut 1 cm sections of the branch bark... 2 Four axillary buds were excised, and each was ground thoroughly with 1 mL of PBS buffer. The mixture was then serially diluted 1000-fold with sterile water. 100 μL of each diluted solution was spread evenly on LB agar plates and incubated at 25°C for 48 h. The number of TA-5 colonies per LB plate was counted, and the number of cells per unit area was expressed as logarithmically. 10 CFU / cm 2 express.

[0074] The results are shown in Table 5 and Figure 5 .

[0075] Table 5. Colonization of Bacillus vesiculosus TA-5 strain on apple branches and buds.

[0076] Results and Analysis: In vitro, strain TA-5 could stably colonize both the surface and buds of apple branches at 25℃. The bacterial count increased rapidly from 0-2 days after inoculation, followed by a slow increase, peaking on the branch surface on day 5 and on the buds on day 10. Subsequently, the colony count decreased slowly, but on day 21, the colony counts on both the branch surface and buds were still significantly higher than the inoculation amount. Furthermore, the trends of increase and decrease in the colony count of strain TA-5 on apple branches and buds were basically consistent.

[0077] Example 3: Study on the control effect of Bacillus vesiculosus TA-5 and chitosan oligosaccharide compound on apple tree canker. Preparation of TA-5 inoculum: Using LB medium, ferment the TA-5 inoculum (5 × 10⁻⁶ cells / day) at a 1% inoculum rate. 9After centrifuging at 5500 rpm for 5 min with 8 L of (CFU / mL) solution, the bacterial cells were collected and resuspended in 400 mL of sterile water. The suspension was then transferred to a 1 L Erlenmeyer flask and incubated at 25 °C with shaking at 180 rpm for 1 h. The bacterial suspension was then poured into a clean beaker and diluted with sterile water to a concentration of 1 × 10⁻⁶. 6 Use CFU / mL.

[0078] Preparation of chitosan oligosaccharide preparation: Chitosan oligosaccharide was purchased from Weifang Dongxing Chitosan Products Factory and dissolved in sterile water. Use at 10µg / mL.

[0079] Preparation of compound bacterial agent: The above 1×10 6 The compound bacterial agent is prepared by mixing CFU / mL TA-5 bacterial agent and 10µg / mL chitosan oligosaccharide preparation at a volume ratio of 1:1.

[0080] Experimental Methods: Healthy one-year-old "Fuji" apple branches were cut and disinfected with 75% ethanol. 200 μL of the compound agent was dripped into the wound area using a punch. TA-5 strain and chitosan oligosaccharide were used as single-agent controls, and sterile water was used as a blank control. The branches were incubated in a 25℃ incubator in the dark for 48 h. Apple tree rot pathogen fungal cakes were inoculated at the wound site. After 120 h of moist incubation, the length of the lesions was measured and photographed. The efficacy of the compound agent against apple tree rot was calculated.

[0081] Calculation of control efficacy: (Length of lesions in control group - Length of lesions in experimental group) × 100% / Length of lesions in control group The results are shown in Table 6 and Figure 6 .

[0082] Table 6. Control efficacy of Bacillus vesiculosus TA-5 combined with chitosan oligosaccharide against apple tree canker.

[0083] Results and Analysis: Indoor efficacy test results showed that 1×10 6 When TA-5 at CFU / mL was combined with chitosan oligosaccharide at 10µg / mL, the control efficacy of TA-5 was 37.12%, that of chitosan oligosaccharide was 24.13%, and the control efficacy of the combined agent was 66.15%, which was significantly greater than the efficacy of either single agent, demonstrating a significant synergistic effect.

[0084] Example 4: Efficacy test of combined Bacillus vesicles TA-5 and Bacillus subtilis in controlling apple and pear ring rot. Preparation of Bacillus subtilis inoculum: 10 billion CFU / g Bacillus subtilis wettable powder was purchased from Yantai Lvyun Biotechnology Co., Ltd., and diluted with sterile water to a concentration of 1×10⁻⁶. 6 Use CFU / mL.

[0085] Preparation of TA-5 inoculant: Following the method described in Example 3, 1×10⁻⁶ inoculants were prepared. 6 CFU / mL TA-5 bacterial agent.

[0086] Preparation of compound bacterial agent: The above 1×10 6 CFU / mL TA-5 bacterial agent and 1×10 6 CFU / mL Bacillus subtilis inoculant, when mixed at a volume ratio of 1:1, becomes a compound inoculant.

[0087] Experimental Methods: Healthy Fuji apples and Crown pears were selected. After disinfection with 75% ethanol, wounds were made on the equatorial surface of the apple and pear fruits and 50 μL of the compound agent was inoculated. TA-5 and Bacillus subtilis were used as single-agent controls, and sterile water was used as the control group. The mixture was incubated at 25°C for 48 hours. Apple and pear ring rot pathogens were then inoculated with fungal cakes and incubated at 25°C. After 3 days, the size of the lesions was observed and counted, and the control effect of the compound agent on apple and pear ring rot was calculated.

[0088] Calculation of control efficacy: (Diameter of lesions in control group - Diameter of lesions in experimental group) × 100% / Diameter of lesions in control group The results are shown in Table 7 and Figure 7 .

[0089] Table 7. Control efficacy of Bacillus vesiculosus TA-5 combined with chitosan oligosaccharide against apple and pear ring rot.

[0090] Results and Analysis: Results of Indoor Efficacy Test for Apple Ring Spot Disease ( Figure 7 As shown in A and Table 7), 1×10 6 CFU / mL TA-5 and 1×10 6 When Bacillus subtilis at CFU / mL was combined, TA-5 showed a control efficacy of 39.2%, Bacillus subtilis showed a control efficacy of 46.2%, and the combined agent showed a control efficacy of 66.1%, which was significantly greater than the effects of either single agent. This indicates that the combination of TA-5 and Bacillus subtilis has a good synergistic effect in the control of apple ring rot.

[0091] Results of indoor pear scab control efficacy test ( Figure 7 B and Table 7 show that 1×10 6 CFU / mL TA-5 and 1×10 6 When Bacillus subtilis at CFU / mL was combined, TA-5 showed a control efficacy of 40.54%, Bacillus subtilis showed a control efficacy of 28.31%, and the combined agent showed a control efficacy of 82.05%, which was significantly greater than the efficacy of either single agent. This indicates that the combination of TA-5 and Bacillus subtilis has a good synergistic effect in the control of pear ring spot disease.

[0092] Example 5: Study on the control effect of compound fungal agents with different compound ratios on apple ring rot. Preparation of compound microbial agents: *Bacillus vesiculosus* TA-5 and *Bacillus subtilis* were compounded in different proportions, and their control effect on apple ring rot was determined. Following the method described in Example 3 above, 1×10⁻⁶ microbial agents were prepared. 6 TA-5 at CFU / mL; prepare 1×10 CFU / mL TA-5 according to the method described in Example 4 above. 6 Bacillus subtilis at CFU / mL can be compounded and used in volume ratios of 4:1, 2:1, 1:1, 1:2, and 1:4.

[0093] Experimental Methods: Following the method described in Example 4, healthy "Fuji" apple fruits were selected, disinfected with 75% ethanol, and wounds were made on the equatorial surface of the apple fruits. 50 μL of the compound agent was inoculated, with TA-5 and Bacillus subtilis as single-agent controls. The control group was replaced with sterile water. The mixture was incubated at 25°C for 48 hours. Apple ring rot fungus was then inoculated with fungal cakes and incubated at 25°C. After 3 days, the size of the lesions was observed and counted, and the control efficacy of different ratios of compound fungal agents against apple ring rot was calculated.

[0094] The synergistic coefficient (SR) was used for calculation, where SR = efficacy of the compound microbial agent / (efficacy of TA-5 alone + efficacy of Bacillus subtilis alone). If SR > 1, it indicates a synergistic effect; SR = 1 indicates an additive effect; and SR < 1 indicates an antagonistic effect.

[0095] The results are shown in Table 8.

[0096] Table 8. Control effects of different ratios of TA-5 and Bacillus subtilis on apple ring rot.

[0097] Note: If SR>1, it indicates a synergistic effect; SR=1 indicates an additive effect; SR<1 indicates an antagonistic effect.

[0098] Results and Analysis: Based on the above experimental results, 1×10 6 CFU / mL TA-5 and 1×10 6 CFU / mL Bacillus subtilis, when compounded in different ratios (2:1, 1:1, 1:2), showed a significant synergistic effect. Among them, when compounded in a volume ratio of 1:1, the synergistic coefficient SR was 1.25 and the control efficacy was as high as 83.24%, showing the best synergistic effect.

[0099] Example 6: Effect of chitosan oligosaccharides on colonization of Bacillus belye TA-5 Preparation of inoculum: Prepare 1×10 according to the method described in Example 3 above. 6TA-5 bacterial agent with CFU / mL and chitosan oligosaccharide preparation with 10µg / mL, as well as compound bacterial agent (1×10 6 (CFU / mL TA-5 and 10µg / mL chitosan oligosaccharide, mixed in a 1:1 volume ratio).

[0100] Experimental method: Fresh Fuji apple branches were cut, disinfected by wiping with 75% ethanol, and then soaked in TA-5 inoculant and compound inoculant for 0.5 hours after drying. After drying, they were placed in a humidification box and incubated at 25℃. 1 cm sections were randomly selected. 2 Branch epidermal tissue was sampled periodically, and each sample was homogenized with 1 mL of PBS buffer. The homogenate was then serially diluted 1000-fold with sterile water. 100 μL of each sample was spread onto LB medium and incubated at 25°C for 48 hours. Colony counts were performed, and population density was expressed as logarithmic values. 10 CFU / cm 2 This indicates that it is repeated three times.

[0101] The results are shown in Table 9 and Figure 8 .

[0102] Table 9. Effects of chitosan oligosaccharides on colonization of Bacillus belye TA-5

[0103] Results and Analysis: Based on the above experimental results, chitosan oligosaccharide significantly promoted the colonization of Bacillus belye TA-5 on the surface of apple branches. On day 3 post-inoculation, the number of bacteria on the branch surface in the compound inoculum treatment group (TA-5 + chitosan oligosaccharide) was significantly higher than that in the TA-5 single-agent control group. Subsequently, the number of colonies increased rapidly, with the TA-5 single-agent treatment group reaching its peak on day 7 post-inoculation, while the compound inoculum treatment group reached its peak on day 5 post-inoculation. The number of bacteria subsequently decreased, but the number of bacteria in the compound inoculum treatment group remained significantly higher than that in the TA-5 single-agent treatment group. These results indicate that chitosan oligosaccharide can effectively improve the colonization rate and number of Bacillus belye TA-5 on the surface of apple branches.

[0104] Example 7: Effects of combined Bacillus belyss TA-5 and Bacillus subtilis on the expression of disease resistance-related genes in apples. Preparation of inoculum: Prepare 1×10 according to the method described in Example 3 above. 6 Prepare 1×10 CFU / mL TA-5 bacterial agent according to the method described in Example 4. 6 CFU / mL Bacillus subtilis inoculum, and compound preparations (1×10 6 CFU / mL TA-5 bacterial agent and 1×10 6 (CFU / mL Bacillus subtilis inoculum, the two were mixed at a volume ratio of 1:1).

[0105] Experimental Methods: Following the method described in Example 4, healthy "Fuji" apple fruits were selected. After disinfection with 75% ethanol, wounds were made on the equatorial surface of the apple fruits, and 50 μL of the compound agent was inoculated. The same volume of TA-5 inoculant and Bacillus subtilis inoculant served as single-agent controls. Sterile water was used as the control group. The mixture was incubated at 25°C for 48 hours. Fruit peel tissue around the inoculation point was collected, and total RNA was extracted and reverse transcribed. RT-qPCR was used to determine the genes of disease-related proteins in the apple fruit tissue. MdPR1 , MdPR5 and MdPR8 The expression of ).

[0106] The results are shown in Table 10 and Figure 9 .

[0107] Table 10 Effects of compound microbial agents on gene expression of disease-related proteins in apple tissues

[0108] Results and Analysis: Based on the above experimental results, compared with Bacillus vesiculosus TA-5 and Bacillus subtilis single agents, the compound inoculant can significantly increase the expression of disease-related protein genes in apple fruit tissue. MdPR1 , MdPR5 and MdPR8 The level of expression. Table 10 and Figure 9 Data from China shows that single-agent treatment with TA-5 and Bacillus subtilis reduced the number of genes in apple fruit tissue. MdPR1 The expression levels were significantly higher than the control, increasing by 6.67 and 3.23 times, respectively. The combined inoculant treatment with these two agents resulted in higher expression levels in the fruit tissue. MdPR1 The expression level increased by 13.52 times; furthermore, compared with the two single-agent treatments, the compound microbial agent treatment increased the expression level in apple fruit tissues. MdPR5 and MdPR8 Gene expression levels were significantly increased.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A compound bacterial agent containing Bacillus belye TA-5 strain, wherein, The active ingredients of the compound bacterial agent include (1) Bacillus belyssus. Bacillus velezensis (2) Live, lyophilized, inactivated cells or cultures of TA-5 strain; (3) Bacillus subtilis and / or chitosan oligosaccharides; The Bacillus vesiculosus TA-5 strain has the accession number CGMCC NO.33034 at the China General Microbiological Culture Collection Center.

2. The compound microbial agent according to claim 1, characterized in that, In the compound bacterial agent, the bacterial strain culture is the Bacillus belesiensis. Bacillus velezensis Fermentation broth, fermentation supernatant, concentrate and / or dried product of TA-5 strain; And / or, the Bacillus subtilis is present in the form of a formulation; preferably a wettable powder; And / or, the chitosan oligosaccharide is an agricultural chitosan oligosaccharide with a degree of polymerization between 2 and 20 and a molecular weight ≤ 3200 Da.

3. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is a liquid or solid preparation. Optionally, the solid dosage form is a powder, a wettable powder, or a dry suspension. Optionally, the liquid formulation is a water dispersible agent or a bacterial suspension; Optionally, when the active ingredient of the compound bacterial agent includes the Bacillus belye... Bacillus velezensis When using TA-5 strain and the aforementioned Bacillus subtilis, the compound bacterial agent is 1×10⁻⁶. 3 ~1×10 8 CFU / mL of Bacillus beles Bacillus velezensis TA-5 strain and 1×10 3 1×10 8 The Bacillus subtilis CFU / mL was prepared at a volume ratio of (0.4~3):

1. Preferably, when the active ingredient of the compound bacterial agent includes the Bacillus belye, Bacillus velezensis When using TA-5 strain and the aforementioned Bacillus subtilis, the compound bacterial agent is 1×10⁻⁶. 5 ~1×10 6 CFU / mL of Bacillus beles Bacillus velezensis TA-5 strain and 1×10 5 ~1×10 6 The Bacillus subtilis CFU / mL was prepared at a volume ratio of (0.5~2):

1. Optionally, when the active ingredient of the compound bacterial agent includes the Bacillus belye... Bacillus velezensis When using TA-5 strain and the chitosan, the compound bacterial agent is 1×10⁻⁶. 3 ~1×10 8 The Bacillus belysinianum CFU / mL Bacillus velezensis TA-5 strain was mixed with chitosan oligosaccharide at a concentration of (5~10) µg / mL at a volume ratio of (1~3):1; Preferably, when the active ingredient of the compound bacterial agent includes the Bacillus belye, Bacillus velezensis When using TA-5 strain and the chitosan, the compound bacterial agent is 1×10⁻⁶. 5 ~1×10 6 The Bacillus belysinianum CFU / mL Bacillus velezensis TA-5 was mixed with chitosan oligosaccharide at a concentration of (8~10) µg / mL at a volume ratio of 1:

1.

4. The compound microbial agent according to claim 3, characterized in that, When the compound bacterial agent is a liquid preparation, the *Bacillus belyssae* is included. Bacillus velezensis The effective viable cell concentration of strain TA-5 is greater than 1×10⁻⁶. 3 CFU / mL, preferably an effective viable bacteria concentration of 1×10⁻⁶. 5 CFU / mL, for example 1×10 5 CFU / mL ~ 1×10 8 CFU / mL; Optionally, in the compound bacterial agent, the Bacillus subtilis and the chitosan can enhance the performance of Bacillus belye. Bacillus velezensis The control effect of TA-5 strain; Preferably, in the compound bacterial agent, the Bacillus subtilis has the ability to enhance the antagonistic effect of the Bacillus belyss TA-5 strain against black rot fungi. Valsa mali and / or Staphylococcus aureus Botryosphaeria dothidea Its function; Preferably, in the compound bacterial agent, the chitosan can promote the growth of Bacillus belye. Bacillus velezensis The TA-5 strain colonized the surface of the crop to be treated.

5. The compound microbial agent according to claim 1, characterized in that, The active ingredient of the compound bacterial agent is Bacillus belysinus. Bacillus velezensis The TA-5 strain consists of live, lyophilized, inactivated cells or strain cultures, and Bacillus subtilis or chitosan oligosaccharides; preferably Bacillus belesiensis. Bacillus velezensis The composition of live, lyophilized, inactivated, or fermentation broth of TA-5 strain with Bacillus subtilis or chitosan oligosaccharides.

6. The compound microbial agent according to any one of claims 1-5, characterized in that, The compound microbial agent also includes excipients, preferably, the excipients are acceptable excipients for pesticide-based microbial agents.

7. The use of a compound microbial agent as described in any one of claims 1-6 in the preparation of biological pesticides.

8. The application according to claim 7, characterized in that, The biopesticide is used for any one or more of the following purposes: (1) Used to antagonize black rot fungus Valsa mali ; (2) Used to prevent and control diseases caused by black rot fungi. Valsa mali This causes apple tree rot disease; (3) Used to antagonize Staphylococcus aureus Botryosphaeria dothidea ; (4) Used to prevent and treat Staphylococcus aureus Botryosphaeria dothidea The resulting ring rot; optionally, the ring rot is apple ring rot or pear ring rot.

9. A species containing Bacillus belesiensis Bacillus velezensis A compound bacterial agent consisting of live, lyophilized, inactivated bacterial cells or bacterial cultures of TA-5 strain and chitosan oligosaccharides was prepared for use against *Heterophyllum oxypetalum*. Valsa mali and / or prevention of black rot caused by scabies Valsa mali Its use in pesticides that treat apple tree rot disease.

10. A species containing Bacillus belesiensis Bacillus velezensis A compound bacterial agent consisting of live, lyophilized, inactivated cells or cultures of TA-5 strain and Bacillus subtilis was prepared for use against Staphylococcus aureus. Botryosphaeria dothidea And / or, prevention and control of Staphylococcus aureus Botryosphaeria dothidea Uses of pesticides that cause ring spot disease; Preferably, the ring rot is apple ring rot or pear ring rot.

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