Bacillus for preventing and treating grape root diseases and application thereof

Abstract

The invention relates to a bacterium for preventing and treating grape root diseases and application thereof. The bacillus velezensis GP50 provided by the invention has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.32909 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. The viable bacteria and metabolites of the strain have efficient prevention and treatment effects on grape root diseases. The strain is separated from grape rhizosphere soil, the fermentation culture time is short, the production cost is low, the control effect is further improved when the strain is matched with a chemical agent for use, and the strain has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of plant protection of agricultural, forestry and horticultural crops, and particularly relates to a Bacillus and its metabolites that have a strong inhibitory effect on grape root rot caused by Rhizoctonia solani and grape canker caused by Neofusicoccum parvum. Technical Background

[0002] Grapes are important economic fruit trees in China. At present, they are mostly planted on a large scale in Gansu, Xinjiang, Ningxia, Hebei, Shandong, Beijing, Tianjin, Yunnan and other places. Root rot is an important disease in grape production, which can be caused by the infection of various pathogens. The typical symptoms are yellowing of leaves, scorching of leaf margins, and blackening of roots. The pathogen invades from the fibrous roots or wounds of grapes, and red-brown round spots appear in the root vascular bundles. In the later stage, the lesions expand and even turn black and rot, the function of absorbing water and nutrients gradually weakens, and finally the whole plant dies. The leaves of the affected plants are scorched at the tips or edges, and the middle of the leaf veins remains green, and the diseased leaves do not fall off quickly.

[0003] There have been many research reports on the control of root diseases by plant rhizosphere microorganisms. Bacillus spp. and Pseudomonas spp. are the biocontrol bacteria reported more frequently. At present, many strains have been applied in practical production, but the field control effect of biocontrol bacteria often shows instability. We also found in the research that the antagonistic ability and disease prevention effect of the same strain on root diseases caused by different pathogens of different plants are different, which may be related to the affinity of biocontrol bacteria for plants and their adaptability to the soil environment. Rhizosphere microorganisms coexist with plants for a long time and benefit each other. The microorganisms in the plant rhizosphere show stronger affinity for the host plant, can better utilize the root exudates of their host plants, and colonize in the plant rhizosphere 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 is very little research on the biological control of grape root diseases. Only the application of Pseudomonas aeruginosa O-C9 to control grape black root rot (Patent CN116836873A) has been reported. Strain O-C9 has inhibitory effects on various pathogenic bacteria, such as Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Coletotrichum viniferum, Botrytis cinerea, etc. The control effect of this strain on the branch canker caused by Lasiodiplodia theobromae reaches 47.44%, and it has an inhibitory effect on the proliferation of the pathogen Dactylonectria torresensis in grape roots, but the control effect on grape root rot caused by Rhizoctonia solani and Neofusicoccum parvum has not been reported. The research group collected grape rhizosphere soil samples from the main wine grape production area on the east slope of Helan Mountain in Ningxia. Aiming at grape root rot caused by Rhizoctonia solani and grape canker caused by Neofusicoccum parvum, a highly efficient antagonistic bacterium GP50 was screened by the method of docking bacteria and pathogenic fungi up and down. Through 16S rRNA gene sequence analysis and whole genome sequencing analysis, it was found that GP50 is Bacillus velezensis. The results of pot control efficacy tests showed that the control effect of the fermentation broth of strain GP50 alone on grape root rot caused by Rhizoctonia solani reached 65.3%, which was 11% higher than that of the chemical pesticide control, 11% metalaxyl-m and fludioxonil. The control effect of the fermentation broth of strain GP50 in combination with 11% metalaxyl-m and fludioxonil on grape canker caused by Neofusicoccum parvum reached 72.2%, which was higher than that of the chemical pesticide control, 11% metalaxyl-m and fludioxonil (control efficacy 54.6%). It is a Bacillus with great application potential. Summary of the Invention

[0005] The object of the present invention is to provide a Bacillus with control effects on grape root rot caused by Rhizoctonia solani and grape canker caused by Neofusicoccum parvum and its application.

[0006] The Bacillus provided by the present invention is specifically Bacillus velezensis GP50. The strain was deposited on December 05, 2024 at the General Microbiology Center of the China Microbial Culture Collection Center (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC No. 32909.

[0007] The present invention also protects a bacterial agent with the active ingredient being the Bacillus velezensis GP50.

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

[0009] More specifically, in an embodiment of the present invention, the adsorption carrier is specifically mixed by a seedling-raising substrate according to a certain volume-weight ratio. Further, the microbial agent is mixed by the fermentation broth of Bacillus velezensis GP50 and the adsorption carrier in a ratio of 1L:1Kg to 1L:5Kg (such as 1L:4Kg). In an embodiment (Embodiment 3) of the present invention, a large amount of fermentation culture medium is used to prepare the fermentation broth of Bacillus velezensis GP50. In the fermentation broth of Bacillus velezensis GP50, the viable count of Bacillus velezensis GP50 is 3 to 5 billion / mL (such as 4 billion / mL) of the fermentation broth, and the ratio of GP50 is 1L:1Kg according to the weight-volume ratio. Among them, the solvent of the large amount of fermentation culture medium is water, and the solute and concentration are as follows: molasses 1.5 - 2.0%, soybean powder 1.2 - 1.8%, KH 2 PO 4 0.5 - 0.85%, MnSO 4 0.05 - 1.0%, pH 7.0; where %, represents mass percentage. Correspondingly, in the prepared microbial agent, the viable count of Bacillus velezensis GP50 is 3 to 5 billion / g (such as 4 billion / g) of the microbial agent; the water content of the microbial agent is within 5 - 8%.

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

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

[0012] (1) Cultivate Bacillus velezensis GP50 to obtain the fermentation broth of Bacillus velezensis GP50; in the fermentation broth, the viable count of Bacillus velezensis GP50 is 3 to 5 billion / mL (such as 4 billion / mL).

[0013] (2) Mix the fermentation broth obtained in step (1) and the adsorption carrier at a ratio of 1 L: 3 kg to 1 L: 5 kg (such as 1 L: 4 kg), or mix the bacterial sludge obtained after centrifuging the fermentation broth and the adsorption carrier at a ratio of 1 L: 3 kg to 1 L: 5 kg (such as 1 L: 4 kg) to obtain the bacterial agent.

[0014] In step (1), the solvent of the medium for culturing the Bacillus velezensis GP50 is water, and the solutes and their concentrations are as follows: molasses 1.5 - 2.0%, soybean powder 1.2 - 1.8%, KH 2 PO 4 0.5 - 0.85%, MnSO 4 0.05 - 1.0%, pH 7.0; wherein, % represents mass percentage.

[0015] More specifically, the solvent of the medium is water, and the solutes and their concentrations are as follows: molasses 1.5 - 2.0%, soybean powder 1.2 - 1.8%, KH 2 PO 4 0.5 - 0.85%, MnSO 4 0.05 - 1.0%, pH 7.0; wherein, % represents mass percentage.

[0016] The fermentation broth of the Bacillus velezensis GP50 also belongs to the protection scope of the present invention.

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

[0018] The application of the Bacillus velezensis GP50 or the bacterial agent or the fermentation broth in the following (1) or (2) also belongs to the protection scope of the present invention:

[0019] (1) Prevent and control grape root rot caused by the pathogenic fungus Rhizoctonia solani and grape canker caused by Neofusicoccum parvum.

[0020] (2) Prepare a product for preventing and controlling the diseases in (1).

[0021] The Bacillus velezensis GP50 CGMCC No. 32909 provided by the present invention has the following advantages:

[0022] 1. This strain is isolated from grape rhizosphere soil, can better adapt to the grape rhizosphere environment, and grows rapidly, which is more conducive to its application in the field.

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

[0024] 3. It has a high control effect against Rhizoctonia solani root rot and Neofusicoccum parvum canker, with a short fermentation cycle, low cost, and is easy to mass-produce and commercially apply.

[0025] The Bacillus velezensis GP50 (CGMCC No. 32909) provided by the present invention is a Bacillus strain that was isolated from grape rhizosphere soil by the inventor in September 2023 and has a high antagonistic effect against the pathogens of grape root rot and grape canker. The characteristics of this strain are as follows: The colonies of GP50 on LB medium are white but opaque, with a rough and wrinkled surface and a dry texture. The strain has a significant disease prevention effect on grape root rot caused by Rhizoctonia solani, reaching 65.3%, which is 11% higher than that of the commonly used chemical pesticide metalaxyl-m + fludioxonil; the disease prevention effect of the strain on grape canker caused by Neofusicoccum parvum reaches 54.6%, and the combined application with 11% metalaxyl-m + fludioxonil can achieve an efficacy of 72.1%, while 11% metalaxyl-m + fludioxonil alone has basically no effect. When strain GP50 is fermented and cultured, it has a fast growth rate and can complete fermentation within 48 hours. After fermentation, the viable cell count can reach 4 - 6 billion / mL, greatly reducing the use cost. Therefore, GP50 is a very promising bacterial combination, and thus the present invention is proposed.

[0026] Deposit Description

[0027] Species name: Bacillus velezensis

[0028] Latin name: Bacillus velezensis

[0029] Strain number: GP50

[0030] Depositary institution: General Microbiological Center of China Committee for Culture Collection of Microorganisms

[0031] Abbreviation of depositary institution: CGMCC

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

[0033] Deposit date: December 05, 2024

[0034] Registration number of the depositary center: CGMCC No. 32909 Detailed Description of the Invention

[0035] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0036] The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0037] Example 1. Antagonistic screening of salt-tolerant antagonistic bacterium GP50 in grape rhizosphere

[0038] More than 300 salt-tolerant bacteria isolated from the rhizosphere soil of Ningxia wine grapes were selected for the screening of antagonistic bacteria. 100 μL of the bacterial suspension cultured in LB was pipetted and spread on a 1 / 2PDA plate, and cultured at 26 °C for 24 - 48 h. The pathogen fungal cake was transferred to a 1 / 2PDA plate and cultured at 28 °C for 24 - 48 h; fresh-cultured 8-mm bacterial and fungal agar columns were punched from the PDA plate and docked on the SWB plate with the bacteria below and the fungi above, with the PDA agar column docked to the pathogen mycelial block as a control. Each strain was replicated on 3 plates. Cultured at 25 - 26 °C for 3 - 5 d. Observe the growth of bacteria on the surface of the SWB medium, and use a vernier caliper to measure the growth radius (mm) of the pathogen colony, with the docking of the pathogen mycelial block as a control, and each strain was replicated on 3 plates. Calculate the inhibition rate.

[0039] Among them, the components and ratios of the LB medium are: 10 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, and made up to 1000 mL with deionized water.

[0040] The components and ratios of the 1 / 2PDA medium are: 100 g of potato, 10 g of glucose, 18 g of agar powder, and 1 L of deionized water.

[0041] The preparation method of the SWB medium: Weigh 28 g of soybean powder, add 500 mL of distilled water, then boil it over high heat, keep boiling over low heat for 10 minutes and then filter it through 8 layers of gauze to obtain 280 mL of medium; 7 g of wheat bran, add 200 mL of distilled water, then boil it over high heat, keep boiling over low heat for 30 minutes, and then filter it through 8 layers of gauze to obtain 70 mL of medium; Combine the filtrates, and then add 5.25 g of agar powder; Autoclave for 30 minutes.

[0042] The results showed that the inhibition rates of strain GP50 against Rhizoctonia solani and Neofusicoccum parvum reached 100% (Table 2).

[0043] Table 2. Inhibition rates of antagonistic bacterium GP50 against pathogens

[0044]

[0045] Example 2. Identification of antagonistic bacterium GP50 strain

[0046] The cell morphology of antagonistic bacterium GP50 was observed microscopically, and Gram staining and H 2 O2 Enzyme activity assay showed that strain GP50 was Gram-positive, and positive for H 2 O 2 enzyme activity. The colonies of strain GP50 on 1 / 10 TSA and LB media were milky white, opaque, with rough and wrinkled surfaces and dry texture. This strain could produce spores and was rod-shaped. The genomic DNA of strain GP50 was extracted, and the 16S rRNA gene sequence was amplified using universal primers 27F and 1492R and sequenced. 27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′; 1492R: 5′-TAC GGC TAC CTT GTT ACG ACT T-3′. The 16S rRNA gene sequence of strain GP50 has been submitted to GenBank with the accession number PV012486; its whole genome sequence has been submitted to GenBank with the accession number SAMN46383039 (BioProject: PRJNA1214700). Based on the 16S rRNA gene sequence and its whole genome sequence of strain GP50, this strain was identified as Bacillus velezensis. Strain GP50 was deposited on December 05, 2024, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number of strain GP50 is CGMCC No. 32909.

[0047] Example 3. Control effect of Bacillus GP50 on grape root diseases

[0048] Preparation of strain GP50 culture solution: Pick a single colony and inoculate it into 100 mL of TSB culture solution, culture at 28 °C and 180 rpm for 24 h, then transfer it to the fermentation culture solution, with a liquid loading of 50 mL / 500 mL, and culture under the conditions of 28 °C and 180 rpm. After 36 h of culture, spores began to be produced by the bacteria in the fermentation broth, and after 48 h, the spore production rate reached over 90%. The bacterial content in the fermentation broth was measured to be 4 billion / mL by the hemocytometer counting method.

[0049] The solvent of the fermentation broth medium described above is water, and the solutes and their concentrations are as follows: molasses 2.0%, soybean powder 1.5%, KH 2 PO 4 0.7%, MnSO 4 0.1%, pH 7.0; where %, represents mass percentage.

[0050] Preparation of solid inoculum of strain GP50: Put the fermentation broth of strain GP50 and the sterilized substrate into a fresh-keeping bag in a ratio of 1 L:4 Kg, mix well by shaking, so that the concentration of GP50 in the substrate reaches 10 7CFU / g.

[0051] Preparation of GP50 bacterial suspension for irrigation: Dilute the GP50 fermentation broth to a concentration of 10 8 CFU / mL, mix in a volume ratio of 1:1, and set aside for use.

[0052] Preparation of pathogen inoculum: Soak millet and rice husk (volume ratio 50:50) in distilled water for 2 h to allow the mixture to fully absorb water, then pour out the water. Then transfer about 160 g of the millet-rice husk medium to a 500 mL conical flask and sterilize at 120 °C. Inoculate 5 pieces each of 5 mm Rhizoctonia solani or Neofusicoccum parvum fungal cakes into the above sterilized medium and culture at 28 °C for 7 d. Mix the Rhizoctonia solani culture and the Neofusicoccum parvum culture with the sterilized substrate in a volume / weight ratio (V:W) of 1% respectively, and mix well in a fresh-keeping bag for standby.

[0053] Pot experiment: The experiment was set with 9 treatments, namely T1, GP50 solid inoculum + Rhizoctonia solani solid inoculum + GP50 fermentation broth for root irrigation; T2, GP50 solid inoculum + Rhizoctonia solani solid inoculum + 11% metalaxyl-mefenoxam (Hebei Green Agriculture Co., Ltd.) for root irrigation; T3, sterile substrate + Rhizoctonia solani solid inoculum + 11% metalaxyl-mefenoxam for root irrigation; CK1, sterile substrate + Rhizoctonia solani solid inoculum + water; T4, GP50 solid inoculum + Neofusicoccum parvum solid inoculum + GP50 fermentation broth for root irrigation; T5, GP50 solid inoculum + Neofusicoccum parvum solid inoculum + 11% metalaxyl-mefenoxam (Hebei Green Agriculture Co., Ltd.) for root irrigation; T6, sterile substrate + Neofusicoccum parvum solid inoculum + 11% metalaxyl-mefenoxam for root irrigation; CK2, sterile substrate + Neofusicoccum parvum solid inoculum + water; CK, water control. There were 5 pots of Cabernet Sauvignon grape seedlings for each treatment, with 3 replicates. The specific operations are as follows:

[0054] Treatment T1: First, fill part of the natural soil in the flower pot, make wounds about 1 cm on the roots of the grape seedlings with a No. 10 scalpel, cover the roots of the seedlings with 50 g of GP50 solid inoculum per pot, and then apply the Rhizoctonia solani inoculum around, 50 g per pot. Then irrigate with a concentration of 10 8150 mL of GP50 fermentation broth at [X] CFU / mL; Treatment T2: The same as Treatment T1, inoculated with 50 g of solid inoculum of antagonistic bacterium GP50, then inoculated with 50 g of Rhizoctonia solani culture, and irrigated with 150 mL of 11% metalaxyl-m + pyrimethanil; Treatment T3: Inoculated with 50 g of sterile substrate and then inoculated with 50 g of Rhizoctonia solani culture, and irrigated with 150 mL of 11% metalaxyl-m + pyrimethanil; CK1: Inoculated with 50 g of sterile substrate and 50 g of Rhizoctonia solani inoculum, and irrigated with 150 mL of clear water; CK: Only inoculated with 50 g of sterile substrate and irrigated with 150 mL of clear water. In Treatments T4, T5, T6 and CK2, except that the pathogen was replaced with the inoculum of Neofusicoccum parvum, the others corresponded to T1, T2, T3 and CK1 one by one. All potted seedlings were placed in the greenhouse of Langfang Base of the Institute of Plant Protection. The temperature was 25 - 30 °C. The disease situation was investigated 2 months after transplantation, and the disease grading criteria refer to Table 3 and Table 4.

[0055] Table 3 Grading criteria for grape root rot caused by Rhizoctonia solani

[0056]

[0057] Table 4 Grading criteria for grape canker caused by Neofusicoccum parvum

[0058]

[0059] The results of the pot experiment showed that: The control effect of the fermentation broth of antagonistic bacterium GP50 against grape root rot caused by Rhizoctonia solani reached 65.3%, which was significantly higher than that of metalaxyl-m + pyrimethanil. The control effect of the fermentation broth of antagonistic bacterium GP50 against grape canker caused by Neofusicoccum parvum reached 54.6%, and the control effect was further improved when used in combination with metalaxyl-m + pyrimethanil, reaching 72.1% (Table 5).

[0060] Table 5 Control effects of GP50 fermentation broth against grape root diseases caused by Rhizoctonia solani and Neofusicoccum parvum

[0061]

Claims

1. Bacillus velezensis GP50, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.32909.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent is the Bacillus velezensis GP50 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 Bacillus velezensis GP50 viable bacteria in the liquid bacterial agent is 3 to 5 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 Bacillus velezensis GP50 and an adsorption carrier in a ratio of 1L:3Kg to 1L:5Kg, or by mixing the bacterial sludge after centrifugation of the fermentation liquid and the adsorption carrier in a ratio of 1L:3Kg to 1L:5Kg.

5. The bacterial agent according to claim 4, characterized in that The preparation method of the solid bacterial agent comprises the following steps: Cultivating Bacillus velezensis GP50 to obtain a fermentation liquid of Bacillus velezensis GP50; The fermentation liquid and the adsorption carrier are mixed in a ratio of 1L:3Kg to 1L:5Kg to obtain the bacterial agent; The culture medium solvent of Bacillus velezensis GP50 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 Bacillus velezensis GP50 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 diseases.

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