Synthetic flora, complex microbial inoculant thereof and application of synthetic flora in prevention and treatment of bacterial wilt

Abstract

The invention discloses a synthetic flora, a complex microbial inoculant thereof and application of the complex microbial inoculant in bacterial wilt prevention and treatment, and aims to solve the technical problems that the current bacterial wilt prevention and treatment effect is poor and the resistance of plants to ralstonia solanacearum is low. According to the application, the Rohe bacillus and the paenibacillus separated from a plant root system subjected to high-calcium treatment and the pseudomonas aeruginosa EZ-35 separated in the early stage can antagonize the ralstonia solanacearum and have different degrees of preference ability to calcium nutrition, and the three strains are matched and combined into a synthetic flora, so that the resistance of the plant to the ralstonia solanacearum can be enhanced; furthermore, the synthetic flora is combined with a calcium fertilizer to prevent and treat bacterial wilt, so that the morbidity can be remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the field of biological control technology, and specifically to a synthetic bacterial community, a composite bacterial agent thereof, and application in the prevention and treatment of bacterial wilt disease. Background Art

[0002] Tomato bacterial wilt is a worldwide soil-borne bacterial disease caused by Ralstonia solanacearum ( Ralstonia solanacearum ), mainly invades through plant roots, blocks vascular bundles, causes plants to wilt due to lack of water and even die. Due to its special infection route, it cannot be controlled by chemical pesticides. At present, the prevention and control methods for bacterial wilt mainly adopt biological control, agricultural control and the use of disease-resistant varieties to prevent and control bacterial wilt. Biological control is to control the development of diseases through beneficial microorganisms and metabolites. It is safe and has no residual toxicity when used, but it also faces the problem of poor prevention and control effects due to the changeable field environment. Agricultural prevention and control is mainly carried out through improving tillage and cultivation measures, soil fumigation and application of organic soil conditioners, but it is often limited by high cost investment and large differences in soil types in different regions, resulting in low prevention and control efficiency. The application of disease-resistant varieties is theoretically considered to be an effective and widely used prevention and control measure for bacterial wilt, but the screening of disease-resistant varieties takes a long time, and there are very few effective tomato variety resources resistant to bacterial wilt in current agricultural production, which is far from meeting the needs of actual agricultural production. Inorganic nutrient management, as a green and safe agricultural management method, is also effective in preventing and controlling crop diseases. However, the beneficial microbial resources in plant roots based on nutrient management have yet to be explored.

[0003] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0004] After long-term research, the inventors of this application found that the application of calcium fertilizer can significantly reduce the incidence of tomato bacterial wilt. Compared with no calcium fertilizer, when the calcium fertilizer application amount is 200 mg / kg, the incidence of tomato bacterial wilt is reduced by 63% in terms of pure calcium. When the calcium fertilizer application amount is greater than or equal to 300 mg / kg, the tomato plants do not show symptoms of bacterial wilt. At the same time, the inventors screened and isolated two strains (Bacillus rhodopsinus and Paenibacillus subtilis) that can antagonize Ralstonia solanacearum from the tomato roots treated with high calcium (800 mg / kg). Both strains were deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC) on November 12, 2024. The deposit number of Bacillus rhodopsinus is CGMCC NO. 32599, and the deposit number of Paenibacillus subtilis is CGMCC NO. 32600. In addition, the inventors found that the deposit number of the strain obtained by screening in the early stage is CGMCC NO. Pseudomonas aeruginosa EZ-35 of 20621 (the strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 9, 2020; see CN112522133A) also has an antagonistic effect on Ralstonia solanacearum, and it has a synergistic effect with the above-mentioned Bacillus rhodioides and Paenibacillus. The combination of the three with calcineurin can also significantly improve the prevention effect against Ralstonia solanacearum.

[0005] Based on this, the present disclosure provides, on the one hand, a synthetic bacterial community and a composite bacterial agent thereof, aiming to solve the technical problem of low plant resistance to bacterial wilt; on the other hand, provides a method for preventing and controlling bacterial wilt, aiming to solve the technical problem of poor current prevention and control effect of bacterial wilt.

[0006] According to one aspect of the present disclosure, there is provided a synthetic bacterial community, the synthetic bacterial community comprising Bacillus roxburghii with a deposit number of CGMCCNO. 32599 ( Rhodanobacter glycinis ), Bacillus sp. with a deposit number of CGMCC NO. 32600 ( Paenibacillus polymyxa ) and Pseudomonas aeruginosa with a deposit number of CGMCC NO. 20621 ( Pseudomonas aeruginosa ).

[0007] In some embodiments of the present disclosure, the concentration ratio of the Bacillus roxburghii, the Bacillus spp. and the Pseudomonas aeruginosa is 0.8~1.2: 0.8~1.2: 0.8~1.2.

[0008] According to a second aspect of the present disclosure, a composite bacterial agent is provided, comprising the above-mentioned synthetic bacterial flora and / or its metabolites.

[0009] According to the third aspect of the present disclosure, the synthetic bacterial community, at least one strain in the synthetic bacterial community, or the composite bacterial agent is used in any one of the following (1) to (6): (1) Preventing and controlling bacterial wilt or preparing products for preventing and controlling bacterial wilt; (2) Inhibiting Ralstonia solanacearum or preparing products that inhibit Ralstonia solanacearum; (3) Enhancing the resistance of plants to Ralstonia solanacearum or preparing products that enhance the resistance of plants to Ralstonia solanacearum; (4) Reducing the colonization of bacterial wilt in the rhizosphere of plants or preparing products that reduce the colonization of bacterial wilt in the rhizosphere of plants; (5) Products used in combination / multiple use with calcium to enhance the control of bacterial wilt or the inhibition of bacterial wilt; (6) Products used in combination with calcium to enhance plant resistance to Ralstonia solanacearum.

[0010] According to a fourth aspect of the present disclosure, the active ingredient is a product of any one of the following A to D: A, at least one strain of the synthetic flora; B, calcitriol and at least one strain of the synthetic flora; C, the composite bacterial agent; D, calcium and the composite bacterial agent; The product has at least one of the following functions (1) to (4): (1) Prevent and control bacterial wilt; (2) Inhibit Ralstonia solanacearum; (3) Enhance plant resistance to Ralstonia solanacearum; (4) Reduce the colonization of bacterial wilt in the plant rhizosphere.

[0011] According to a fifth aspect of the present disclosure, there is provided a method for preventing and controlling bacterial wilt, comprising the following steps: Before sowing crops, apply calcium fertilizer to the soil topsoil at a rate of 80-100 mg / kg·soil of pure calcium; When the crop grows to the three-leaf stage, the synthetic bacterial flora or the composite bacterial agent is inoculated or applied to the root of the crop.

[0012] In some embodiments of the present disclosure, the calcium fertilizer includes at least one of CaCl2, Ca(NO3)2, CaSO4, and slaked lime.

[0013] In some embodiments of the present disclosure, the bacterial concentration of the synthetic bacterial flora or composite bacterial agent is 10 8 ~10 9 CFU / mL.

[0014] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. The application of calcium fertilizer can significantly reduce the incidence of tomato bacterial wilt. Some beneficial microbial resources (Rochebacterium rotundum) were isolated from the roots of plants treated with high calcium (800 mg / kg). Rhodanobacter glycinis , PaenibacillusPaenibacillus polymyxa ), and Pseudomonas aeruginosa EZ-35, can all antagonize Ralstonia solanacearum and have a certain preference for calcium. Combining the three into a synthetic bacterial community or further forming a composite bacterial agent and applying it to plants can resist the invasion of Ralstonia solanacearum and enhance the plant's resistance to Ralstonia solanacearum.

[0015] 2. Since Bacillus, Paenibacillus and Pseudomonas aeruginosa have different degrees of preference for calcium nutrition, the combination of calcium and synthetic bacteria significantly improved the control effect of bacterial wilt disease.

[0016] 3. This application study has guiding significance for strengthening calcium nutrient management in agricultural production and preventing and controlling crop diseases and insect pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the statistical results of the incidence of tomato bacterial wilt under different calcium application rates in one embodiment of the present application.

[0018] Figure 2 In one embodiment of the present application, the Rhodanobacter glycinis , Paenibacillus Paenibacillus polymyxa and Pseudomonas aeruginosa Pseudomonas aeruginosa Comparison of the effects against Ralstonia solanacearum; Figure 3 In one embodiment of the present application, the Rhodanobacter glycinis , Paenibacillus Paenibacillus polymyxa and Pseudomonas aeruginosa Pseudomonas aeruginosa Statistical results of calcium nutrition preference; different lowercase letters in the figure represent significant differences between the two. p <0.05.

[0019] Figure 4 The statistical results of the incidence of bacterial wilt of tomatoes under the treatment of calcium supplementation and synthetic bacterial flora in one embodiment of the present application; different lowercase letters in the figure represent significant differences between the two. p <0.05. DETAILED DESCRIPTION

[0020] The specific implementation methods of the present invention are described below in conjunction with examples, but the following examples are only used to illustrate the present application in detail and do not limit the scope of the present application in any way.

[0021] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents unless otherwise specified; the detection methods involved are all conventional methods unless otherwise specified.

[0022] Example 1: Effect of applying calcium fertilizer on the prevention and treatment of tomato bacterial wilt The pot experiment was conducted at the experimental base of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Langfang City, Hebei Province from April 29 to June 26, 2021. Seven calcium fertilizer application gradients were set up: Ca0 (0 mg / kg), Ca50 (50 mg / kg), Ca100 (100 mg / kg), Ca200 (200 mg / kg), Ca300 (300 mg / kg), Ca400 (400 mg / kg), Ca800 (800 mg / kg), and each treatment was repeated 12 times. The calcium fertilizer used was calcium chloride. In addition, 150 mg / kg of urea and KH2PO4 were added. All fertilizers were added to each pot in a water-soluble form at one time. After one night, the soil was mixed. After one week, the tomato seedlings in the three-leaf stage were transplanted into the pots, and one tomato seedling was planted in each pot. The pathogenic strain P380 (provided by the laboratory of Wei Hailei, a researcher at the Institute of Resource Planning, Chinese Academy of Agricultural Sciences) was selected for solanacearum expansion culture using NB liquid medium (3 g beef extract, 10 g glucose, 5 g tryptone, 0.5 g yeast extract, 1 L deionized water, pH 7.0) and cultured at 28°C and 200 rpm for 48 h. The OD 600 value to 1, making about 10 9 CFU / mL of bacterial suspension for later use. When the tomato plant grows the sixth leaf, use a blade to pierce the soil layer about 2 cm away from the root of the plant to injure the root, and then pour in 50 mL of bacterial wilt suspension. From the first appearance of disease symptoms in the tomato plant, the incidence rate of tomato plants is counted every 2 days. In the later stage, the statistical interval is shortened to 1 day depending on the disease situation. Tomato plant incidence rate (%) = (number of diseased plants / total number of surveyed plants) × 100%. The statistical results of the incidence rate of tomato bacterial wilt under different calcium application rates are shown in the following figure. Figure 1 The results showed that compared with no calcium fertilizer application, when the calcium fertilizer application amount was 200 mg / kg, the incidence of tomato bacterial wilt was reduced by 63%. When the calcium fertilizer application amount was greater than or equal to 300 mg / kg, the tomato plants did not show symptoms of bacterial wilt, indicating that the application of calcium fertilizer can significantly reduce the incidence of tomato bacterial wilt.

[0023] Example 2: Isolation, identification and functional verification of beneficial microorganisms 1. Separation: Take the roots of healthy tomato plants treated with Ca800 in Example 1, rinse the surface soil with deionized water, and then wipe the moisture on the surface of the roots with absorbent paper. Take 1 g of fresh tomato roots, disinfect them with 75% ethanol for 30 seconds and 5% sodium hypochlorite solution for 2 minutes, and then wash them with sterile water for 3 times; then cut the roots into small pieces, put them in a mortar, add 9 mL of PBS solution, grind them thoroughly, and then filter the homogenate with 4 layers of sterile gauze and let it stand for 15 minutes. Use the gradient dilution coating method to dilute the supernatant after standing step by step and spread it on a 1 / 4 R2A plate, pick a single colony and purify it on LB medium to obtain a single colony.

[0024] 2. Identification: The selected strains were cultured on LB solid medium, and their physiological and biochemical characteristics such as colony characteristics and color were observed according to the Manual of Identification of Common Bacterial Systems; PCR amplification was performed using bacterial universal primers GM3F and GM4R to amplify a 1200 bp product, which was sequenced and sequenced using Blast.

[0025] 3. Functional Verification (1) Verification of antagonism to Ralstonia solanacearum: Prepare a culture medium containing Ralstonia solanacearum, add 10 mL of overnight cultured Ralstonia solanacearum suspension to 250 mL of NA solid culture medium that has been sterilized at high temperature and cooled to about 55°C, shake well and pour the plate, wait for the plate to solidify, add 3 μL of overnight cultured suspension of the above isolated bacteria and Pseudomonas aeruginosa EZ-35 suspension in the center of the plate, and culture at 28°C for 2 days. Two of the isolated bacteria (collected by Bi Jingjing; collected at Langfang City, Hebei Province; collected in July 2021) have antagonistic effects on Ralstonia solanacearum, and were identified as Rhodococcus ( Rhodanobacter glycinis )、Paenibacillus( Paenibacillus polymyxa ); The overall verification results showed that Bacillus roxburghii, Paenibacillus and Pseudomonas aeruginosa could inhibit the growth of Ralstonia solanacearum to varying degrees, and all showed antagonistic ability to Ralstonia solanacearum (such as Figure 2 as shown).

[0026] (2) Verification of calcium preference: NB liquid culture medium containing 0 (Ca0), 0.692 (Ca100), 1.385 (Ca200), 2.769 (Ca400), 4.154 (Ca600), and 5.539 (Ca800) g / L CaCl2 solution was prepared and cultured in a 96-well culture plate. 2 μL of the above-mentioned bacterial suspension cultured overnight and Pseudomonas aeruginosa EZ-35 suspension were added to 198 μL of the culture medium, respectively. The culture was carried out at 30°C and 160 rpm for 4, 12, and 24 h, and the OD was measured using a multifunctional microplate reader. 600Value, the result is Figure 3 As shown, different strains all showed a preference for calcein.

[0027] The raw material formula of the above culture medium is: 1 / 4 R2A medium: yeast extract 0.125 g, tryptone 0.125 g, casein hydrolysate 0.125 g, glucose 0.125 g, soluble starch 0.125 g, sodium pyruvate 0.075 g, K2HPO4 0.075 g, MgSO4·7H2O 0.0125 g, agar 15 g, deionized water 1 L, pH 7.2±0.2.

[0028] LB medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 15 g agar, 1 L deionized water, pH 7.0.

[0029] NA solid medium: 3 g beef extract, 10 g glucose, 5 g tryptone, 0.5 g yeast extract, 15 g agar, 1 L deionized water, pH 7.0.

[0030] Example 3: Effect of calcium and synthetic bacteria combined to control tomato bacterial wilt This example is based on the beneficial microorganisms (Rochebacterium Rhodanobacter glycinis :Bacillus Paenibacillus polymyxa :Pseudomonas aeruginosa Pseudomonas aeruginosa =1:1:1) functional verification results, in this example, the three strains were combined into a synthetic bacterial consortium at a concentration ratio of 1:1:1, and used in combination with calcium to prevent and treat tomato bacterial wilt.

[0031] Specifically, tomato seeds were germinated at 28°C for 2 days and then sown in seedling trays containing 80 g of seedling medium. Four treatments were set up: Ca0 (no calcium fertilizer), Ca0 + synthetic flora, Ca100 (100 mg / kg calcium fertilizer), and Ca100 + synthetic flora. The synthetic flora was based on the Rhodeobacterium spp. Rhodanobacter glycinis :Bacillus Paenibacillus polymyxa :Pseudomonas aeruginosa Pseudomonas aeruginosa The calcium fertilizer was applied before sowing tomatoes. When tomatoes reached the three-leaf stage, the roots of all treated tomato plants were inoculated with a suspension of Ralstonia solanacearum. One week later, the synthetic bacterial flora (10 9 CFU / mL), the statistical results of the incidence of tomato bacterial wilt are as follows Figure 4 The results showed that the application of synthetic bacteria or calcium fertilizer (CaCl2) alone can significantly reduce the incidence of bacterial wilt. If calcium fertilizer is applied in combination with synthetic bacteria, the incidence of tomato plants can be extremely significantly reduced.

[0032] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0033] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A synthetic bacterial community, characterized in that The synthetic bacterial flora includes Luohe Bacillus with a deposit number of CGMCC NO. 32599 ( Rhodanobacter glycinis ), Bacillus with the deposit number of CGMCC NO.32600 ( Paenibacillus polymyxa ) and Pseudomonas aeruginosa with a deposit number of CGMCC NO. 20621 ( Pseudomonas aeruginosa ).

2. The synthetic bacterial flora according to claim 1, characterized in that The concentration ratio of the Bacillus roxburghii, the Bacillus spp. and the Pseudomonas aeruginosa is 0.8~1.2: 0.8~1.2: 0.8~1.

2.

3. A composite bacterial agent, characterized in that: Containing the synthetic bacterial flora and / or its metabolites as claimed in claim 1.

4. Use of the synthetic bacterial flora of claim 1, at least one strain of the synthetic bacterial flora, or the composite bacterial agent of claim 2 in any one of the following (1) to (6): (1) Preventing and controlling bacterial wilt or preparing products for preventing and controlling bacterial wilt; (2) Inhibiting Ralstonia solanacearum or preparing products that inhibit Ralstonia solanacearum; (3) Enhancing the resistance of plants to Ralstonia solanacearum or preparing products that enhance the resistance of plants to Ralstonia solanacearum; (4) Reducing the colonization of bacterial wilt in the rhizosphere of plants or preparing products that reduce the colonization of bacterial wilt in the rhizosphere of plants; (5) Products used in combination / multiple use with calcium to enhance the control of bacterial wilt or the inhibition of bacterial wilt; (6) Products used in combination with calcium to enhance plant resistance to Ralstonia solanacearum.

5. Products whose active ingredient is any one of the following A to D: A. at least one strain of the synthetic bacterial flora according to claim 1; B, calcium and at least one strain of the synthetic bacterial flora according to claim 1; C. The composite bacterial agent according to claim 3; D, calcium and the composite bacterial agent according to claim 3; The product has at least one of the following functions (1) to (4): (1) Prevent and control bacterial wilt; (2) Inhibit Ralstonia solanacearum; (3) Enhance plant resistance to Ralstonia solanacearum; (4) Reduce the colonization of bacterial wilt in the plant rhizosphere.

6. A method for preventing and treating bacterial wilt, characterized in that: The following steps are involved: (1) Apply calcium fertilizer to the soil topsoil at a rate of 80-100 mg / kg of pure calcium per kg of soil before sowing crops; (2) When the crop grows to the three-leaf stage, the synthetic bacterial flora according to claim 1 or the composite bacterial agent according to claim 2 is inoculated or applied to the root of the crop.

7. The method according to claim 6, characterized in that The calcium fertilizer includes at least one of CaCl2, Ca(NO3)2, CaSO4 and slaked lime.

8. The method according to claim 6, characterized in that The bacterial concentration of the synthetic bacterial flora or composite bacterial agent is 10 8 ~10 9 CFU / mL.

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