Synthetic bacterial flora, its complex microbial agent and application in prevention and treatment of bacterial wilt

Abstract

This invention discloses a synthetic microbial community, its compound microbial agent, and its application in the control of bacterial wilt, aiming to solve the technical problems of poor control effect and low plant resistance to bacterial wilt fungus. The present application isolates *L. romaejasminoides* and *Bacillus subtilis* from the roots of plants treated with high calcium, as well as *Pseudomonas aeruginosa* EZ-35 isolated previously, all of which can antagonize bacterial wilt fungus and exhibit varying degrees of preference for calcium nutrition. The combination of these three strains into a synthetic microbial community can enhance plant resistance to bacterial wilt fungus. Furthermore, the combined use of this synthetic microbial community with calcium fertilizer for the control of bacterial wilt can significantly reduce the incidence rate.

Description

Technical Field

[0001] This invention application relates to the field of biological control technology, specifically to a synthetic microbial community, its compound microbial agent, and its application in the control of bacterial wilt. Background Technology

[0002] Bacterial wilt of tomatoes is a worldwide soil-borne bacterial disease caused by Ralstonia solanaceae (Solanaceae). Ralstonia solanacearum Bacterial wilt is caused primarily by invasion through the plant roots, blocking the vascular bundles and leading to water shortage, wilting, and even death. Due to its unique infection route, it cannot be controlled with chemical pesticides. Currently, the main methods for controlling bacterial wilt include biological control, agricultural control, and the use of resistant varieties. Biological control uses beneficial microorganisms and metabolites to control the disease's development; it is safe and leaves no toxic residues, but its effectiveness is limited by variable field environments. Agricultural control mainly involves improving cultivation practices, soil fumigation, and applying organic soil conditioners, but these methods are often limited by high investment costs and significant differences in soil types across regions, resulting in low efficiency. The application of resistant varieties is theoretically considered an effective and widely applicable control measure for bacterial wilt; however, the selection of resistant varieties is time-consuming, and currently, there are very few effective resistant tomato varieties available for agricultural production, far from meeting the needs of actual agricultural production. Inorganic nutrient management, as a green and safe agricultural management method, has also shown some effectiveness in the prevention and control of crop diseases. However, the beneficial microbial resources in plant roots based on nutrient management still need to be explored.

[0003] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0004] Through long-term research, the inventors of this application discovered that applying calcium fertilizer can significantly reduce the incidence of bacterial wilt in tomatoes. Compared with no calcium fertilizer application, when the calcium fertilizer application rate is 200 mg / kg, the incidence of bacterial wilt in tomatoes is reduced by 63%. When the calcium fertilizer application rate 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 (L. roxithorax bacillus and Bacillus subtilis) that can antagonize Ralstonia solanacearum from tomato roots treated with high calcium (800 mg / kg). Both strains were deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 12, 2024. The preservation number of the L. roxithorax bacillus strain is CGMCC NO. 32599, and the preservation number of Bacillus subtilis is CGMCC NO. 32600. In addition, the inventors found that the preservation number obtained in the previous screening was CGMCC NO. Pseudomonas aeruginosa EZ-35 of strain 20621 (this strain was deposited at the China General Microbiological Culture Collection Center on September 9, 2020; see CN112522133A) also has an antagonistic effect against Ralstonia solanacearum, and it has a synergistic effect with the above-mentioned Lorobacterium and Bacillus subtilis. The combination of the three with calcium can also significantly improve the control efficacy against bacterial wilt.

[0005] Based on this, this disclosure provides, on the one hand, a synthetic microbial community and its compound microbial agent, which aims to solve the technical problem of low plant resistance to bacterial wilt; on the other hand, it provides a method for controlling bacterial wilt, which aims to solve the technical problem of poor control effect of current bacterial wilt.

[0006] According to one aspect of this disclosure, a synthetic microbial community is provided, said synthetic microbial community comprising Lorobacterium tumefaciens with accession number CGMCC NO. 32599. Rhodanobacter glycinis Bacillus subtilis with accession number CGMCC NO. 32600 Paenibacillus polymyxa ) and Pseudomonas aeruginosa with accession number CGMCC NO. 20621 ( Pseudomonas aeruginosa ).

[0007] In some embodiments of this disclosure, the concentration ratio of the *Lonicera japonica*, 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 this disclosure, a compound microbial agent is provided, containing the aforementioned synthetic microbial flora and / or its metabolites.

[0009] According to the third aspect of this disclosure, the synthetic microbial community, at least one strain of the synthetic microbial community, or the compound microbial agent is applied in any of the following (1) to (6): (1) To prevent and control bacterial wilt or to prepare products for the prevention and control of bacterial wilt; (2) Inhibit Ralstonia solanacearum or prepare products that inhibit Ralstonia solanacearum; (3) Enhance plant resistance to Ralstonia solanacearum or prepare products that enhance plant resistance to Ralstonia solanacearum; (4) Reduce the amount of bacterial wilt colonization in the rhizosphere of plants or prepare products that reduce the amount of bacterial wilt colonization in the rhizosphere of plants; (5) Products used in combination with calcium to enhance the prevention and control of bacterial wilt or the inhibitory effect of bacterial wilt fungus; (6) Products used in combination with calcium to enhance plant resistance to Ralstonia solanacearum.

[0010] According to the fourth aspect of this disclosure, the product has an active ingredient of any one of the following A to D: A. At least one strain of the synthetic microbial community; B. Calcium and at least one strain of the synthetic microbial community; C. The aforementioned compound microbial agent; D. Calcium and the aforementioned compound microbial agent; The product has at least one of the following functions (1) to (4): (1) Prevention and control of bacterial wilt; (2) Inhibits Ralstonia solanacearum; (3) Enhance plant resistance to Ralstonia solanacearum; (4) Reduce the amount of bacterial wilt colonization in the plant rhizosphere.

[0011] According to a fifth aspect of this disclosure, a method for preventing bacterial wilt is provided, comprising the following steps: Apply calcium fertilizer to the topsoil layer at a rate of 80-100 mg / kg of pure calcium in the soil before sowing the crop; When the crop reaches the three-leaf stage, the synthetic microbial community or the compound microbial agent is inoculated or applied to the roots of the crop.

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

[0013] In some embodiments of this disclosure, the bacterial concentration of the synthetic microbial community or compound microbial agent is 10. 8 ~10 9 CFU / mL.

[0014] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Applying calcium fertilizer can significantly reduce the incidence of bacterial wilt in tomatoes. Beneficial microbial resources (Lorva oryzae) were isolated from the roots of plants treated with high calcium (800 mg / kg). Rhodanobacter glycinis Bacillus subtilisPaenibacillus polymyxa Both *Ralstonia solanacearum* and *Pseudomonas aeruginosa* EZ-35 can antagonize *Ralstonia solanacearum* and have a certain preference for calcium. Combining these three into a synthetic microbial community or further forming a compound microbial agent and applying it to plants can help them resist the invasion of *Ralstonia solanacearum* and enhance their resistance to it.

[0015] 2. Since *L. roximately 2,000*, *Bacillus subtilis*, and *Pseudomonas aeruginosa* have varying degrees of preference for calcium nutrition, combining calcium supplements with synthetic microbial groups significantly improved the control effect of bacterial wilt.

[0016] 3. This research provides guidance for strengthening calcium nutrient management in agricultural production to control crop diseases and pests. Attached Figure Description

[0017] Figure 1 This is a statistical result of the incidence of bacterial wilt in tomatoes under different calcium application rates in one embodiment of this application.

[0018] Figure 2 Loropetalum paraguayense is an example of this application. Rhodanobacter glycinis Bacillus subtilis Paenibacillus polymyxa and Pseudomonas aeruginosa Pseudomonas aeruginosa Comparison of the effects of antagonizing Ralstonia solanacearum; Figure 3 Loropetalum paraguayense is an example of this application. Rhodanobacter glycinis Bacillus subtilis Paenibacillus polymyxa and Pseudomonas aeruginosa Pseudomonas aeruginosa Statistical results on calcium nutrition preferences; different lowercase letters in the figure represent significant differences between the two groups. p <0.05.

[0019] Figure 4 This figure shows the statistical results of the incidence of bacterial wilt in tomatoes under the combined treatment of calcium and synthetic microbial flora in one embodiment of this application; different lowercase letters in the figure represent significant differences between the two treatments. p <0.05. Detailed Implementation

[0020] The specific implementation of this application will be described below with reference to the embodiments. However, the following embodiments are only used to illustrate this application in detail and do not limit the scope of this 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; and the detection methods involved are all conventional methods unless otherwise specified.

[0022] Example 1: The effect of calcium fertilizer application on the prevention and control of bacterial wilt in tomatoes. The pot experiment was conducted from April 29 to June 26, 2021, at the experimental base of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Langfang City, Hebei Province. 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), and Ca800 (800 mg / kg), with 12 replicates for each treatment. 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 water-soluble form at once. After letting it sit overnight, the fertilizer was mixed into the soil. One week later, tomato seedlings at the three-leaf stage were transplanted into flowerpots, with one tomato seedling per pot. Ralstonia solanacearum strain P380 (provided by the laboratory of Researcher Wei Hailei, Institute of Resource Regionalization, Chinese Academy of Agricultural Sciences) was selected for large-scale cultivation 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). The culture was carried out at 28℃ and 200 rpm with shaking for 48 h, and the OD was adjusted. 600 Value up to 1, making approximately 10 9 Prepare a bacterial suspension of CFU / mL. When the tomato plant has grown its sixth leaf, make a cut in the soil about 2 cm from the root to damage the root, and then infuse 50 mL of the bacterial suspension. From the first appearance of symptoms, count the incidence rate of tomato plants every 2 days. Later, shorten the interval to 1 day depending on the severity of the disease. Incidence rate (%) = (number of infected plants / total number of plants surveyed) × 100%. The statistical results of the incidence rate of bacterial wilt in tomatoes under different calcium application rates are shown below. Figure 1 As shown, the results indicate that compared with no calcium fertilizer application, the incidence of bacterial wilt in tomatoes decreased by 63% when the calcium fertilizer application rate was 200 mg / kg, and no bacterial wilt symptoms were observed in tomato plants when the calcium fertilizer application rate was greater than or equal to 300 mg / kg. This suggests that the application of calcium fertilizer can significantly reduce the incidence of bacterial wilt in tomatoes.

[0023] Example 2: Isolation, Identification and Functional Verification of Beneficial Microorganisms 1. Isolation: Roots from healthy tomato plants treated with Ca800 in Example 1 were taken, and the surface soil was rinsed with deionized water. The roots were then wiped dry with absorbent paper. 1 g of fresh tomato roots were surface-sterilized with 75% ethanol for 30 seconds and 5% sodium hypochlorite solution for 2 minutes, followed by rinsing three times with sterile water. The roots were then cut into small pieces, placed in a mortar, and 9 mL of PBS solution was added. The mixture was thoroughly ground, and the homogenate was filtered through four layers of sterile gauze and allowed to stand for 15 minutes. The supernatant was serially diluted and plated onto 1 / 4 R2A plates using a serial dilution method. Single colonies were picked and cultured on LB agar for purification.

[0024] 2. Identification: The selected strains were cultured on LB solid medium, and their colony characteristics and physiological and biochemical features such as color were observed in accordance with the "Handbook of Systematic Identification of Common Bacteria". PCR amplification was performed using universal bacterial primers GM3F and GM4R to amplify a 1200 bp product. Sequencing was performed, and the sequencing results were compared with the sequence using BLAST.

[0025] 3. Functional Verification (1) Verification of antagonistic ability against Ralstonia solanacearum: A culture medium containing Ralstonia solanacearum was prepared. 10 mL of Ralstonia solanacearum suspension cultured overnight was added to 250 mL of NA solid medium that had been sterilized at high temperature and cooled to about 55℃. After shaking well, the medium was poured onto a plate. After the plate solidified, 3 μL of the above-mentioned isolated bacterial suspension cultured overnight and Pseudomonas aeruginosa EZ-35 bacterial suspension were added to the center of the plate. The plate was cultured at 28℃ for 2 days. Two isolates (collector: Bi Jingjing; collection location: Langfang City, Hebei Province; collection time: July 2021) showed antagonistic activity against Ralstonia solanacearum. They were identified as Lorobacterium tumefaciens (Lorobacterium tumefaciens) ... Rhodanobacter glycinis ), Bacillus subtilis ( Paenibacillus polymyxa Overall validation results showed that *Rhizobium glomeratum*, *Bacillus subtilis*, and *Pseudomonas aeruginosa* all inhibited the growth of *Rhizobium glomeratum* to varying degrees, and all exhibited antagonistic ability against *Rhizobium glomeratum* (e.g., *Rhizobium glomeratum*). Figure 2 (As shown).

[0026] (2) Verification of calcium preference ability: NB liquid medium containing 0 (Ca0), 0.692 (Ca100), 1.385 (Ca200), 2.769 (Ca400), 4.154 (Ca600), and 5.539 (Ca800) g / L CaCl2 solutions were prepared and cultured in 96-well plates. 2 μL of the above-mentioned bacterial suspensions and Pseudomonas aeruginosa EZ-35 bacterial suspensions, which had been cultured overnight, were added to 198 μL of the medium, respectively. The plates were incubated at 30℃ and 160 rpm for 4, 12, and 24 h, and the OD was measured using a multifunctional microplate reader. 600Value, result as Figure 3 As shown, different strains all exhibited a preference for calcium.

[0027] The raw material formula for the above culture medium is as follows: 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: The control effect of combined treatment with calcium and synthetic microbial flora on bacterial wilt in tomatoes. This example is based on the beneficial microorganisms (Lornorum bacillus) in Example 2. Rhodanobacter glycinis Bacillus subtilis Paenibacillus polymyxa Pseudomonas aeruginosa Pseudomonas aeruginosa Based on the results of functional verification (1:1:1), this example combines the three strains in a 1:1:1 concentration ratio to form a synthetic bacterial group, and uses it in combination with calcium to control bacterial wilt in tomatoes.

[0031] Specifically, tomato seeds were germinated at 28℃ for 2 days, and then sown in seedling trays containing 80 g of seedling substrate. Four treatments were set up: Ca0 (no calcium fertilizer), Ca0 + synthetic microorganisms, Ca100 (100 mg / kg calcium fertilizer), and Ca100 + synthetic microorganisms. The synthetic microorganisms were classified according to *L. oryzae*. Rhodanobacter glycinis Bacillus subtilis Paenibacillus polymyxa Pseudomonas aeruginosa Pseudomonas aeruginosa The solution was prepared at a concentration ratio of 1:1:1. Calcium fertilizer was applied before tomato sowing. When the tomatoes reached the three-leaf stage, all treated tomato plants were inoculated with a suspension of *Ralstonia solanacearum* at the base of their roots. One week later, synthetic microorganisms (10...) were inoculated. 9 (CFU / mL), statistical results of the incidence of bacterial wilt in tomatoes are as follows: Figure 4 As shown, the results indicate that the application of synthetic mycotoxins or calcium fertilizer (CaCl2) alone can significantly reduce the incidence of bacterial wilt. If calcium fertilizer is applied in combination with synthetic mycotoxins, the incidence of disease in tomato plants can be reduced significantly.

[0032] Although some preferred embodiments of this invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0033] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A complex microbial agent, characterized in that, It consists of the following three strains with a preference for calcium: *Lycium oryzae* with accession number CGMCC NO.32599 (… Rhodanobacter glycinis Bacillus subtilis with accession number CGMCC NO.32600 Paenibacillus polymyxa ) and Pseudomonas aeruginosa with accession number CGMCC NO.20621 ( Pseudomonas aeruginosa All three strains are deposited at the China General Microbiological Culture Collection Center of the China Microbiological Culture Collection Management Committee; the concentration ratio of the *Lonicera japonica*, the *Bacillus* spp., and the *Pseudomonas aeruginosa* is 0.8~1.2:0.8~1.2:0.8~1.2; the compound microbial agent is used in combination with calcium fertilizer.

2. The compound microbial agent according to claim 1, in any one of the following (1) to (3): (1) To prevent and control bacterial wilt or to prepare products for the prevention and control of bacterial wilt; (2) Inhibit Ralstonia solanacearum or prepare products that inhibit Ralstonia solanacearum; (3) Enhance the resistance of plants to Ralstonia solanacearum or prepare products that enhance the resistance of plants to Ralstonia solanacearum.

3. The product whose active ingredient is the compound microbial agent of claim 1 has at least one of the following functions (1) to (3): (1) Prevention and control of bacterial wilt; (2) Inhibits Ralstonia solanacearum; (3) Enhance the plant’s resistance to Ralstonia solanacearum.

4. A method of controlling bacterial wilt, characterized by, Includes the following steps: (1) Apply calcium fertilizer to the soil tillage layer at a rate of 80-100 mg / kg of pure calcium in the soil before sowing the crop; (2) When the crop grows to the three-leaf stage, the compound microbial agent of claim 1 is inoculated or applied to the roots of the crop.

5. The method of claim 4, wherein, The calcium fertilizer includes at least one of CaCl2, Ca(NO3)2, CaSO4, and slaked lime.

6. The method of claim 4, wherein, The concentration of the bacterial body of the complex microbial agent is 10 8 ~10 9 CFU / mL.

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