Soil-borne disease biocontrol inoculant and preparation method and application thereof

Abstract

The invention belongs to the technical field of biocontrol microbial agents, and particularly relates to a soil-borne disease biocontrol microbial agent as well as a preparation method and application thereof. The soil-borne disease biocontrol microbial inoculum is prepared by compounding a bacillus velezensis fermentation broth and a brevibacillus beautiful fermentation broth according to a volume ratio of 1: (0.5-1.5). Wherein the fermentation liquor contains thalli. The bacillus velezensis is a YCH-H7 strain, the preservation number of the bacillus velezensis is CGMCC (China General Microbiological Culture Collection Center) No.33510, and the preservation date of the bacillus velezensis is February 11, 2025. The brevibacillus beautiful is an NF2 strain. The corrected death rate of second-instar larvae treated by the soil-borne disease biocontrol microbial inoculum for 48 hours is 98.85%, the nematode egg incubation inhibition rate is 88%, and the corrected death rate of ditylenchus destructor treated for 4 days is 97.7%. The hypha growth inhibition rates of the soil-borne disease biocontrol microbial inoculum on kiwi fruit root rot pathogenic bacteria, pepper phytophthora blight pathogenic bacteria, tomato neck and root rot, cucumber root rot pathogenic bacteria and wheat stem rot pathogenic bacteria exceed 90%, and the soil-borne disease biocontrol microbial inoculum has obvious broad spectrum.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biocontrol agents, and particularly relates to a soil-borne disease biocontrol agent and a preparation method and application thereof. BACKGROUND

[0002] Soil-borne diseases are plant diseases caused by pathogenic organisms in the soil, which often spread rapidly throughout the planting area once they occur. Soil-borne diseases have the following characteristics: (1) complex pathogens: a variety of pathogenic bacteria, including erwinia, monosporic bacteria, etc.; pathogenic fungi, including fusarium, pythium, etc.; plant parasitic nematodes, including root knot nematodes, rot stem nematodes, cyst nematodes, etc. (2) concealment: pathogenic organisms usually invade from the root or stem base of the plant, and early symptoms are not easy to be found. When obvious symptoms appear on the aboveground part, the disease is usually in a serious stage. (3) pathogen accumulation: pathogenic organisms can survive in the soil for a long time, even without host plants, and can survive for many years in the form of dormant spores, cysts, egg capsules or other forms, making it particularly difficult to prevent and control soil-borne diseases as the pathogenic organisms continue to accumulate. (4) fast transmission and wide range: pathogenic organisms are transmitted over long distances through water flow, soil, farm tools, seedlings, etc., thereby expanding the damage area. Therefore, soil-borne diseases have become an important threat to the production of melons, fruits, vegetables, food crops and other crops.

[0003] Chemical control of soil-borne diseases is currently the main control method, but the problems of drug resistance and environmental pollution are prominent. Microbial biocontrol agents can effectively overcome the problems of drug resistance and environmental pollution of chemical control, but the pathogens of soil-borne diseases are relatively complex, for example, root knot nematodes and fungal diseases often occur in combination, and current biocontrol agents mainly target single pathogenic bacteria species for control, such as B. subtilis with a control efficiency of only about 60% on nematodes, and Trichoderma with a control efficiency of only about 50% on fungi, and the control effect is very limited. SUMMARY

[0004] To solve the above technical problems, the application provides a soil-borne disease biocontrol agent and its application in preventing and controlling soil-borne diseases.

[0005] The first object of the application is to provide a soil-borne disease biocontrol agent, which is prepared by compounding Bacillus velezensis and Brevibacillus brevis according to a volume ratio of 1:0.5-1.5.

[0006] The Bacillus velezensis is YCH-H7 strain, with a preservation number of CGMCC No. 33510 and a preservation date of February 11, 2025. Bacillus velezensis The Brevibacillus brevis is NF2 strain.

[0007] The Brevibacillus brevis is NF2 strain.

[0008] The viable count of Bacillus vesiculosus in the culture was greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL.

[0009] The viable count of Bacillus brevis in the bacterial culture was greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL.

[0010] Preferably, the viable cell count in both the *Bacillus bellsii* culture and the *Bacillus brevis* culture is 1.0 × 10⁻⁶. 8 CFU / mL ~1.0×10 9 CFU / mL.

[0011] Preferably, the volume ratio of Bacillus vesiculosus suspension to Bacillus brevis suspension is 1:1.

[0012] The second objective of this invention is to provide a method for preparing a soil-borne disease biocontrol agent, comprising the following steps: Bacillus belye was activated and cultured on a large scale to obtain a Bacillus belye fermentation broth. The viable cell concentration of the Bacillus belye fermentation broth was adjusted to be greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL was used to obtain Bacillus vesiculosus culture.

[0013] Bacillus belye was activated and cultured on a large scale to obtain a Bacillus belye fermentation broth. The viable cell concentration of the Bacillus belye fermentation broth was adjusted to be greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL was used to obtain Bacillus vesiculosus culture.

[0014] Mix Bacillus vesiculosus and Bacillus brevis in a volume ratio of 1:0.5~1.5 to obtain a soil-borne disease biocontrol agent.

[0015] Preferably, the beef extract peptone liquid culture medium contains 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.2.

[0016] Preferably, the fermentation medium has the following formulation: 20 g / L sucrose, 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 0.3 g / L MnSO4, 0.5 g / L CaCl2, 5 g / L yeast extract, and pH 7.2.

[0017] Preferably, the activation culture temperature is 36℃~37℃, the rotation speed is 150r / min~250r / min, and the time is 16h~20h.

[0018] Preferably, the culture temperature is 36℃~37℃, the rotation speed is 150r / min~250r / min, and the time is 46h~50h.

[0019] A third object of the present application is to provide an application of the biocontrol agent for soil-borne diseases in preventing and controlling soil-borne diseases.

[0020] Preferably, the soil-borne diseases include wheat basal stem rot, kiwifruit root rot, pepper bacterial wilt, tomato collar and root rot, cucumber root rot and root-knot nematode disease.

[0021] Preferably, the pathogenic bacteria of the soil-borne diseases include kiwifruit root rot pathogen Fusarium solani , pepper bacterial wilt pathogen Phytophthora capsici , tomato collar and root rot pathogen Fusarium oxysporum f. sp. radicis- lycopersici , cucumber root rot pathogen Phytophthora nicotianae , and wheat basal stem rot pathogen Fusarium pseudograminearum .

[0022] Biological information preservation instructions The strains involved in the present application are as follows: The bacillus velezensis is YCH-H7 strain, which was preserved in China General Microbiological Culture Collection Center on February 11, 2025, and the recommended classification name is Bacillus velezensis , the preservation number is CGMCC No. 33510, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing.

[0023] Compared with the prior art, the present application has the following beneficial effects: 1. The biocontrol agent for soil-borne diseases of the present application is prepared by compounding the fermentation liquor of bacillus velezensis and the fermentation liquor of b. formosanus at a volume ratio of 1:0.5-1.5. The fermentation liquor contains bacterial cells. The bacillus velezensis is YCH-H7 strain, the preservation number is CGMCC No. 33510, and the preservation date is February 11, 2025. The b. formosanus is NF2 strain. The bacillus velezensis fermentation liquor has good killing effect on root-knot nematode and stem rot nematode, the b. formosanus fermentation liquor has obvious inhibition effect on root-knot nematode eggs, and the combination of the bacillus velezensis fermentation liquor and the b. formosanus fermentation liquor has good killing effect on nematodes and good incubation inhibition effect on nematode eggs. The corrected mortality rate of second instar larvae of southern root-knot nematode is 98.85% after 48h treatment, the nematode egg hatching inhibition rate is 88%, and the corrected mortality rate of stem rot nematode is 97.7% after 4d treatment, which realizes the dual synergy of “adult killing” and “egg inhibition”, and the prevention and control efficiency is significantly improved compared with the prior art. On the other hand, the mycelial growth inhibition rates of the biocontrol agent for soil-borne diseases of the present application on kiwifruit root rot pathogen, pepper bacterial wilt pathogen, tomato collar and root rot pathogen, cucumber root rot pathogen and wheat basal stem rot pathogen all exceed 90%, and the broad-spectrum effect is obvious.

[0024] 2. The preparation method of the soil-borne disease biocontrol agent of the present application comprises the following steps: picking single colonies of B. bell push and B. formosseum respectively, inoculating them into beef extract protein peptone liquid medium for culture, and obtaining B. bell push seed liquid and B. formosseum seed liquid respectively. The B. bell push seed liquid and the B. formosseum seed liquid are respectively inoculated into fermentation medium for fermentation at a volume fraction of 2% to 5% of inoculation amount, and B. bell push fermentation liquid and B. formosseum fermentation liquid are obtained respectively. The B. bell push fermentation liquid and the B. formosseum fermentation liquid are diluted with fermentation medium, and B. bell push bacterial liquid and B. formosseum bacterial liquid are obtained. The B. bell push bacterial liquid and the B. formosseum bacterial liquid are mixed to obtain the soil-borne disease biocontrol agent. The preparation method of the soil-borne disease biocontrol agent of the present application is simple, and the B. bell push fermentation liquid and the B. formosseum fermentation liquid meet the agricultural microbial agent execution standard GB20287-2006. Therefore, the soil-borne disease biocontrol agent of the present application has a very broad application prospect in the prevention and control of soil-borne diseases. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a colony morphology diagram of YCH-H7 of the present application.

[0026] Figure 2 It is a gram staining diagram of YCH-H7 of the present application.

[0027] Figure 3 It is a phylogenetic tree diagram constructed on the basis of 16S rDNA sequence of YCH-H7 bacteria of the present application.

[0028] Figure 4 It is a NF2 bacterial body and spore diagram of the present application.

[0029] Figure 5 It is a NF2 flagellum staining diagram of the present application.

[0030] Figure 6 It is a phylogenetic tree constructed on the basis of 16S rDNA sequence of NF2 bacteria of the present application. DETAILED DESCRIPTION

[0031] In order for those skilled in the art to better understand the technical solutions of the present application and to implement them, the technical solutions in the embodiments of the present application are described clearly and completely in combination with the preferred embodiments and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0032] It should be noted that all the professional terms used in the present application are only for the purpose of describing the specific embodiments and are not intended to limit the protection scope of the present application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present application can be purchased from the market or prepared by the existing method.

[0033] The materials used in the present application mainly include beef extract, peptone, agar and yeast extract powder.

[0034] Among them, the beef extract is purchased from Beijing Aoboxing Biotechnology Co., Ltd., with the item number 01-009. The peptone is purchased from Beijing Aoboxing Biotechnology Co., Ltd., with the item number 01-001. The yeast extract powder is purchased from Beijing Aoboxing Biotechnology Co., Ltd., with the item number 01-012.

[0035] The NF2 strain used in the present application is classified and named as Brevibacillus formosus, The preservation number is CGMCC No. 12749, which is disclosed in the research of Fu Bo, Li Yanqi, Li Zhongling, et al. A strain of Brevibacillus chagmatus NF2 antagonistic to kiwi leaf blight He-Ne laser mutagenesis breeding [J]. Journal of Northwest University (Natural Science Edition), 2014, 44(01): 66-70. DOI:10.16152 / j.cnki.xdxbzr.2014.01.024. The patent applicant promises to issue the NF2 strain material to the public within twenty years from the application date.

[0036] Example 1 1. Strain classification identification The Brevibacillus haynesii YCH-H7 strain of the present application is isolated from the petals of rape flowers in Dali County, Weinan City, Shaanxi Province. The Brevibacillus chagmatus NF2 strain is isolated from the rhizosphere soil of kiwi in Zhouzhi County, Shaanxi Province. In order to clarify the category of the bacteria used in the present application, the YCH-H7 bacteria and the NF2 bacteria used are subjected to cell morphology observation, physiological and biochemical tests and 16S rDNA molecular detection.

[0037] 2. Strain classification identification results The cell morphology of YCH-H7 bacteria is shown in Figure 1 and Figure 2 . Figure 1 It is the colony streaking diagram of YCH-H7 on beef extract peptone solid plate, and the single colony is milky white, smooth and round. Figure 2 It is a gram staining diagram, and the cell morphology is long rod-shaped, and the gram staining is positive, which belongs to gram-positive bacteria.

[0038] The phylogenetic tree constructed based on the 16S rDNA sequence of YCH-H7 bacteria is shown in Figure 3The YCH-H7 strain was identified as Bacillus velezensis by 16S rDNA molecular detection Bacillus velezensis , and the patent deposit number is CGMCC No. 33510.

[0039] The physiological and biochemical characteristics of YCH-H7 strain are shown in Table 1. By analyzing the metabolic characteristics, carbon source utilization ability and enzyme activity of YCH-H7 strain through 9 core biochemical indicators, the results showed that the V-P reaction was negative, indicating that YCH-H7 strain lacked the key enzyme for converting pyruvate to acetyl methyl methyl alcohol, so the glucose concentration in the culture medium needed to be controlled during fermentation culture to avoid excessive accumulation of organic acids, which would cause a sudden drop in pH, affecting the growth of the strain and the secretion of active substances. The negative of mannitol and the positive of D-mannitol indicated that YCH-H7 strain could not utilize mixed mannitol, but could utilize D-mannitol, indicating that it had strict selectivity for the stereochemical configuration of carbon sources. The negative of rhamnose indicated that YCH-H7 strain lacked the key enzyme system for decomposing rhamnose, such as rhamnose isomerase and rhamnose kinase, which suggested that YCH-H7 strain had weak ability to decompose plant residues containing rhamnose components in soil. The positive of D-xylose and L-arabinose indicated that YCH-H7 strain had a complete five-carbon sugar metabolic enzyme system and could utilize D-xylose and L-arabinose produced by plant residue degradation to colonize in soil without the need for additional complex carbon sources. In fermentation production, agricultural waste such as corn cob, wheat bran and raw materials rich in hemicellulose can be used to prepare cheap carbon sources, reducing production costs. Its carbon source acquisition relies more on free simple sugars such as D-xylose and L-arabinose in soil. The positive of gelatin liquefaction indicated that YCH-H7 strain could synthesize and secrete a large amount of gelatinase, which could break the peptide bonds in gelatin, making solid gelatin into liquid. From a functional point of view, gelatinase is essentially a broad-spectrum protease that not only can degrade gelatin, but also can decompose proteinaceous organic matter in soil such as proteins in plant residues and protein components in pathogen cell walls, providing nitrogen source for strain growth. More importantly, the body wall of decaying stem nematodes contains 20%~30% protein. In the early stage of soil-borne disease biocontrol agents, protease can destroy the protein structure of nematode body wall, assist chitinase in enhancing insecticidal effect, and also can inhibit the growth of pathogenic fungi, such as glycoproteins in fungal cell walls, to enhance broad-spectrum disease prevention ability. The positive of contactase indicated that the strain had high contactase activity, which had physiological significance in two aspects: one was to enhance environmental adaptability. In aerobic fermentation or aerobic soil environment, the strain metabolism would produce a large amount of hydrogen peroxide, which could be quickly decomposed by contactase to protect cells from oxidative stress and ensure normal growth of the strain in high oxygen environment. The second was to improve the stability of biocontrol. In field application, contactase could reduce the damage of hydrogen peroxide induced by environmental factors such as ultraviolet light and high temperature to active substances such as antibacterial peptides, prolonging the effective period of the agent. The positive of starch hydrolysis indicated that the Bacillus strain had the ability to degrade starch, and amylase could act on the α-1, 4 glycosidic bond of starch to gradually decompose large molecular weight starch into small molecular weight sugars that could be utilized, providing energy for the strain. This property is particularly important in fermentation process.Starch is an inexpensive and readily available carbon source, such as corn starch and potato starch. The strains can directly utilize starch as a fermentation carbon source without first converting it to glucose, simplifying the culture medium preparation process. Simultaneously, in the soil, amylase helps the strains decompose starchy substances in plant residues, further enhancing their colonization ability in the soil and ensuring their continued biocontrol effect.

[0040] In summary, the biochemical reaction results of YCH-H7 bacteria clearly reflect its carbon metabolism, enzyme activity, and environmental adaptability characteristics. This not only provides a basis for the identification of YCH-H7 bacteria and the optimization of fermentation processes, but also further verifies its functional potential in the field of biocontrol, such as a biocontrol agent for soil-borne diseases, laying the foundation for the subsequent application and performance improvement of YCH-H7 bacteria.

[0041] Table 1 Physiological and biochemical characteristics of YCH-H7 bacteria In the table, "-" indicates negative and "+" indicates positive.

[0042] The cell morphology of NF2 bacteria is as follows: Figure 4 and Figure 5 As shown. Figure 4 This is a Gram staining image. The bacterial cells are rod-shaped and Gram-positive, indicating they are Gram-positive bacteria that can produce spores in the later stages of growth. Figure 5 This is a staining image of flagella. The bacterial cell has multiple long flagella attached around it, indicating strong motility.

[0043] A phylogenetic tree constructed based on the 16S rDNA sequence of NF2 bacteria is as follows: Figure 6 As shown in Table 2, the physiological and biochemical characteristics of NF2 bacteria are as follows. NF2 bacteria is *Bacillus brevis*. Brevibacillus formosus The patent accession number is CGMCC No. 12749.

[0044] The physiological and biochemical characteristics of NF2 bacteria are shown in Table 2. The negative oxidase test result indicates that NF2 bacteria lack typical cytochrome oxidases, and its terminal oxidases in the aerobic respiratory chain may be other types, such as catalase and peroxidase, rather than relying on oxidases for electron transport. The positive catalase test result indicates that NF2 bacteria obtain oxygen by decomposing hydrogen peroxide through catalases, compensating for the aerobic metabolic demand caused by the lack of oxidases, while avoiding oxidative stress damage.

[0045] The negative MR test result confirms that NF2 bacteria do not produce large amounts of organic acids or acetylmethyl methanol during glucose metabolism. The negative glucose fermentation result indicates that NF2 bacteria cannot obtain energy through glucose fermentation and has a weak ability to utilize glucose.

[0046] The nitrate reduction test results were negative, indicating that NF2 bacteria lack nitrate reductase system and cannot use nitrate as an electron acceptor for anaerobic respiration, so sufficient oxygen supply is required during fermentation culture, and loose and well-ventilated plough layer soil is more suitable for soil application.

[0047] The results of glucose fermentation, mannitol fermentation and xylitol fermentation were all negative, indicating that the strain not only cannot ferment glucose, consistent with the MR test results, but also cannot utilize mannitol and xylitol for fermentation metabolism. From the structure of carbon source, glucose, mannitol and xylitol contain 6 or 5 carbon atoms, but the position or configuration of hydroxyl group is different, and NF2 bacteria cannot utilize these carbon sources.

[0048] The citrate utilization test results were negative, indicating that NF2 bacteria lack citrate transport enzyme or isocitrate lyase, and cannot convert citrate into a usable carbon source, indicating that the carbon metabolism of NF2 bacteria does not depend on the intermediate products of the tricarboxylic acid cycle, so there is no need to add citrate carbon source in the fermentation medium.

[0049] The starch hydrolysis test results were negative, indicating that NF2 bacteria lack amylase α-amylase and β-amylase, and cannot degrade starch. The gelatin hydrolysis test results were positive, confirming that NF2 bacteria can secrete gelatinase and can degrade protein substances, providing evidence for its utilization of plant residue protein to obtain nitrogen source in soil and destruction of nematode body wall protein in biocontrol. This property has high stability and is not affected by the detection conditions.

[0050] The tyrosine hydrolysis test results were negative, indicating that NF2 bacteria lack tyrosinase and cannot degrade aromatic amino acids. This property suggests that the strain has weak ability to decompose complex organic nitrogen, such as protein containing aromatic amino acids, in soil, and its nitrogen source acquisition is more dependent on simple amino acids such as glutamic acid and alanine or ammonium salt, so protein peptone can be added as a nitrogen source in fermentation medium to improve the growth efficiency of the strain.

[0051] The temperature growth test results showed that NF2 bacteria can grow at low temperature of 15℃, but cannot survive at high temperature of 50℃, belonging to a low-temperature adaptive strain. Field application is suitable for low-temperature environment in spring and autumn in temperate or subtropical regions, but attention should be paid to shading or selection of early and late application to avoid high temperature inhibition of its activity in summer when the temperature exceeds 35℃.

[0052] The result of the 0.001% mass fraction lysozyme growth test is positive. Lysozyme can destroy the peptidoglycan structure of the bacterial cell wall, leading to cell lysis. The strain can grow in the culture medium containing 0.001% mass fraction of lysozyme, indicating that the cell wall structure has strong resistance to lysozyme. This property is of great significance in biocontrol application. There is a small amount of lysozyme secreted by plants or other microorganisms naturally existing in the soil, and the anti-lysozyme ability of the strain can avoid its lysis by lysozyme in the soil, and improve the colonization stability.

[0053] H2S is toxic and can inhibit plant growth and other beneficial microbial activities. The result of the H2S production test is negative. The mass fraction does not produce H2S, indicating that it will not release toxic gases when colonizing in soil, and will not have a negative impact on crop roots or soil microecology, and has high safety, meeting the application requirements of "green and environmentally friendly" biocontrol agents.

[0054] Table 2 physiological and biochemical characteristics of NF2 strain "-" in the table represents negative, "+" represents positive.

[0055] Example 2 A preparation method of a soil-borne disease biocontrol agent, comprising the following steps: The single colonies of YCH-H7 strain and NF2 strain were respectively inoculated in beef extract protein peptone liquid medium at a temperature of 36℃ and a rotation speed of 200r / min for 18h to obtain YCH-H7 seed liquid and NF2 seed liquid. In the beef extract protein peptone liquid medium, beef extract was 3g / L, protein peptone was 10g / L, NaCl was 5g / L, and pH was 7.2. The YCH-H7 seed liquid and the NF2 seed liquid were respectively inoculated into the fermentation medium at a volume fraction of 2% at a temperature of 36℃ and a rotation speed of 200r / min for 48h to obtain YCH-H7 fermentation liquid and NF2 fermentation liquid. In the fermentation medium, the concentration of sucrose was 20g / L, beef extract was 3g / L, protein peptone was 10g / L, NaCl was 5g / L, MnSO4 was 0.3g / L, CaCl2 was 0.5g / L, yeast extract powder was 5g / L, water was supplemented to 1L, and pH was 7.2. The YCH-H7 fermentation liquid and the NF2 fermentation liquid were diluted with the fermentation medium to obtain YCH-H7 bacterial liquid and NF2 bacterial liquid. After sterilization, the YCH-H7 bacterial liquid and the NF2 bacterial liquid were mixed in a volume ratio of 1:1 to obtain a soil-borne disease biocontrol agent.

[0056] Example 3 A preparation method of a soil-borne disease biocontrol agent, comprising the following steps: Single colonies of YCH-H7 and NF2 were picked and inoculated into beef extract peptone liquid medium at 37℃ and 150 r / min for 16h to obtain YCH-H7 seed liquid and NF2 seed liquid, respectively. In the beef extract peptone liquid medium, beef extract was 3g / L, peptone was 10g / L, NaCl was 5g / L, and pH was 7.2. YCH-H7 seed liquid and NF2 seed liquid were inoculated into fermentation medium at 5% mass fraction at 37℃ and 150 r / min for 46h to obtain YCH-H7 fermentation liquid and NF2 fermentation liquid, respectively. In the fermentation medium, sucrose was 20g / L, beef extract was 3g / L, peptone was 10g / L, NaCl was 5g / L, MnSO4 was 0.3g / L, CaCl2 was 0.5g / L, yeast extract powder was 5g / L, and pH was 7.2. YCH-H7 fermentation liquid and NF2 fermentation liquid were diluted with fermentation medium to obtain YCH-H7 bacterial liquid and NF2 bacterial liquid. After sterilization, YCH-H7 bacterial liquid and NF2 bacterial liquid were mixed at a volume ratio of 1:0.5 to obtain the soil-borne disease biocontrol agent.

[0057] Example 4 A preparation method of a soil-borne disease biocontrol agent includes the following steps: Single colonies of YCH-H7 and NF2 were picked and inoculated into beef extract peptone liquid medium at 36.5℃ and 250 r / min for 20h to obtain YCH-H7 seed liquid and NF2 seed liquid, respectively. In the beef extract peptone liquid medium, beef extract was 3g / L, peptone was 10g / L, NaCl was 5g / L, and pH was 7.2. YCH-H7 seed liquid and NF2 seed liquid were inoculated into fermentation medium at 4% mass fraction at 36.5℃ and 250 r / min for 50h to obtain YCH-H7 fermentation liquid and NF2 fermentation liquid, respectively. In the fermentation medium, sucrose was 20g / L, beef extract was 3g / L, peptone was 10g / L, NaCl was 5g / L, MnSO4 was 0.3g / L, CaCl2 was 0.5g / L, yeast extract powder was 5g / L, and pH was 7.2. YCH-H7 fermentation liquid and NF2 fermentation liquid were diluted with fermentation medium to obtain YCH-H7 bacterial liquid and NF2 bacterial liquid. After sterilization, YCH-H7 bacterial liquid and NF2 bacterial liquid were mixed at a volume ratio of 1:1.5 to obtain the soil-borne disease biocontrol agent.

[0058] Comparative Example 1 A preparation method of a soil-borne disease biocontrol agent includes the following steps: Single colonies of *Bacillus cereus* YCH-H7 were inoculated into beef extract peptone liquid medium and cultured at 36℃ and 200 rpm for 18 h to obtain the YCH-H7 seed culture. The beef extract peptone liquid medium contained 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.2. The YCH-H7 seed culture was then inoculated into fermentation medium at a 2% (w / w) inoculation rate and fermented at 36℃ and 200 rpm for 48 h to obtain the YCH-H7 fermentation broth. The fermentation broth contained 20 g / L sucrose, 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 0.3 g / L MnSO4, 0.5 g / L CaCl2, 5 g / L yeast extract, and pH 7.2. The YCH-H7 fermentation broth was diluted with fermentation medium to obtain YCH-H7 bacterial solution, which is the soil-borne disease biocontrol agent.

[0059] Comparative Example 2 A method for preparing a soil-borne disease biocontrol agent includes the following steps: Single colonies of NF2 bacteria were inoculated into beef extract peptone liquid medium and cultured at 36℃ and 200 rpm for 18 h to obtain NF2 seed culture. The beef extract peptone liquid medium contained 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.2. The NF2 seed culture was then inoculated into fermentation medium at a 2% (w / w) inoculation rate and fermented at 36℃ and 200 rpm for 48 h to obtain NF2 fermentation broth. The fermentation broth contained 20 g / L sucrose, 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 0.3 g / L MnSO4, 0.5 g / L CaCl2, 5 g / L yeast extract, and pH 7.2. The NF2 fermentation broth was diluted with fermentation medium to obtain NF2 bacterial solution, which is the soil-borne disease biocontrol agent.

[0060] To illustrate the beneficial effects of the present invention, the following experiments were also conducted.

[0061] I. Experimental Methods 1. Nematicidal effect of sterile fermentation broth Single colonies of YCH-H7 and NF2 bacteria were inoculated into beef extract peptone liquid medium and cultured at 36℃ and 200 rpm for 18 h to obtain fermentation seed cultures for YCH-H7 and NF2 bacteria, respectively. The beef extract peptone liquid medium contained 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.2.

[0062] The fermentation seed liquid of YCH-H7 bacteria and the fermentation seed liquid of NF2 bacteria were respectively inoculated into the fermentation medium at an inoculation amount of 2% mass volume fraction, at a temperature of 36°C, a rotation speed of 200 r / min, and for 48 h of fermentation, to obtain the fermentation liquid of YCH-H7 bacteria and the fermentation liquid of NF2 bacteria. The fermentation medium has the following formulation: 20 g / L of sucrose, 3 g / L of beef extract, 10 g / L of peptone, 5 g / L of NaCl, 0.3 g / L of MnSO4, 0.5 g / L of CaCl2, 5 g / L of yeast extract, and a pH of 7.2. The live bacteria count in the fermentation liquid of YCH-H7 bacteria was 6.2 x 10 9 CFU / mL, and the live bacteria count in the fermentation liquid of NF2 bacteria was 3.6 x 10 9 CFU / mL, which met the agricultural microbial inoculant execution standard GB20287-2006.

[0063] Preparation of potato dextrose agar medium: 200 g of peeled and diced potatoes were boiled in 900 g of water for 30 min, filtered, and the filtrate was collected. 20 g of glucose and 20 g of agar were added to the filtrate, heated and dissolved, and water was added to make up to 1000 mL. The medium was sterilized by high-pressure steam at 115°C for 30 min and was ready for use. The potato dextrose agar medium is abbreviated as PDA medium.

[0064] The live bacteria count of the fermentation liquid of YCH-H7 bacteria and the fermentation liquid of NF2 bacteria was diluted to 1.0 x 10 8 CFU / mL using sterile fermentation medium, to obtain YCH-H7 bacteria liquid and NF2 bacteria liquid. The YCH-H7 group is denoted as YCH-H7, the NF2 group is denoted as NF2, the YCH-H7 supernatant and the NF2 supernatant mixed at a volume ratio of 1:1 are denoted as YCH-H7+NF2, and the sterile fermentation liquid without inoculation is used as a blank control and is denoted as CK. 30 mL of YCH-H7 bacteria liquid and NF2 bacteria liquid were centrifuged at 10000 rpm / min for 3 min, the supernatant was filtered to remove bacteria using a 0.22 μm cell filter, to obtain YCH-H7 supernatant and NF2 supernatant.

[0065] In a 24-well cell culture plate, 1.5 mL of YCH-H7 supernatant, NF2 supernatant, and YCH-H7 supernatant and NF2 supernatant mixed at a volume ratio of 1:1 were added to sterile fermentation liquid. 100 second instar larvae of southern root-knot nematode, 100 egg masses of southern root-knot nematode, and 100 stem decaying nematodes were added to each treatment, with 3 replicates. The plate was placed at 28°C, and after 48 h, the mortality rate of southern root-knot nematode second instar larvae and the egg hatching rate of southern root-knot nematode were counted, and the mortality rate of stem decaying nematodes was counted after 4 days. The corrected mortality rate and egg hatching inhibition rate were calculated according to the following formula:

[0066] Formula (1): Corrected mortality (%) = (treatment mortality - control mortality) / (1 - control mortality) x 100% Formula (2): Egg hatch inhibition rate (%) = (control hatch rate - treatment hatch rate) / control hatch rate x 100%.

[0067] 2. Inhibition of multiple soil-borne pathogenic fungi by sterile fermentation broth Take 1 mL of YCH-H7 supernatant and NF2 supernatant, respectively, and add them to 100 mL of PDA medium heated and cooled to 50℃, pour flat plates, and obtain YCH-H7 flat plates and NF2 flat plates. Use a 5mm punch to take the cultured plant soil-borne pathogenic fungi, kiwifruit root rot pathogen, Latin name Fusarium solani , pepper blight pathogen, Latin name Phytophthora capsici , tomato neck and root rot pathogen, Latin name Fusarium oxysporum f.sp. radicis- , cucumber root rot pathogen, Latin name lycopersici , and wheat basal stem rot pathogen, Latin name Phytophthora nicotianae Fusarium pseudograminearum , and place them in flat plates containing YCH-H7 fermentation filtrate and NF2 fermentation filtrate. Take PDA culture plates without fermentation filtrate as controls, and incubate them at 28℃ for 5d, count the colony diameters, and calculate the mycelial growth inhibition rate. The mycelial growth inhibition rate is calculated according to the following formula:

[0068] Formula (3): Mycelial growth inhibition rate (%) = (control mycelial growth - treatment mycelial growth) / control mycelial growth x 100% 3. Field control Carry out carrot root-knot nematode disease control tests in Huangfu Village, Qiangbai Town, Dali County, carry out wheat basal stem rot disease control tests in Dingcun, Huayi District, and carry out kiwifruit root rot disease control tests in Xifurao Village, Mazhao Town, Zhouzhi County. Dilute the fermentation broth of YCH-H7 and NF2 bacteria to 1.0 x 10 8 CFU / mL, and apply the composite biocontrol agent according to the volume ratio 1:1 soil-borne disease before planting and at the seedling stage, with water as the blank control CK, and cooperate with normal field management. Calculate the incidence, disease index, and control effect. The incidence, disease index, and control effect are calculated according to formulas (4) to (6), respectively.

[0069] Formula (4): Incidence = diseased plants / total plants x 100% Formula (5): Disease index = ∑ (grade x number of plants at that grade) / (highest grade x total plants) x 100% Formula (6): Control effect = (control disease index - treatment disease index) / control disease index x 100% II. Experimental results 1. Nematicidal effect of the fermentation broth The nematicidal effect of YCH-H7 fermentation broth and NF2 fermentation broth is shown in Table 3. The results show that the fermentation broth of YCH-H7 and NF2 has obvious lethal effect on southern root-knot nematode, the corrected mortality rate of nematodes after 48h treatment is 97.61% and 92.5% respectively, and the egg hatching inhibition rate is 81.03% and 83.2% respectively; the mortality rate of rotten stem nematodes after 4d treatment is 96.72% and 93.5% respectively. The nematicidal effect of the compound microbial agent is the highest, the corrected mortality rate of second instar larvae after 48h treatment is 98.85%, the egg hatching inhibition rate of nematodes is 88%, and the corrected mortality rate of rotten stem nematodes after 4d treatment is 97.7%. Therefore, the compound microbial agent of the application can inhibit the hatching of nematode eggs and kill nematode individuals.

[0070] Table 3 Lethal effect of fermentation broth on parasitic nematodes 2. Bacteriostatic effect of the fermentation broth on various soil-borne pathogenic fungi The bacteriostatic effect of YCH-H7 bacteria and NF2 bacteria on various soil-borne pathogenic fungi is shown in Table 4. The results show that the fermentation filtrate of YCH-H7, NF2 and the mixed liquid of YCH-H7 and NF2 fermentation filtrate v:v = 1:1 has obvious inhibitory effect on the growth of plant soil-borne pathogenic fungi, after 5d treatment, the mycelial growth inhibition rate of single microbial agent filtrate on kiwi root rot pathogen, pepper blight pathogen, tomato neck rot and root rot pathogen, cucumber root rot pathogen and wheat stem base rot pathogen is more than 87%, and the inhibition rate of mixed filtrate is more than 90%, and the broad-spectrum inhibitory effect is obvious.

[0071] Table 4 Inhibitory effect of fermentation broth on soil-borne pathogenic fungi 3. Field control The field control effect of YCH-H7 bacteria and NF2 bacteria is shown in Table 5. The results show that the field control effect of the compound biocontrol microbial agent on wheat stem base rot, pepper blight and root knot nematode can reach more than 80%.

[0072] Table 5 Field control effect of the compound biocontrol microbial agent In the table, " / " indicates that there is no such item.

[0073] IV. Results and discussion The soil-borne disease biocontrol agent of the present application corrects the mortality rate of the second instar larvae for 48h to be 98.85%, the nematode egg hatching inhibition rate is 88%, the rotten stem nematode correction mortality rate is 97.7% after 4d of treatment. And the mycelial growth inhibition rate of the soil-borne disease biocontrol agent on the pathogen of kiwi root rot disease, pepper blight pathogen, tomato neck rot and root rot disease, cucumber root rot disease pathogen, and wheat stem base rot disease pathogen all exceeds 90%, the broad-spectrum inhibition effect is obvious.

[0074] It should be noted that when the numerical range is involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Since the same steps and examples are used, the preferred embodiments are described in the present application to prevent redundancy. Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the inventive concept of the present application, and these changes and modifications all fall within the scope of the present application.

[0075] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. If these modifications and variations of the present application fall within the scope of the equivalent technology of the present application, the present application also intends to include these modifications and variations.

Claims

1. A soil-borne disease biocontrol agent, characterized in that, It is prepared by compounding Bacillus vesiculosus and Bacillus brevis in a volume ratio of 1:0.5~1.5; Among them, the Bacillus belyssus ( Bacillus velezensis The strain is YCH-H7, with accession number CGMCC No. 33510 and accession date of February 11, 2025. The *Bacillus brevis* strain is NF2. The viable count of Bacillus vesiculosus in the culture was greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL; The viable count of Bacillus brevis in bacterial culture is greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL.

2. The soil-borne disease biocontrol agent according to claim 1, characterized in that, The viable cell counts in both the *Bacillus vesiculosus* and *Bacillus brevis* bacterial suspensions were 1.0 × 10⁻⁶. 8 CFU / mL ~1.0×10 9 CFU / mL.

3. The soil-borne disease biocontrol agent according to claim 1, characterized in that, The volume ratio of Bacillus vesiculosus suspension to Bacillus brevis suspension was 1:

1.

4. The method for preparing a soil-borne disease biocontrol agent according to claim 1, characterized in that, Includes the following steps: Bacillus berberis was activated and cultured on a large scale to obtain Bacillus berberis fermentation broth. The viable cell concentration of the Bacillus berberis fermentation broth was adjusted to be greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL was used to obtain Bacillus vesiculosus culture; Bacillus berberis was activated and cultured on a large scale to obtain Bacillus berberis fermentation broth. The viable cell concentration of the Bacillus berberis fermentation broth was adjusted to be greater than or equal to 1.0 × 10⁻⁶. 8 CFU / mL was used to obtain Bacillus vesiculosus culture; Mix Bacillus vesiculosus and Bacillus brevis in a volume ratio of 1:0.5~1.5 to obtain a soil-borne disease biocontrol agent.

5. The method for preparing a soil-borne disease biocontrol agent according to claim 4, characterized in that, The activation culture was carried out at a temperature of 36℃~37℃, a rotation speed of 150r / min~250r / min, and a time of 16h~20h.

6. The method for preparing a soil-borne disease biocontrol agent according to claim 4, characterized in that, The culture was carried out at a temperature of 36℃~37℃, a rotation speed of 150r / min~250r / min, and a time of 46h~50h.

7. The application of the soil-borne disease biocontrol agent according to claim 1 in the control of soil-borne diseases.

8. The application of the soil-borne disease biocontrol agent according to claim 7 in the control of soil-borne diseases, characterized in that, The soil-borne diseases mentioned include wheat stem base rot, kiwi root rot, pepper blight, tomato neck rot and root rot, cucumber root rot, and root-knot nematode disease.

9. The application of the soil-borne disease biocontrol agent according to claim 7 in the control of soil-borne diseases, characterized in that, The pathogens causing the soil-borne diseases include the pathogen causing kiwifruit root rot. Fusarium solani Pathogen of pepper blight Phytophthora capsici Tomato neck rot and root rot Fusarium oxysporum f. sp. radicis- lycopersici Cucumber root rot pathogen Phytophthora nicotianae wheat stem rot pathogen Fusarium pseudograminearum .

Images