Microbial biocontrol agent, preparation method and application thereof

Abstract

The present application provides a kind of microbial biocontrol agent and its preparation method and application, belong to the field of microorganism technology, the microbial biocontrol agent includes bacillus velezensis R_RT64 and bacillus licheniformis LX2501, not only can inhibit the growth of chain sporomycetes in ganoderma lucidum cultivation process, simultaneously can promote the increase of polysaccharide, triterpenoid, sterol, nucleoside and other beneficial active substances in ganoderma lucidum, greatly improve the quality of ganoderma lucidum, has broad application prospect in ganoderma lucidum cultivation field.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a microbial biocontrol agent, its preparation method, and its application. Background Technology

[0002] Ganoderma lucidum ( Ganoderma lucidum Known as the "immortal herb" or "resurrection herb," ​​Ganoderma lucidum is a precious traditional Chinese medicine. As a type of large, higher basidiomycete, it mainly grows in tropical, subtropical, and temperate regions and is widely distributed in China.

[0003] In recent years, with the strengthening of national ecological environmental protection and the development of Ganoderma lucidum products, the number of wild Ganoderma lucidum has been decreasing year by year, and it can no longer meet the current market demand. Therefore, the scale of artificial cultivation of Ganoderma lucidum is getting larger and larger. In the process of artificial cultivation of Ganoderma lucidum, due to the lack of attention to the control of germplasm resources, the quality and yield of Ganoderma lucidum have declined significantly. At the same time, Ganoderma lucidum has insufficient resistance to diseases and pests and is susceptible to pathogens, which seriously hinders the healthy development of the Ganoderma lucidum industry.

[0004] Neurospora ( Neurospora *Neurospora* is the main pathogen causing severe yield reduction in Ganoderma lucidum. In its early growth stages, it appears as a fluffy, white or gray fungus, with conidia that are oval or nearly spherical. It easily grows and reproduces in high temperature and humidity environments. *Neurospora* spores germinate and mycelial grow extremely quickly, spreading rapidly and with strong infectivity. Therefore, once *Neurospora* outbreaks during the growth of Ganoderma lucidum, not only will the quality be severely affected, but the yield will also be significantly reduced.

[0005] Reishi mushroom is a fungus with extremely high medicinal value. It contains active ingredients such as polysaccharides, triterpenoids, sterols, nucleosides, and trace elements. These active ingredients have various effects such as immune regulation and anti-oxidation. Among them, Ganoderma lucidum polysaccharides are natural polymers formed by monosaccharides linked by α or β glycosidic bonds to form the main chain and side chains. They are the main active ingredients of Ganoderma lucidum and have a variety of pharmacological effects such as anti-oxidation, immunomodulation, liver protection, hypoglycemia, anti-aging, and anti-cancer. Ganoderma lucidum triterpenes are a class of highly oxidized lanosterane derivatives with strong antioxidant effects, as well as various physiological functions such as anti-inflammatory, antiviral, immune regulation, blood pressure reduction, and blood lipid reduction. Ganoderma lucidum sterols contain four terpene rings in their structure and have effects such as enhancing immunity, anti-inflammation, anti-oxidation, anti-tumor, blood lipid regulation, and hypoglycemia reduction. Ganoderma lucidum nucleosides are a class of water-soluble components with broad physiological activities, including adenine, adenine nucleoside, ganoderic purine, and uracil nucleoside. They have strong inhibitory effects on platelet aggregation and sedative and anti-hypoxic effects.

[0006] The state has explicitly banned the use of highly toxic pesticides, and currently there is still no safe and effective method to control *Neurospora* fungus affecting Ganoderma lucidum. Developing a novel ecological preparation that can both effectively inhibit *Neurospora* fungus and beneficially promote the growth of Ganoderma lucidum will have significant application value in the field of Ganoderma lucidum cultivation. Summary of the Invention

[0007] This invention provides a microbial biocontrol agent, its preparation method, and its application.

[0008] The technical solution of this invention is as follows:

[0009] A microbial biocontrol agent, including Bacillus belye ( Bacillus velezensis R_RT64 and Bacillus licheniformis ( Bacillus licheniformis LX2501, where:

[0010] The Bacillus belyssus R_RT64 was deposited on April 10, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.34162.

[0011] The Bacillus licheniformis LX2501 was deposited on July 23, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251673.

[0012] Preferably, the microbial biocontrol agent comprises an amino acid polymerized zinc mixture.

[0013] More preferably, the volume percentage of the amino acid-polymerized zinc mixture in the microbial biocontrol agent is 8% to 12%.

[0014] More preferably, the preparation method of the amino acid-polymerized zinc mixture includes the following steps:

[0015] (1) Prepare a polyglutamic acid aqueous solution of 90~110g / L;

[0016] (2) Prepare a 0.45~0.55 mol / L zinc sulfate aqueous solution;

[0017] (3) Mix the polyglutamic acid aqueous solution and the zinc sulfate aqueous solution to obtain a mixed solution, wherein the molar ratio between polyglutamic acid and zinc ions is 1:(0.5~0.7).

[0018] (4) Adjust the pH of the mixture to 5.8~6.2 and react at 75~85℃ for 1.5~2.5h to obtain amino acid polymerized zinc mixture.

[0019] The method for preparing the microbial biocontrol agent involves mixing a bacterial suspension of Bacillus belye R_RT64 with a bacterial suspension of Bacillus licheniformis LX2501 to obtain the microbial biocontrol agent.

[0020] Preferably, the bacterial concentration in the microbial biocontrol agent is (1~5)×10⁻⁶. 9 cfu / mL.

[0021] Preferably, an amino acid polymeric zinc mixture is added to the microbial biocontrol agent.

[0022] The application of the microbial biocontrol agent in inhibiting Neurospora.

[0023] The application of the microbial biocontrol agent in Ganoderma lucidum cultivation.

[0024] Preferably, the microbial biocontrol agent is used to increase the active substances in Ganoderma lucidum, and the active substances include polysaccharides, triterpenoids, sterols, nucleosides, organic acids and their derivatives, phenylpropanoids and polyketides, and organic nitrogen-containing compounds.

[0025] Beneficial effects:

[0026] This invention provides a microbial biocontrol agent that can not only inhibit the growth of *Neurospora*, but also promote the increase of beneficial active substances such as polysaccharides, triterpenoids, sterols, and nucleosides in Ganoderma lucidum, greatly improving the quality of Ganoderma lucidum and having broad application prospects in the field of Ganoderma lucidum cultivation. Attached Figure Description

[0027] Figure 1 The inhibitory effect of microbial biocontrol agents on Neurospora;

[0028] Figure 2 A pie chart classifying the metabolomic compounds of Ganoderma lucidum in group A;

[0029] Figure 3 A pie chart classifying the metabolomic compounds of Ganoderma lucidum in group B;

[0030] Figure 4 A pie chart showing the classification of metabolomic compounds in Ganoderma lucidum from the CK group. Detailed Implementation

[0031] The following description is based on specific embodiments:

[0032] Explanation of the source of experimental materials:

[0033] Bacillus belye R_RT64: deposited on April 10, 2025 at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number CGMCC No. 34162;

[0034] Bacillus licheniformis LX2501 was deposited on July 23, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251673.

[0035] Neurospora: isolated from a contaminated fungal package and identified as Neurospora ( Neurospora crassa Its gene fragment information has been recorded in the NCBI database, accession number MH790416.1.

[0036] Example 1: Preparation of microbial biocontrol agents

[0037] Follow these steps:

[0038] I. Preparation of Bacillus belyssus R_RT64 bacterial culture

[0039] (1) Bacillus belye R_RT64 was spread on LB solid medium and activated at 32℃ for 48h to grow single colonies;

[0040] (2) Pick a single colony and inoculate it into LB liquid medium. Shake and culture at 180 rpm and 30 ℃ for 48 h to obtain seed culture;

[0041] (3) Inoculate the seed culture into a new LB liquid medium at a volume percentage of 1.5%, and culture at 180 rpm and 30°C for 48 h with shaking to obtain Bacillus belysinus R_RT64 bacterial culture.

[0042] II. Preparation of Bacillus licheniformis LX2501 bacterial culture

[0043] (1) Bacillus licheniformis LX2501 was spread on LB solid medium and activated at 50°C for 48 h to grow single colonies;

[0044] (2) Pick a single colony and inoculate it into LB liquid medium. Shake and culture at 200 rpm and 50 ℃ for 48 h to obtain seed culture;

[0045] (3) Inoculate the seed culture into a new LB liquid medium at a volume percentage of 1.5%, and culture at 200 rpm and 50°C for 48 h with shaking to obtain Bacillus licheniformis LX2501 bacterial culture.

[0046] III. Preparation of Microbial Biocontrol Agents

[0047] A microbial biocontrol agent was prepared by mixing *Bacillus belyeis* R_RT64 bacterial suspension and *Bacillus licheniformis* LX2501 bacterial suspension at a volume ratio of 1:1, with a bacterial concentration of 5 × 10⁻⁶.9 cfu / mL.

[0048] Example 2: Preparation of microbial biocontrol agents

[0049] Unlike Example 1, an amino acid polymerized zinc mixture was added to the prepared microbial biocontrol agent, with the volume percentage of the amino acid polymerized zinc mixture in the microbial biocontrol agent being 10%.

[0050] The preparation method of the amino acid-polymerized zinc mixture is as follows:

[0051] (1) Prepare a 100 g / L aqueous solution of polyglutamic acid (degree of polymerization 25);

[0052] (2) Prepare a 0.5 mol / L zinc sulfate aqueous solution;

[0053] (3) Mix the polyglutamic acid aqueous solution and the zinc sulfate aqueous solution to obtain a mixed solution, wherein the molar ratio between polyglutamic acid and zinc ions is 1:0.6;

[0054] (4) Adjust the pH of the mixture to 6.0 and react at 80°C for 2 hours to obtain an amino acid polymerized zinc mixture.

[0055] Example 3: Inhibitory effect of microbial biocontrol agents on Neurospora.

[0056] Follow these steps:

[0057] (1) Prepare Bacillus licheniformis LX2501 bacterial suspension and Bacillus belyssus R_RT64 bacterial suspension respectively according to the same method as in Example 1, with a bacterial concentration of 5×10⁻⁶. 9 cfu / mL.

[0058] (2) Bacillus licheniformis LX2501 bacterial suspension, Bacillus belysinus R_RT64 bacterial suspension, the microbial biocontrol agent prepared in Example 1, and the microbial biocontrol agent prepared in Example 2 were diluted 10 times with sterile water and then added to PDA solid culture medium at a volume ratio of 1% (added in a molten state at room temperature) to prepare PDA solid mixed drug culture medium; at the same time, sterile PDA solid culture medium was used as the control group CK, and three replicates were set for each group.

[0059] (3) Inoculate the activated Trichoderma into the above culture medium, and then place each group of culture medium in a constant temperature incubator and incubate in the dark at 25°C for 1 day. Take pictures to record the growth of Trichoderma and measure the hyphae length in each group of culture medium.

[0060] The results are as follows Figure 1As shown in the figure, Figure A shows the growth of *Neurospora* in each group of culture media, and Figure B shows the hyphal length of *Neurospora* in each group of culture media. Figure 1 It can be seen that Bacillus licheniformis LX2501 has no significant inhibitory effect on Neurospora, while Bacillus belyss R_RT64 has a significant inhibitory effect on Neurospora. Compared with single strains of Bacillus licheniformis LX2501 and Bacillus belyss R_RT64, the microbial biocontrol agents prepared in Examples 1 and 2 have a more significant inhibitory effect on Neurospora. The microbial biocontrol agent prepared in Example 2 can inhibit the growth of Neurospora by 100%, which is of great significance for curbing the outbreak of Neurospora in the cultivation of Ganoderma lucidum and improving the quality and yield of Ganoderma lucidum.

[0061] Example 4: Application of microbial biocontrol agents in Ganoderma lucidum cultivation

[0062] Follow these steps:

[0063] The microbial biocontrol agents prepared in Examples 1 and 2 were used for Ganoderma lucidum cultivation. Specifically, after inoculating the Ganoderma lucidum spawn bags with Ganoderma lucidum spawn, 10 mL of the microbial biocontrol agent diluted 10 times with sterile water was added to each spawn bag. The group treated with the agent from Example 1 was designated as Group A, and the group treated with the agent from Example 2 was designated as Group B. An equal amount of water was added as the control group (CK). Each group had three replicates.

[0064] The experiment lasted for 20 days. During the mycelial stage of Ganoderma lucidum, mycelial portions were harvested, placed in containers, and flash-frozen in liquid nitrogen. The samples were then sent to Shanghai Meiji Biomedical Technology Co., Ltd. for metabolomics analysis.

[0065] The pie charts showing the classification of metabolomic compounds in Ganoderma lucidum from groups A, B, and CK are as follows: Figure 2 , Figure 3 , Figure 4 As shown (the Arabic numerals in the figure represent the order of the compounds). From Figures 2-4 The results show that the proportion of active ingredients in Ganoderma lucidum was 76.47% in group A, 80.02% in group B, and 65.13% in group CK. These active ingredients refer to polysaccharides (organic oxygen compounds), triterpenoids (lipids and lipid-like molecules), sterols (lipids and lipid-like molecules), nucleosides (nucleosides, nucleotides, and their analogues), organic acids and their derivatives, phenylpropanoids and polyketides, and organic nitrogen-containing compounds. Specifically, compared to the control group (CK), the active ingredient content in group A was significantly increased by approximately 17.41%. p <0.05), the active ingredient content in Ganoderma lucidum in group B was significantly increased by approximately 22.86% ( p <0.05).

[0066] The above results indicate that the microbial biocontrol agent provided by this invention can significantly increase the beneficial active substances such as polysaccharides, triterpenoids, sterols, and nucleosides in Ganoderma lucidum, thereby improving the quality of Ganoderma lucidum.

Claims

1. A microbial biocontrol agent, characterized in that, Including Bacillus belysinus ( Bacillus velezensis R_RT64 and Bacillus licheniformis ( Bacillus licheniformis LX2501, where: The Bacillus belyssus R_RT64 was deposited at the China General Microbiological Culture Collection Center on April 10, 2025, with accession number CGMCC No. 34162. The Bacillus licheniformis LX2501 was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number CCTCC NO: M 20251673.

2. The microbial biocontrol agent as described in claim 1, characterized in that, The microbial biocontrol agent includes a mixture of amino acid-polymerized zinc.

3. The microbial biocontrol agent as described in claim 2, characterized in that, The volume percentage of the amino acid-polymerized zinc mixture in the microbial biocontrol agent is 8% to 12%.

4. The microbial biocontrol agent as described in claim 2, characterized in that, The preparation method of the amino acid polymerized zinc mixture includes the following steps: (1) Prepare a polyglutamic acid aqueous solution of 90~110g / L; (2) Prepare a 0.45~0.55 mol / L zinc sulfate aqueous solution; (3) Mix the polyglutamic acid aqueous solution and the zinc sulfate aqueous solution to obtain a mixed solution, wherein the molar ratio between polyglutamic acid and zinc ions is 1:(0.5~0.7). (4) Adjust the pH of the mixture to 5.8~6.2 and react at 75~85℃ for 1.5~2.5h to obtain amino acid polymerized zinc mixture.

5. The method for preparing the microbial biocontrol agent according to claim 1, characterized in that, The microbial biocontrol agent is prepared by mixing the bacterial suspension of Bacillus belye R_RT64 with the bacterial suspension of Bacillus licheniformis LX2501.

6. The preparation method according to claim 5, characterized in that, The bacterial concentration in the microbial biocontrol agent is (1~5)×10⁻⁶. 9 cfu / mL.

7. The preparation method according to claim 5, characterized in that, An amino acid polymerized zinc mixture was added to the microbial biocontrol agent.

8. The microbial biocontrol agent according to claim 1 inhibits the growth of *Neurospora* (…). Neurospora crassa Applications in ).

9. The application of the microbial biocontrol agent according to claim 1 in Ganoderma lucidum cultivation.

10. The application as described in claim 9, characterized in that, The microbial biocontrol agent is used to increase the active substances in Ganoderma lucidum.

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