Plant disease-resistant probiotic complex microbial inoculant based on metabolism and syntrophy as well as preparation method and application of plant disease-resistant probiotic complex microbial inoculant

Abstract

The invention provides a plant disease-resistant probiotic complex microbial inoculant based on metabolism nursing and a preparation method and application thereof.The complex microbial inoculant is composed of bacillus amyloliquefaciens SQR9 and lysobacter enzymogenes XL8, and the strain XL8 is obtained by being separated from rhizosphere of cucumber and can grow by means of metabolite of the strain SQR9; the double-bacterium fermentation broth can significantly inhibit the growth of pathogenic bacteria fusarium oxysporum. The plant disease-resistant probiotic complex microbial inoculant not only can significantly promote cucumber growth, but also can significantly prevent and control plant fusarium wilt caused by fusarium oxysporum.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural microorganisms, and in particular relates to a plant disease-resistant probiotic composite bacterial agent based on metabolic syntrophy, and a preparation method and application thereof. Background Art

[0002] A major challenge facing modern agriculture is the control of plant diseases. Fusarium oxysporum is a ubiquitous soil-borne pathogen that is well known for causing wilt in many crops, resulting in severe yield losses worldwide. In agricultural production, farmers mostly rely on chemical products such as fertilizers and chemical pesticides to achieve the purpose of disease control and crop yield increase. However, the long-term application of chemical products has caused serious damage to the soil, such as decreased fertility, soil compaction, continuous cropping, and poor pest control. In addition, chemical pesticide residues on crops also threaten human health. Therefore, it is particularly important to develop an environmentally friendly, efficient and sustainable method for plant disease control.

[0003] In recent years, progress in the study of microbial communities has highlighted the potential of synthetic microbiota in enhancing plant disease resistance. Composite microbial agents are composed of two or more microorganisms. Through artificial design, a new microbial ecosystem is created to form a composite product with specific functions. Compared with single microbial agents or natural microbial communities, composite microbial agents have significant advantages due to their lower complexity, high controllability and excellent stability. In the field of agriculture, the application research of composite microbial agents is still in its infancy. Future research will focus on how to use existing ecological theories to design and construct stable and efficient composite microbial agents to ensure the long-term performance of their biological functions. Summary of the invention

[0004] In view of the deficiencies in the prior art, the first object of the present invention is to provide a plant disease-resistant probiotic composite bacterial agent that can not only inhibit plant wilt disease, but also significantly promote plant growth and health through metabolic symbiosis and the formation of biofilms, thereby providing theoretical and technical support for the scientific design of composite bacterial agent products.

[0005] Another object of the present invention is to provide an application of the composite bacterial agent.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In the first aspect, the present invention protects a plant disease-resistant probiotic composite bacterial agent based on metabolic symbiosis, wherein the active ingredient of the bacterial agent is a mixture of Bacillus velezensis SQR9 and Lysobacterenzymogenes XL8; the Bacillus velezensis SQR9 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee, with a deposit date of February 27, 2012 and a deposit number of CGMCC NO.5808. Since Bacillus velezensis SQR9 has been disclosed many times in the prior art, this application does not provide a deposit certificate; the Lysobacterenzymogenes XL8 is deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a deposit date of July 29, 2024 and a deposit number of GDMCC NO: 64920.

[0008] In a specific embodiment, the composite bacterial agent is prepared from bacterial liquids of Bacillus velez SQR9 and Bacillus enzymolyticus XL8 in a volume ratio of 1:1.

[0009] Since Bacillus Velez SQR9 was deposited as Bacillus amyloliquefaciens SQR9 and was later identified as Bacillus Velez SQR9 after technological advances, in this article, whether it is Bacillus Velez SQR9 or Bacillus amyloliquefaciens SQR9, it refers to the strain with the deposit number CGMCC NO.5808.

[0010] In the second aspect, the plant disease-resistant composite bacterial agent of the present invention is mainly prepared by the following method: Bacillus amyloliquefaciens SQR9 and Bacillus enzymolyticus XL8 are inoculated into tryptone soy broth (TSB) medium respectively, and liquid fermentation is carried out under the following conditions: pH 7.0, fermentation temperature 30°C, rotation speed 170r / min, and fermentation time 24h; centrifugation and washing to obtain bacterial cells, and resuspending the bacterial cells with an equal amount of 0.9% sodium chloride solution; adjusting the bacterial solution OD 600 to 2, and mixing the two bacterial liquids in equal amounts is the plant disease-resistant composite bacterial agent.

[0011] The preparation method of the TSB culture medium used is, taking the preparation of 1L culture medium as an example: 30g of TSB powder, dilute to 1000mL, and sterilize at 115℃ for 30min.

[0012] In a third aspect, the present invention protects the use of the plant disease-resistant probiotic composite bacterial agent described above in promoting the growth of cucumbers.

[0013] In a fourth aspect, the present invention protects the use of the plant disease-resistant probiotic composite bacterial agent described above in the preparation of a product that promotes cucumber growth.

[0014] In a fifth aspect, the present invention protects the use of the plant disease-resistant probiotic composite bacterial agent described above in preventing and controlling cucumber wilt disease.

[0015] In a sixth aspect, the present invention protects the use of the plant disease-resistant probiotic composite bacterial agent described above in the preparation of products for preventing and controlling cucumber wilt disease.

[0016] Beneficial effects: Application of a composite inoculant (BLF) consisting of Bacillus amyloliquefaciens SQR9 and Bacillus enzymolyticus XL8 to the rhizosphere of cucumbers infected with Fusarium oxysporum can significantly improve the growth and health of cucumbers. Specifically, compared with the control group F inoculated with Fusarium oxysporum alone, after application of the composite inoculant, the plant height and fresh weight of cucumbers increased by 100% and 127%, respectively, and the incidence rate decreased by 80%. In contrast, the inoculant (BF) inoculated with Bacillus amyloliquefaciens SQR9 alone increased the plant height and fresh weight of cucumbers by 55% and 97%, respectively, and reduced the incidence rate by 46%; while the inoculant (LF) inoculated with Bacillus enzymolyticus XL8 alone increased the plant height and fresh weight of cucumbers by 64% and 112%, respectively, and reduced the incidence rate by 60%.

[0017] Biomaterial Preservation Information

[0018] Bacterial species name: Lysobacterium zymogenes;

[0019] Latin name: Lysobacter enzymogenes;

[0020] Strain ID: XL8;

[0021] Depository: Guangdong Microbiological Culture Collection Center;

[0022] Abbreviation of depository institution: GDMCC;

[0023] Deposit address: Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou;

[0024] Deposit date: July 29, 2024;

[0025] The registration number of the Depository and Clearing Corporation is GDMCC NO: 64920. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Phylogenetic tree of Bacillus amyloliquefaciens SQR9 and Bacillus enzymolyticus XL8.

[0027] Figure 2 The metabolic symbiotic pattern of Bacillus amyloliquefaciens SQR9 and Bacillus enzymolyticus XL8, the metabolites secreted by Bacillus amyloliquefaciens can be metabolized and utilized by Bacillus enzymolyticus XL8.

[0028] Figure 3 The fermentation broth of Bacillus amyloliquefaciens SQR9 and Bacillus enzymolyticus XL8 synergistically antagonized Fusarium oxysporum FOC.

[0029] Figure 4 Pot plant test of plant disease-resistant composite bacterial agent.

[0030] Among them, CTL: no inoculation, F: inoculation with pathogenic Fusarium oxysporum FOC; BF: inoculation with Bacillus amyloliquefaciens SQR9 and Fusarium oxysporum FOC, LF: inoculation with Bacillus enzymolyticus XL8 and Fusarium oxysporum FOC; BLF: inoculation with Bacillus amyloliquefaciens SQR9, Bacillus enzymolyticus XL8 and Fusarium oxysporum FOC. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0032] Example 1: Isolation and identification of disease-resistant strains

[0033] The cucumber rhizosphere growth-promoting Bacillus SQR9, with a strain collection number of CGMCC No. 5808, was isolated in the laboratory in the early stage. Enzyme-producing Lysozyme Bacillus XL8 was screened from the healthy cucumber rhizosphere in soil with Fusarium wilt: the cucumber root system was taken out of the soil, and the rhizosphere soil was collected and shaken at 30°C and 170r / min for 20 minutes to form a soil suspension, and 10 -6 ,10 -7 ,10 -8 0.1 mL of the diluted soil suspension was spread on a TSB plate, with 3 replicates for each concentration. After culturing at 30°C for 3 days, the colonies were selected and streaked on the plate for purification. A bacterial strain named XL8 was obtained. The colonies on the TSB plate were small, light yellow, Gram-negative, and the bacteria were evenly colored. According to the 16S rDNA sequence of the strain, strain XL8 was identified as an enzyme-producing lysozyme bacillus. It was preserved in the Guangdong Provincial Microbial Culture Collection Center with the collection number GDMCC 64920. The preparation method of the TSB culture plate used is, taking the preparation of 1L culture medium as an example: TSB 30g, agar 20g, dilute to 1000mL, pH natural, and sterilize at 115°C for 30min.

[0034] Example 2: Metabolic symtrophic relationship of composite bacterial agents

[0035] The SQR9 and XL8 strains stored in -80℃ glycerol tubes were streaked on TSB solid medium plates for activation and cultured in a 30℃ incubator for 2 days. A single SQR9 colony was picked up and placed in a 5mL liquid TSB test tube, cultured overnight at 30℃ and 170rpm, and 2mL of cells were collected. Centrifuged at 6000rpm for 2min, and the cells were resuspended in 0.9% sodium chloride solution and the OD was adjusted. 600=1, inoculated with 1% (v / v) inoculation in 100mL M9 glucose medium, 30℃, 170rpm shaking culture for 4 days. Collect 80mL SQR9 fermentation broth, centrifuge at 6000rpm for 5min, filter and sterilize the supernatant, which is the SQR9 metabolite, and store it in a 4℃ refrigerator. Pick XL8 single colony in 5mL liquid TSB test tube, 30℃, 170rpm shaking culture overnight, collect 2mL of bacteria, centrifuge at 6000rpm for 2min, resuspend the bacteria with 0.9% sodium chloride solution, and adjust OD 600 =1, inoculated with 1% (v / v) inoculum in 10mL SQR9 metabolites, and cultured at 30°C and 170rpm for 4 days. Collect 8mL XL8 fermentation broth, centrifuge at 6000rpm for 5min, filter and sterilize the supernatant, which is the metabolite of XL8 ​​after growth using SQR9 metabolites, and temporarily store in a 4°C refrigerator. The metabolites were sent to Beijing Novogene Technology Co., Ltd. for non-targeted metabolomics detection to compare differential metabolites. The preparation method of the M9 glucose culture medium used is as follows: taking the preparation of 1L culture medium as an example: 11.28g of 5×M9 inorganic salt powder, 2g of glucose, dilute to 1000mL, natural pH, and sterilize at 115°C for 30min. See the results. Figure 2 Lysobacterium XL8 can utilize a variety of metabolites of Bacillus SQR9 for growth, including 2-morpholine-1-phenyl-1-ethanol, D-phenylalanine, benzathine penicillin G, sodium oleoyl-L-α-lysophosphatidic acid, 5-hydroxyindole, L-prolyl-L-leucine, methyl-3-hydroxybutyric acid, deoxycholic acid, prolylvaline, bisphenol G, ergocrine, norbuprenorphine, etc.

[0036] Example 3: Test on the antagonism of pathogenic bacteria by fermentation broth of composite bacterial agent

[0037] Preparation of fermentation broth: SQR9 and XL8 strains stored in -80℃ glycerol tubes were streaked on TSB solid medium plates for activation and cultured in a 30℃ incubator for 2 days. Single colonies of SQR9 and XL8 were picked up in 5mL liquid TSB test tubes, cultured at 30℃, 170rpm and shaking overnight, 2mL of cells were collected, centrifuged at 6000rpm for 2min, resuspended in 0.9% sodium chloride solution, and OD was adjusted. 600=1. SQR9 and XL8 were inoculated into 100mL Landy medium and 100mL NA medium at 1% (v / v), respectively, and cultured at 30°C and 170rpm for 36h. The SQR9 bacterial solution was centrifuged at 4°C and 11000rpm for 15min, filtered and sterilized with a 0.45μm sterile filter membrane, and 6mol / L hydrochloric acid was added to adjust the pH to 2.0, and placed at 4°C overnight. Then centrifuged at 8000rpm for 10min, the precipitate was dried, extracted with 3mL methanol, and filtered and sterilized with a 0.22μm sterile filter membrane to obtain SQR9 fermentation liquid. The XL8 bacterial solution was centrifuged at 4°C and 11000rpm for 15min, filtered and sterilized with a 0.22μm sterile filter membrane to obtain XL8 fermentation liquid. The Landy medium used is prepared as follows: 20 g glucose, 5 g L-glutamic acid, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.5 g potassium chloride, 1 g yeast powder, 5 mg manganese sulfate, 0.16 mg copper sulfate, 0.15 mg iron sulfate, 2 mg L-phenylalanine, 1 L distilled water, pH 7.0. The NA medium used is prepared as follows: 10 g glucose, 3 g beef extract, 0.5 g yeast powder, 5 g peptone, 1 L distilled water, pH 7.0.

[0038] Oxford cup confrontation experiment: 2 μL of Fusarium oxysporum FOC spore liquid was drawn from the glycerol tube and spotted in the center of the PDA plate. After culturing at 28°C for 2 days, an Oxford cup was placed 3 cm away from the colony. Four treatments were set up: (1) CTL treatment: 100 μL of methanol was added to the Oxford cup as a negative control; (2) SQR9 treatment: 100 μL of SQR9 fermentation liquid was added to the Oxford cup; (3) XL8 treatment: 100 μL of XL8 ​​fermentation liquid was added to the Oxford cup; (4) SQR9+XL8 treatment: 50 μL of SQR9 fermentation liquid and 50 μL of XL8 ​​fermentation liquid were added to the Oxford cup. The plate was cultured at 28°C for 4 to 7 days, and photos were taken to record the antagonism of the strain to pathogens, and the size of the inhibition zone was measured. Figure 3 As shown, both SQR9 fermentation broth and XL8 fermentation broth can inhibit the mycelial growth of the pathogen Fusarium oxysporum to form an antagonistic zone, and the inhibitory effect of the double-strain fermentation broth is significantly higher than that of the single-strain fermentation broth.

[0039] Example 4: Plant disease resistance test of composite bacterial agent

[0040] The cucumber pot experiment was conducted at the Baima Experimental Base of Nanjing Agricultural University from July 1 to August 31, 2023. The cucumber variety for the test was Jinchun No. 4, purchased from Jiangsu Academy of Agricultural Sciences. The cucumber seeds were surface disinfected with 2% sodium hypochlorite solution for 10 min, then soaked in a 30°C incubator for 8 h, and then placed in a 30°C constant temperature box for germination.

[0041] After the seeds germinate, select plump seeds with uniform germination and sow them in a plug tray filled with seedling medium, one seed per hole. After sowing, place the plug tray in a greenhouse with good light, and keep water regularly according to the state of the medium to create an environment suitable for cucumber growth. After 14 days, select seedlings with uniform growth for potted experiments. The experiment set up five treatments: (1) CTL treatment, no inoculation; (2) F treatment, inoculation with pathogen Fusarium oxysporum FOC; (3) BF treatment, adding single bacteria Bacillus amyloliquefaciens SQR9 inoculant and pathogen Fusarium oxysporum FOC; (4) LF treatment, adding single bacteria enzyme-producing Bacillus lysozyme XL8 inoculant and pathogen Fusarium oxysporum FOC; (5) BLF treatment, adding composite bacteria (Bacillus SQR9 + Bacillus lysozyme XL8) and pathogen Fusarium oxysporum FOC. Each treatment had 10 pots, each pot was filled with 2 kg of soil, and the application rate of pathogens was 1×10 6 CFU / g soil, the dosage of the microbial agent is 5×10 7 CFU / g soil. Routine management was followed. After 40 days, plant height and fresh weight were measured, plant morbidity was counted, and rhizosphere soil was collected. 0.25 g of rhizosphere soil was taken, soil bacterial DNA was extracted using the QIAGEN kit, and the number of Fusarium oxysporum in the rhizosphere soil was quantified using the qPCR method.

[0042] Results Figure 4 , applying a composite inoculant (BLF) consisting of Bacillus amyloliquefaciens SQR9 and Bacillus lysozyme XL8 to the rhizosphere of cucumber can significantly improve the growth and health of cucumber under Fusarium oxysporum infection. Compared with the control group (F) inoculated with Fusarium oxysporum alone, the inoculation of Bacillus amyloliquefaciens SQR9 alone (BF) and the inoculation of Bacillus lysozyme XL8 alone (LF) can significantly increase the plant height and fresh weight of cucumber and reduce the incidence of Fusarium wilt. In contrast, after the application of the composite inoculant, the plant height and fresh weight of cucumber were not significantly different from those of the single inoculation, but the incidence rate was reduced to 0%, indicating that the composite inoculant has the best disease resistance effect.

[0043] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A plant disease-resistant probiotic composite bacterial agent based on metabolic syntrophy, characterized in that: The composite bacterial agent is composed of a mixture of bacterial liquids of Bacillus velezensis SQR9 and Lysobacter enzymogenes XL8, wherein the Bacillus velezensis SQR9 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on February 27, 2012, with a deposit number of CGMCC NO.5808, and the Lysobacter enzymogenes XL8 is deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 29, 2024, with a deposit number of GDMCC NO: 64920.

2. The composite bacterial agent according to claim 1, characterized in that The composite bacterial agent is prepared from bacterial liquids of Bacillus velez SQR9 and Bacillus enzymolyticus XL8 in a volume ratio of 1:0.5-5.

3. The composite bacterial agent according to claim 2, characterized in that: The composite bacterial agent is prepared from bacterial liquids of Bacillus velez SQR9 and Bacillus enzymolyticus XL8 in a volume ratio of 1:

1.

4. Use of the plant disease-resistant probiotic composite bacterial agent according to claim 1 in promoting the growth of cucumbers.

5. Use of the plant disease-resistant probiotic composite bacterial agent according to claim 1 in the preparation of a product for promoting cucumber growth.

6. Use of the plant disease-resistant probiotic composite bacterial agent according to claim 1 in preventing and controlling cucumber wilt disease.

7. Use of the plant disease-resistant probiotic composite bacterial agent according to claim 1 in the preparation of products for preventing and controlling cucumber wilt disease.

8. The method for preparing the plant disease-resistant probiotic composite bacterial agent according to claim 1, characterized in that: The plant probiotic composite bacterial agent is prepared by the following method: the activated strains SQR9 and XL8 are inoculated into a tryptone soy broth liquid culture medium at 25-35°C and 150-200 r min-1 for 15-30 hours; the bacterial cells are obtained by centrifugation and washing, and the bacterial cells are resuspended with an equal amount of 0.9% sodium chloride; the bacterial solution OD600 is adjusted to 2, and the two bacterial solutions are mixed in equal amounts to obtain the plant growth-promoting and disease-resistant composite bacterial agent.

9. The preparation method according to claim 7, characterized in that: The tryptone soy broth liquid culture medium is as follows: 30 g of TSB powder, fixed to 1000 mL, and sterilized at 115° C. for 30 min.

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