Bacterial composition for preventing and treating grape root rot and application thereof

Abstract

The invention relates to a bacterial composition for preventing and treating grape root rot and application thereof. The preservation numbers of the paenibacillus sp. GP28-1 and the bacillus velezensis GP28-2, which are provided by the invention, in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms are CGMCC (China General Microbiological Culture Collection Center) No.32906 and CGMCC No.32907 respectively, and the preservation number of the paenibacillus sp. GP28-1 and the preservation number of the bacillus velezensis GP28-2, which are provided by the invention, in the China General Microbiological Culture Collection Center are CGMCC No.32906 and CGMCC No.32907 respectively. The two strains are separated from grape rhizosphere soil, and viable bacteria and metabolites of the two strains have efficient prevention and treatment effects on grape root rot caused by fusarium. The bacterial composition is separated from grape rhizosphere soil, has strong affinity with grapes, short strain fermentation culture time and low production cost, has more advantages than other microbial agents, is beneficial to application to prevention and treatment of grape root rot, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of agricultural, forestry and horticultural crop plant protection, and particularly relates to a bacillus combination having a strong inhibitory effect on grape root rot caused by Fusarium and its metabolites. Technical Background

[0002] Grapes are an important economic fruit tree in my country. They have health and anti-aging effects. Not only are the fruits juicy and delicious, and they are helpful for digestion, but grapes also contain a large amount of substances such as resveratrol and polyphenols, which can prevent cancer and hypertension. At present, they are mostly planted on a large scale in Gansu, Xinjiang, Ningxia, Hebei, Shandong, Beijing and Tianjin. Root rot is an important disease in grape production, which can be caused by infection by a variety of fungi, such as Fusarium, Rhizoctonia, Fusobacterium spp., and Diplodia spp. The typical symptoms are yellowing of leaves, scorching of leaf edges, and blackening of roots. Grape root rot bacteria invade from fibrous roots or wounds, and reddish-brown circular spots appear in the root vascular bundles. In the later stage, the spots expand and even turn black and rot, and the ability to absorb water and nutrients gradually weakens, and finally the whole plant dies. The tips or edges of the leaves of the affected plants are scorched, and the middle of the veins remain green, and the diseased leaves will not fall off quickly.

[0003] Bacillus spp. and Pseudomonas spp. are the most reported biocontrol bacteria. Currently, many strains have been used in practical production, but the field efficacy of biocontrol bacteria is often unstable. We found that the same strain has different antagonistic ability and disease prevention effects on root diseases caused by different pathogens of different plants, which may be related to the affinity of biocontrol bacteria to plants and their adaptability to the soil environment. Rhizosphere microorganisms coexist with plants for a long time and benefit each other. Microorganisms in the rhizosphere of plants show stronger affinity for host plants, can better utilize the root secretions of their host plants, and colonize in the rhizosphere of plants to play their disease prevention and growth promotion functions. Therefore, it is very necessary to screen specific and efficient biocontrol microorganisms for different fungal diseases of different plants, which has important practical significance for enriching biocontrol resources and preventing and controlling diseases.

[0004] At present, there are very few studies on biological control of grape root diseases, and only Pseudomonas aeruginosa O-C9 is used to control grape black root disease (patent CN116836873A). Strain O-C9 has inhibitory effects on a variety of pathogens, such as Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Coletotrichum viniferum, Botrytis cinerea, etc. The strain has a control effect of 47.44% on branch canker caused by Lasiodiplodia theobromae, and has an inhibitory effect on the proliferation of Dactylonectriator resensis pathogens in grape roots, but no reports have been made on its control effect on grape root rot caused by Fusarium. Our research group collected grape rhizosphere soil samples from the main wine grape producing area on the eastern foot of Helan Mountain in Ningxia. Since most of the soil in Ningxia is saline-alkali soil, we first screened salt-tolerant bacteria, and then used the confrontation culture method and the upper and lower docking method of bacteria and pathogenic fungi to screen out a highly effective antagonistic bacterium GP28 for grape root rot caused by Fusarium. It was later found that the strain was a mixed strain. After further separation and purification, strains GP28-1 and GP28-2 were obtained. 16S rRNA gene sequence analysis and whole genome sequencing analysis showed that GP28-1 was different from the reported strains of Paenibacillus and was a new species of the genus; GP28-2 strain was Bacillus velez. The results of the potted plant control test showed that the combination of GP28-1 and GP28-2 had a control effect of 88.6% on grape root rot caused by Fusarium, which was much higher than the chemical pesticide control, and was a Bacillus combination with great application potential. Summary of the invention

[0005] The purpose of the present invention is to provide a bacillus combination having a preventive and therapeutic effect on grape root rot caused by Fusarium and its application.

[0006] The Bacillus combination provided by the present invention is specifically composed of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2. The strains have been deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 5, 2024, with the deposit numbers CGMCC No. 32906 and CGMCC No. 32907.

[0007] The present invention also protects a bacterial agent whose active ingredients are the Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2.

[0008] The microbial agent may further include an adsorption carrier; the adsorption carrier may be at least one of a seedling medium, diatomaceous earth, peat, vermiculite, rice husk powder, straw powder, perlite and calcium carbonate.

[0009] More specifically, in one embodiment of the present invention, the adsorption carrier is specifically formed by mixing the seedling substrate in a certain volume-to-weight ratio. Further, the bacterial agent is formed by mixing the fermentation liquid of the Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 with the adsorption carrier in a ratio of 1L:4Kg to 1L:6Kg (e.g., 1L:4Kg). In one embodiment of the present invention (Example 3), a large amount of fermentation culture medium is used to prepare the fermentation broth of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2, and the number of viable bacteria of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 in the fermentation broth is 3 to 5 billion / mL (such as 4 billion / mL), wherein GP28-1 and GP28-2 are mixed in a ratio of 1:1 (V / V). The solvent of the large amount of fermentation culture liquid is water, and the solutes and concentrations are as follows: molasses 1.5-2.0%, soybean powder 1.2-1.8%, KH2PO4 0.5-0.85%, MnSO4 0.05-1.0%, pH 7.0; wherein % represents the mass percentage. Correspondingly, in the prepared bacterial agent, the number of live bacteria of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 is 3-5 billion / g (such as 4 billion / g); the water content of the bacterial agent is within 5-8%.

[0010] The preparation method of the bacterial agent also belongs to the protection scope of the present invention.

[0011] The preparation method of the bacterial agent may specifically include the following steps:

[0012] (1) Cultivating the Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 to obtain fermentation broth of the Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2; in the fermentation broth, the number of viable bacteria of the Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 is 3 to 5 billion / mL (e.g., 4 billion / mL).

[0013] (2) The fermentation liquid obtained in step (1) and the adsorption carrier are mixed in a ratio of 1L:4Kg to 1L:6Kg (e.g., 1mL:4Kg) or the bacterial sludge obtained after centrifugation of the fermentation liquid and the adsorption carrier are mixed in a ratio of 1L:4Kg to 1L:6Kg (e.g., 1L:4Kg) to obtain the bacterial agent.

[0014] In step (1), the solvent of the culture medium for culturing the Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 is water, and the solutes and concentrations are as follows: molasses 1.5-2.0%, soybean powder 1.2-1.8%, KH2PO4 0.5-0.85%, MnSO4 0.05-1.0%, pH 7.0; wherein % represents the mass percentage.

[0015] More specifically, the solvent of the culture medium is water, and the solutes and concentrations are as follows: molasses 1.5-2.0%, soybean powder 1.2-1.8%, KH2PO4 0.5-0.85%, MnS04 0.05-1.0%, pH 7.0; wherein % represents the mass percentage.

[0016] The fermentation broth of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 also belongs to the protection scope of the present invention.

[0017] The invention also provides a product for preventing and treating grape root rot diseases.

[0018] The use of the Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 or the bacterial agent or the fermentation broth in the following (1) or (2) also falls within the protection scope of the present invention:

[0019] (1) Prevent and control grape root rot caused by infection with the pathogenic fungus Fusarium solani.

[0020] (2) Prepare products for preventing and controlling diseases described in (1).

[0021] The Paenibacillus sp. GP28-1 CGMCC No. 32906 and Bacillus velezensis GP28-2 CGMCC No. 32907 provided by the present invention have the following advantages:

[0022] 1. The strain was isolated from the rhizosphere soil of grapes, which makes it more adaptable to the rhizosphere environment of grapes and grows rapidly, making it more suitable for field application.

[0023] 2. This strain was obtained by screening using high-salt culture medium and has very good adaptability to salt stress.

[0024] 3. It has a highly effective prevention and control effect on Fusarium root rot, short fermentation cycle, low cost, and is easy to mass produce and commercialize.

[0025] The Paenibacillus sp. GP28-1 CGMCC No. 32906 and Bacillus velezensis GP28-2 CGMCC No. 32907 provided by the present invention are a combination of Bacillus strains with highly effective antagonism to grape root rot pathogens isolated by the inventor from grape rhizosphere soil in September 2023. The characteristics of these two strains are: the colonies of strain GP28-1 on LB medium (tryptone 10g, NaCl 10g, yeast extract 5g, agar 18g, distilled water 1L, pH 7.0) are milky white, round, transparent, with irregular edges, rough surface, and viscous texture; the colonies of GP28-2 on LB medium are white, but opaque, with a rough and wrinkled surface and a dry texture. The strain combination has a significant disease prevention effect on grape root rot caused by Fusarium punctatum, which can reach 88.9%, and is higher than the commonly used chemical pesticide 11% methylpyrifos, and is safe for humans and the environment. When the strains GP28-1 and GP28-2 are fermented and cultured, the growth rate is fast and the fermentation can be completed within 48 hours. After the fermentation, the number of live bacteria can reach 4 to 6 billion / mL, which greatly reduces the cost of use. Therefore, GP28-1 and GP28-2 are a very potential bacterial combination, and the present invention is proposed.

[0026] Collection Instructions

[0027] Bacteria name: Paenibacillus

[0028] Latin name: Paenibacillus sp.

[0029] Strain ID: GP28-1

[0030] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0031] Abbreviation of depository institution: CGMCC

[0032] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0033] Deposit date: December 5, 2024

[0034] CGMCC Registration Number: CGMCC No.32906

[0035] Bacteria name: Bacillus velez

[0036] Latin name: Bacillus velezensis

[0037] Strain ID: GP28-2

[0038] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0039] Abbreviation of depository institution: CGMCC

[0040] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0041] Deposit date: December 5, 2024

[0042] CGMCC Registration Number: CGMCC No.32907 DETAILED DESCRIPTION

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] Example 1. Isolation and screening of antagonistic bacteria GP28-1 and GP28-2

[0046] (1) Isolation and screening of salt-tolerant bacteria in the grape rhizosphere

[0047] Grape rhizosphere soil was collected from the wine grape planting area of ​​Ningxia, placed in a ziplock bag, and stored in a refrigerator at 4°C for later use. Take 0.5g of rhizosphere soil, add it to 9.5mL of sterile water, shake and mix, let it settle for 1 minute, take 1mL of the supernatant and add it to 9mL of high-salt culture solution, shake and culture at 28°C and 180rpm for 24 to 48 hours, observe the growth, serially dilute the turbid culture solution, and spread it on 1 / 10TSA culture medium. Culture at 28°C for 48 to 72 hours. According to the size, morphology, color and isolation frequency of the bacterial colony, pick a single colony and transfer it to 1 / 10TSA culture medium, and purify it by streaking. The purified strain is stored in 30% glycerol and stored in a refrigerator at -20°C.

[0048] The components and proportions of the high-salt culture medium are: NaCl 230 g / L, MgSO4.7H2O 20 g / L, sodium citrate 3 g / L, KCl 2 g / L, tryptone 5 g / L, yeast extract 3 g / L and ion solution 0.1 mL / L, wherein the composition of the ion solution is ZnSO4·H2O 1.32 g / L, MgSO4·H2O 0.34 g / L, Fe(NH4)2SO4·6H2O0.82 g / L, and CuSO4·5H2O0.14 g / L.

[0049] The components and proportions of the 1 / 10 TSA culture medium are: 1.5 g of tryptone, 0.05 g of yeast extract, 0.05 g of KH2PO4, 2.5 g of NaCl, 18.0 g of agar powder, and the volume is adjusted to 1000 mL with deionized water, and the pH is 7.0.

[0050] Results: The above method was used to isolate and pick bacteria, and more than 300 salt-tolerant bacterial strains were obtained by streaking.

[0051] (2) Plate confrontation method to determine the antibacterial effect of bacteria

[0052] The plate confrontation culture method was used to determine the inhibitory activity of bacteria against Fusarium. First, the bacteria were streaked 1 cm away from the edge of the plate, and 4 strains were inoculated on each plate. After culturing at 28°C for 2-3 days, a fungal cake with a diameter of 5 mm was inoculated in the center of the culture medium, and only a 5 mm fungal cake was inoculated in the control group. The culture was continued at 28°C for 4-5 days, and the width of the inhibition zone (mm) was measured with a vernier caliper.

[0053] When the salt-tolerant bacteria were selected for antagonistic assay, it was found that strain GP28 was a mixed strain, and then strains GP28-1 and GP28-2 were obtained after separation and purification. The results of the antagonistic assay showed that strains GP28-1 and GP28-2 had good antagonistic effects on a variety of Fusarium (Table 1).

[0054] Table 1 The width of the inhibition zone of antagonistic bacteria GP28-1 and GP28-2 against pathogens (mm)

[0055]

[0056] (3) Inhibition rate of mixed bacterial suspension of strains GP28-1 and GP28-2 against Fusarium

[0057] The antagonistic bacteria that can antagonize the growth of pathogens through nutrient competition were further screened by using the upper and lower docking method of pathogens and antagonistic bacteria. 100 μL of the mixed bacterial suspension of GP28-1 and GP28-2 cultured in LB was applied to a 1 / 2 PDA plate and cultured at 26°C for 24-48 hours. The pathogenic fungal cake was transferred to a 1 / 2 PDA plate and cultured at 28°C for 24-48 hours; freshly cultured 8mm bacterial and fungal agar columns were punched from the PDA plate with a hole puncher, and the bacteria were docked on the SWB plate with the fungi at the bottom and the fungi at the top, and the PDA agar column was docked with the pathogen mycelium block as a control. Three plates were repeated for each strain. Cultured at 25-26°C for 3-5 days. Observe the growth of bacteria on the surface of the SWB culture medium, and measure the growth radius (mm) of the pathogen colony with a vernier caliper, with the docked pathogen mycelium block as a control. Calculate the inhibition rate.

[0058] The LB medium components and proportions are as follows: 10 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, and deionized water to a volume of 1000 mL.

[0059] The components and proportions of the 1 / 2 PDA culture medium are: 100 g potato, 10 g glucose, 18 g agar powder, and 1 L deionized water.

[0060] The preparation method of the SWB culture medium comprises the following steps: weighing 28 g of soybean powder, adding 500 mL of distilled water, boiling over high heat, keeping boiling over low heat for 10 minutes, and filtering through 8 layers of gauze to obtain 280 mL of culture medium; adding 200 mL of distilled water to 7 g of wheat bran, boiling over high heat, keeping boiling over low heat for 30 minutes, and filtering through 8 layers of gauze to obtain 70 mL of culture medium; combining the filtrate, and then adding 5.25 g of agar powder; and sterilizing by high pressure for 30 minutes.

[0061] The results showed that the inhibition rates of the mixed bacterial suspension of strains GP28-1 and GP28-2 against the four Fusarium species were all above 80%, and the inhibition rates against Fusarium solani and Fusarium acuminata could reach 100% (Table 2).

[0062] Table 2 Inhibitory effect of mixed bacterial suspension of antagonistic bacteria GP28-1 and GP28-2 on pathogenic bacteria

[0063]

[0064] Example 2: Identification of Antagonistic Bacteria GP28-1 and GP28-2 Strains

[0065] The bacterial morphology of antagonistic bacteria GP28-1 and GP28-2 was observed microscopically, and Gram staining and H2O2 enzyme activity were determined. The results showed that both strains GP28-1 and GP28-2 were Gram-positive and H2O2 enzyme activity was positive. The colonies of strain GP28-1 on 1 / 10 TSA medium were milky white and opaque, transparent on LB medium, with irregular edges, liquid, close to the medium, viscous, and not easy to pick up. The colonies of strain 28-2 on TSA medium were white, round, with irregular edges; on LB medium, the colonies were white, opaque, with rough and wrinkled surfaces and dry texture. Both strains can produce spores, and the bacteria are rod-shaped. The length of GP28-1 bacteria is 1.0μm to 6μm, and the length of GP28-2 bacteria is 1.0μm to 3μm.

[0066] The genomic DNA of strains GP28-1 and GP28-2 was extracted, and the 16S rRNA gene sequences were amplified and sequenced using universal primers 27F and 1492R. 27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′; 1492R: 5′-TAC GGC TACCTT GTT ACG ACT T-3′. The 16S rRNA gene sequences of strains GP28-1 and GP28-2 have been submitted to GenBank with accession numbers PQ857664 and PQ857669, respectively; their complete genome sequences have also been submitted to GenBank with submission numbers SUB15004840 (BioProject: PRJNA1208703) and SUB15004401 (BioProiect: PRJNA1208735), respectively. According to the 16S rRNA gene sequences of strains GP28-1 and GP28-2, they were identified as Paenibacillus and Bacillus, respectively. The homology of the whole genome sequence of strain GP28-1 and the type species of Paenibacillus was less than 95%, so it could only be identified as Paenibacillus sp. According to the whole genome sequence of strain GP28-2, it was identified as Bacillus velezensis. Strains GP28-1 and GP28-2 were deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​December 5, 2024. The deposit numbers of strains GP28-1 and GP28-2 are CGMCC No. 32906 and CGMCC No. 32907, respectively.

[0067] Example 3: Effect of the combination of Bacillus sp. GP28-1 and GP28-2 on the prevention of grape root rot

[0068] Preparation of culture medium of strains GP28-1 and GP28-2: Pick a single colony and culture it in 100 mL TSB culture medium at 28°C and 180 rpm for 24 h, then transfer it to M2 fermentation culture medium and culture it at 28°C and 180 rpm for 2-3 days, and measure the bacterial content in the inoculum by dilution coating method / hemocytometer counting method, and set aside.

[0069] The solvent of the M2 fermentation medium is water, and the solutes and concentrations are as follows: 2.0% molasses, 1.5% soybean powder, 0.7% KH2PO4, 0.1% MnSO4, pH 7.0; wherein % represents the mass percentage.

[0070] Preparation of solid inoculum of strains GP28-1 and GP28-2: Put the fermentation liquid of strains GP28-1 and GP28-2 and the sterilized matrix into a fresh-keeping bag, shake and mix, so that the concentration of GP28-1 and GP28-2 in the matrix reaches 10 7 Pieces / g.

[0071] Preparation of GP28-1 and GP28-2 bacterial suspension for irrigation: dilute the GP28-1 and GP28-2 fermentation liquid to a concentration of 10 8 / mL, mix in a volume ratio of 1:1 and set aside.

[0072] Preparation of Fusarium acuminate inoculum: Soak millet and rice husk (volume 50:50) in distilled water for 2 hours to allow the mixture to fully absorb water, then pour out the water. Then transfer about 160g of millet and rice husk culture medium to a 500mL conical flask and sterilize at 120℃. Inoculate 5 pieces of 5mm Fusarium acuminate cake into the above sterilized culture medium and culture at 28℃ for 7 days. Mix the Fusarium acuminate culture with the sterilized matrix at a volume / weight ratio (V:W) of 1%, mix well in a fresh-keeping bag, and set aside.

[0073] Potted test: The test has five treatments, namely T1, GP28-1 and GP28-2 solid inoculum + GP28-1 and GP28-2 bacterial suspension root irrigation; T2, GP28-1 and GP28-2 solid inoculum + 11% methylcarbazine (Hebei Green Agricultural Company) root irrigation, T3, sterile matrix + 11% methylcarbazine root irrigation; CK1, only inoculated with pathogens, CK, water control. Each treatment has 5 pots of Cabernet Sauvignon grape seedlings, repeated 3 times. The specific operations are as follows: Treatment T1: First, fill the flowerpot with some natural soil, use a No. 10 scalpel to make a wound of about 1 cm on the roots of the grape seedlings, cover the roots of the seedlings with GP28-1 and GP28-2 solid inoculum at a rate of 50g per pot, and then sprinkle the acute top Fusarium inoculum around, 50g per pot. Then water at a concentration of 10 8150mL of GP28-1 and GP28-2 bacterial suspension with a concentration of 100 g / mL; Treatment T2: Same as Treatment T1, inoculated with 50g of antagonistic bacteria GP28-1 and GP28-2 solid inoculum, then inoculated with 50g of pathogen culture, and irrigated with 150mL of 11% methylcarbazine; Treatment T3: Inoculated with 50g of sterile substrate and then inoculated with 50g of pathogen culture, and irrigated with 150mL of 11% methylcarbazine; CK1: Inoculated with 50g of sterile substrate and 50g of pathogen inoculum, and irrigated with 150mL of water; CK: Inoculated with only 50g of sterile substrate, and irrigated with 150mL of water. All potted seedlings were placed in the greenhouse of the Langfang base of the Institute of Plant Protection. The temperature is 25-30℃. The disease condition was investigated after 2 months, and the disease level was graded as follows: Level 0, no disease; Level 1, slight yellowing of root leaves; Level 2, entire root leaves yellowing; Level 3, severe yellowing and withering of root leaves; Level 4, death of root leaves; Level 5, death of the entire plant.

[0074] The results of the pot experiment (Table 3) showed that the control effects of treatment T3 with seroquelamide alone and treatment T2 with seroquelamide plus GP28-1 and GP28-2 were lower than those of treatment T1 with GP28-1 and GP28-2 alone, with the control effect of T1 reaching 88.9%.

[0075] Table 3 The efficacy of GP28-1 and GP28-2 mixed fungi against grape root rot

[0076]

Claims

1. Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 are deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit numbers CGMCC No.32906 and CGMCC No.32907, respectively.

2. A bacterial agent, characterized in that The active ingredients of the bacterial agent are Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 as described in claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is a liquid bacterial agent, and the number of live bacteria of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 in the liquid bacterial agent is 4 to 6 billion / mL.

4. The bacterial agent according to claim 2, characterized in that: The bacterial agent is a solid bacterial agent, which is obtained by mixing the fermentation liquid of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 and an adsorption carrier in a ratio of 1L:4Kg to 1L:6Kg, or by mixing the bacterial sludge after centrifugation of the fermentation liquid and the adsorption carrier in a ratio of 1L:4Kg to 1L:6Kg.

5. The bacterial agent according to claim 4, characterized in that The preparation method of the solid bacterial agent comprises the following steps: Cultivating Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 to obtain fermentation broth of Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2; The fermentation liquid and the adsorption carrier are mixed in a ratio of 1L:4Kg to 1L:6Kg to obtain the bacterial agent; The culture medium solvent for culturing Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 is water, and the culture medium solutes and concentrations are as follows: molasses 1.5-2.0%, soybean powder 1.2-1.8%, KH2PO4 0.5-0.85%, MnSO4 0.05-1.0%, pH 7.0; wherein % represents the mass percentage content.

6. The Paenibacillus sp. GP28-1 and Bacillus velezensis GP28-2 according to claim 1, the bacterial agent according to any one of claims 2 to 4, and their use in the prevention and treatment of grape root rot.

Images