Paenibacillus polymyxa and application thereof
By using complex bacterial agents, including Bacillus Bacillus and Bacillus polyamide, the secondary salinization and disease prevention and control of facilities' vegetables and fruits have been solved, and the effects of soil improvement and yield improvement have been achieved.
Patent Information
- Application Number
- CN202411503217.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-02-25
AI Technical Summary
The continuous cropping barriers between facilities, including secondary salinization of soil and soil-borne diseases of plants, lead to a decrease in yield and quality, and the long-term use of chemical pesticides has led to increased resistance to pathogenic bacteria and deterioration of soil microbial ecology.
A compound bacterial agent, including Bacillus velezensis Bv-HR6-1 and Bacillus polymyxa XY8-23, was prepared by seed liquid culture, expansion culture and fermentation culture. agent, used to treat secondary salinization of soil and prevent and treat epidemics.
This compound bacteria agent can not only effectively improve the secondary salinization problem of soil, but also significantly improve the disease prevention and control capabilities of facilities vegetables and fruits, improve yield and quality, and at the same time improve soil microecology and reduce the use of chemical pesticides.
Smart Images
Figure CN119506126A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Paenibacillus polymyxa and application thereof. Background Art
[0002] In recent years, the cultivation area of greenhouse tomatoes has been increasing in greenhouse vegetable production. However, many years of continuous cropping will lead to soil continuous cropping obstacles. Continuous cropping obstacles are mainly manifested in secondary soil salinization and plant soil-borne diseases, which seriously affect the yield and quality of tomatoes and seriously restrict the development of the tomato industry. Salinized soils are widely distributed in my country, and the phenomenon of blind fertilization is relatively common, leading to the deterioration of soil physical and chemical properties. For example, soil hardening, unreasonable tillage and irrigation cause soil salinization and other problems, which cause serious harm to plant growth and seriously affect production. Tomato blight is a soil-borne disease caused by Phytophthora capsici and Phytophthora parasitica. It is characterized by rapid occurrence and strong destructiveness, which seriously restricts the development of the tomato industry.
[0003] Nowadays, the management of saline soil requires multiple measures such as controlling moisture, improving structure, soil fertilization, and biological improvement. The core is still the close combination of water and fertilizer, which complement each other. In production, the prevention and control of tomato blight adopts comprehensive prevention and control measures with agricultural prevention as the main and chemical prevention as the auxiliary, which can effectively control the occurrence and spread of the disease. However, the long-term and large-scale use of pesticides has caused the resistance of pathogens to be enhanced, the pesticide residues in vegetables have increased, and the soil microbial ecology has deteriorated. Long-term continuous cropping will seriously damage the physical and chemical properties of the soil, resulting in the continuous accumulation of pathogens in the continuous cropping soil. For more than half a century, the long-term and large-scale use of chemical pesticides has caused great harm to the development of the tomato industry. At present, in order to solve the problem of obstacles to continuous cropping of facility vegetables, research on environmentally friendly biological control is imminent.
[0004] Invention application CN114276965B discloses a Bacillus velezensis Bv-HR6-1, which is deposited in the "General Microbiological Center of China Microbiological Culture Collection Committee" with a deposit number of CGMCC No. 14469; the Bacillus velezensis can be used to prepare microbial agents for improving soil, especially for treating secondary salinization and heavy metal pollution of soil, and can improve plant immunity and regulate soil microecology. Although the above strain can improve secondary salinization of soil, it cannot effectively prevent and treat greenhouse vegetable blight. Summary of the invention
[0005] In view of this, the first object of the present invention is to provide a strain of Paenibacillus polymyxa to effectively improve the disease prevention and control of greenhouse vegetables, fruits and other crops.
[0006] To achieve the above purpose, the technical solution provided by the present invention is: a strain of Paenibacillus polymyxa XY8-23, which was deposited in the "General Microbiological Center of China Microbiological Culture Collection Committee" on August 20, 2024, with a deposit number of CGMCC No. 31696, and a deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The above-mentioned Paenibacillus polymyxa XY8-23 was isolated and screened from the root system of Chlorophytum comosum in Huangchuan County, Henan Province.
[0007] The above-mentioned Paenibacillus polymyxa XY8-23 (hereinafter referred to as "XY8-23") has the following biological characteristics: it forms beige, irregular, translucent, and irregular-edged colonies on TSA culture medium. The bacteria are straight rod-shaped, with flagella around them, are Gram-positive, have a growth temperature of 4 to 50°C, have a maximum salt tolerance of 4% NaCl by mass, can utilize sucrose, glucose, glycerol, maltose, and mannitol, but cannot utilize galactose and tannic acid, are positive in starch hydrolysis, nitrate reduction reaction, and VP determination, and are negative in gelatin liquefaction and H2S production tests. The 16S rRNA gene amplified sequence of Paenibacillus polymyxa XY8-23 is 1492 bp in length. The BLAST comparison results using NCBI show that the homology with the corresponding 16S rRNA sequence of Paenibacillus polymyxa reaches 99%. The antibacterial spectrum of Bacillus polymyxa XY8-23 strain is relatively broad, and it has strong antibacterial activity against a variety of plant pathogens. In addition, Bacillus polymyxa XY8-23 strain can decompose organic phosphorus, protein, starch and cellulose, and has the ability to produce siderophore, glucanase and indoleacetic acid.
[0008] The second purpose of the present invention is to provide a composite bacterial agent to control the problems of secondary salinization of soil and continuous cropping of vegetable and fruit plants in facilities such as blight. Specifically, a composite bacterial agent includes Bacillus velezensis Bv-HR6-1 and the above-mentioned Paenibacillus polymyxa XY8-23, wherein the taxonomic name of the Bacillus velezensis Bv-HR6-1 is Bacillus velezensis, and the deposit number is CGMCC No.14469, deposited in the General Microbiology Center of China Microbiological Culture Collection Committee; deposit date: July 27, 2017; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.
[0009] Furthermore, while solving the problem of continuous cropping of greenhouse vegetables, the yield and quality of greenhouse vegetables, fruits and other crops are effectively improved. In the composite microbial agent, the concentration of Bacillus velezensis Bv-HR6-1 is 2×10 10 -1×10 12 cfu / g, preferably 2×10 11 -1×10 12 / cfu / g; the colony forming unit concentration of Paenibacillus polymyxa XY8-23 is 2×10 9 -6×10 10 cfu / g, preferably 3×10 10 -6×10 10 cfu / g.
[0010] Preferably, the composite bacterial agent is in powder form, so that it is convenient to apply. In order to further improve the vitality and growth of the strain and have a stable storage capacity; the carrier used to culture the strain in the composite bacterial agent is bean dregs, which contains about 45-55% dietary fiber, 20-30% protein, 7-12% lipid and 1%-2% carbohydrate; preferably, the pH value of the carrier is 6.5-7.5.
[0011] The third object of the present invention is to provide a method for preparing a composite bacterial agent, which is simple to operate, saves time and effort, has low cost, and has wide applicability. Specifically, a method for preparing a composite bacterial agent comprises the steps of:
[0012] Seed solution culture: Bacillus velez Bv-HR6-1 and Paenibacillus polymyxa XY8-23 were cultured separately to obtain Bv-HR6-1 seed solution and XY8-23 seed solution;
[0013] Propagation culture: performing propagation culture on the Bv-HR6-1 seed solution and the XY8-23 seed solution to obtain Bv-HR6-1 fermentation solution and XY8-23 fermentation solution, respectively;
[0014] Preparation of composite bacterial agent: ferment the Bv-HR6-1 fermentation broth and the XY8-23 fermentation broth separately to obtain Bv-HR6-1 fermentation bacterial agent and XY8-23 fermentation bacterial agent, and evenly mix the Bv-HR6-1 fermentation bacterial agent and the XY8-23 fermentation bacterial agent to obtain the composite bacterial agent.
[0015] The step of seed liquid culture comprises: inoculating Bv-HR6-1 mother strain and XY8-23 mother strain into seed liquid culture medium respectively, and culturing for 16-24 hours at a temperature of 25-30°C and 140-200r / min, to obtain Bv-HR6-1 seed liquid and XY8-23 seed liquid respectively. Preferably, the seed liquid culture medium comprises: 20g / L peptone, 5g / L yeast extract, 0.5g / L sodium chloride, 10mL / L potassium chloride (0.25mol / L), 5mL / L magnesium chloride (2mol / L), 20mL / L glucose (1mol / L); preferably, the pH value of the seed liquid culture medium is 6.0-8.0.
[0016] Wherein, the Bv-HR6-1 mother strain and the XY8-23 mother strain are both obtained by streaking culture on a solid culture medium at a temperature of 28°C for 24-96 hours; preferably, the solid culture medium comprises: 17 g / L trypticase, 3 g / L soybean papain hydrolysate, 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, 18 g / L agar, and a pH value of 7.3±0.2.
[0017] The multiplication culture includes two multiplication cultures, specifically including:
[0018] First propagation: inoculating the Bv-HR6-1 seed solution and the XY8-23 seed solution into the first liquid culture medium respectively, and the inoculation amount is 0.5%-2.0% of the volume of the liquid culture medium, and fermenting and culturing at 28-32° C. for 36-50 hours to obtain Bv-HR6-1 bacterial solution and XY8-23 bacterial solution;
[0019] Secondary propagation: The Bv-HR6-1 bacterial solution and the XY8-23 bacterial solution are inoculated into the second liquid culture medium respectively, and the inoculation amount is 5%-15% of the volume of the liquid culture medium, and fermented and cultured at 22-35°C and 120-200rpm for 16-48h to obtain Bv-HR6-1 fermentation liquid and XY8-23 fermentation liquid. The pH value of the second liquid culture medium is preferably 6-8, more preferably 6.5-7.5.
[0020] In the propagation culture, the first liquid culture medium and the second liquid culture medium both include: 20 g / L peptone, 5 g / L yeast extract, 0.5 g / L sodium chloride, 10 mL / L potassium chloride (0.25 mol / L), and 5 mL / L magnesium chloride (2 mol / L); preferably, the pH value of the liquid culture medium is 6.0-8.0.
[0021] Preferably, during the secondary expansion culture process, the inoculum amount of Bv-HR6-1 is 12.5% of the volume of the second liquid culture medium, and the inoculum amount of XY8-23 is 15% of the volume of the second liquid culture medium.
[0022] When the composite bacterial agent is a powder, the steps of preparing the composite bacterial agent include: fully mixing the Bv-HR6-1 fermentation liquid and the XY8-23 fermentation liquid with bean dregs, respectively, and fermenting and culturing for 10-15 days, drying, and crushing to obtain Bv-HR6-1 fermentation bacterial powder and XY8-23 fermentation bacterial powder; uniformly mixing the Bv-HR6-1 fermentation bacterial powder and the XY8-23 fermentation bacterial powder to obtain the composite bacterial agent. Preferably, the number of viable bacteria in the Bv-HR6-1 fermentation bacterial powder is 1×10 12 -1×10 13 cfu / g; the viable count of the XY8-23 fermentation bacteria powder is 1×10 10 -1×10 11 Preferably, the mass ratio of the Bv-HR6-1 fermentation bacteria powder to the XY8-23 fermentation bacteria powder is 1:2-2:1.
[0023] The fourth object of the present invention is to provide a composite bacterial agent seedling matrix, which is mainly prepared by uniformly mixing the composite bacterial agent and the seedling matrix at a ratio of 6-18 g / L. The seedling matrix is an existing seedling matrix for crops such as vegetables and fruits.
[0024] The fifth object of the present invention is to provide an application of the above-mentioned composite microbial agent or the above-mentioned composite microbial agent seedling substrate in the seedling raising and cultivation of greenhouse vegetables and fruits. The application is manifested in the application in the treatment of secondary salinization of soil and the prevention and control of diseases. Among them, the vegetables described in the present invention are preferably tomatoes, cucumbers, peppers, eggplants, etc., and the fruits are preferably melons, watermelons, etc.
[0025] The above-mentioned composite bacterial agent has the effects of controlling soil secondary salinization and preventing and controlling crop diseases, so the above-mentioned seedling substrate or microbial composite bacterial fertilizer can also improve plant immunity, control heavy metal pollution and regulate soil microecology. In addition, the application method of the composite bacterial agent is simple, easy to operate, time-saving and labor-saving, and has wide universality. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a colony morphology of Paenibacillus polymyxa XY8-23;
[0027] Figure 2 This is a bacterial morphology diagram of Paenibacillus polymyxa XY8-23;
[0028] Figure 3 This is the phylogenetic tree diagram of XY8-23 constructed based on 16S rRNA sequences;
[0029] Figure 4 The results of the effect of environmental conditions on the growth of Bv-HR6-1 are shown in Figure A. Growth curve under culture time; Figure B. Culture time-OD 600 Figure C. pH-growth change curve of the culture medium; Figure D. Inoculation amount-growth change curve; Figure E. Culture temperature-growth change curve; Figure F. Culture speed-growth change curve;
[0030] Figure 5 The results of the effect of environmental conditions on the growth of XY8-23 are shown in Figure A. The growth rate change curve under culture time; Figure B. The culture time-OD 600 Figure C. pH-growth change curve of the culture medium; Figure D. Inoculation amount-growth change curve; Figure E. Culture temperature-growth change curve; Figure F. Culture speed-growth change curve;
[0031] Figure 6 It is the growth amount diagram of Bv-HR6-1 (A) and XY8-23 (B) in different carriers under fermentation conditions;
[0032] Figure 7 This is the phenotype of tomato seedlings treated with different compound ratios of compound fungi;
[0033] Figure 8 This is a graph showing the effects of different compound proportions of compound fungi on tomato seedlings;
[0034] Fig. 9 This is the root phenotype of tomato seedlings under the treatment of different compound proportions of compound fungi;
[0035] Fig.10 This is a graph showing the effects of different compound proportions of compound microbial agents on the root growth of tomato seedlings;
[0036] Fig.11 This is a graph showing the growth of tomato seedlings under different dosages of the composite microbial agent.
[0037] Fig.12 This is a graph showing the effects of different dosages of the composite microbial agent on tomato seedlings;
[0038] Fig.13 This is a graph showing the effects of the composite microbial agent and the Bv-HR6-1 microbial agent on the growth of tomato seedlings;
[0039] Fig.14 This is a graph showing the effects of the compound bacterial agent and the XY8-23 bacterial agent on the growth of tomato seedlings.
[0040] The different letters in the above figures indicate that the differences between treatments reached a 5% significance level. DETAILED DESCRIPTION
[0041] The technical solution of the present invention is further described in detail below through specific implementation methods.
[0042] Unless otherwise specified, the terms used in the present invention are commonly used terms in the relevant field. The raw materials, equipment, preparation processes, testing methods, etc. used in the embodiments are all prior arts in the relevant field unless otherwise specified.
[0043] The solid culture medium includes: 30 g / L tryptic soy bean liquid medium (TSB) (17 g / L tryptic soy bean liquid medium, 3 g / L soy papain hydrolyzate, 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate), 18 g / L agar (gel temperature: 35-37°C, sol temperature: 87-89°C)
[0044] Example 1 Paenibacillus polymyxa XY8-23
[0045] 1.1 Source and screening of Paenibacillus polymyxa
[0046] Paenibacillus polymyxa XY8-23 was isolated and screened from the roots of Chlorophytum comosum in Huangchuan County, Henan Province. Paenibacillus polymyxa XY8-23 was induced and cultured on a medium containing rifampicin, and the concentration of rifampicin was gradually increased. Finally, a stable rifampicin-resistant (100 mg·L -1 ) strains and save them for future use; after culturing on TSA medium plates for 72 h, the colonies were scraped and sterilized with 0.1 mol·L -1 MgSO4 solution was prepared into 10 8 CFU·mL -1 of bacterial suspension.
[0047] 1.2 Identification
[0048] like Figure 1 and 2 As shown, the Paenibacillus polymyxa XY8-23 forms beige, translucent, rough-surfaced, and irregular-edge colonies on the NA medium. The XY8-23 bacteria are straight rod-shaped, have flagella around them, produce spores, and are Gram-positive.
[0049] 1.3 Physical, chemical and biochemical properties
[0050] The growth temperature range of Paenibacillus polymyxa XY8-23 is 5-50℃, the highest salt tolerance is 5% NaCl mass fraction, the starch hydrolysis and nitrate reduction reactions are positive, the gelatin liquefaction and H2S production tests are negative, and it can utilize sucrose, glucose, glycerol, maltose and mannitol, but cannot utilize galactose and tannic acid.
[0051] like Figure 3 As shown, 16S rRNA sequence homology analysis: The 16S rRNA gene amplified sequence of XY8-23 is 1487bp in length. Using NCBI for BLAST comparison, it was found that the homology with the corresponding 16S rRNA sequence of Paenibacillus polymyxa reached 99%. Based on the 16S rRNA sequence, the phylogenetic tree was constructed using MEGA software. The results showed that Paenibacillus polymyxa was clustered in one branch. According to the results of colony morphology, physiological and biochemical characteristics and molecular biology, the strain belongs to Paenibacillus polymyxa of the genus Paenibacillus, and it was named Paenibacillus polymyxa XY8-23.
[0052] Furthermore, the above strain XY8-23 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), classified and named Paenibacillus polymyxa, with the deposit number CGMCC No.31696, the deposit date: August 20, 2024, and the deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.
[0053] Example 2 Composite bacterial agent
[0054] The present embodiment provides a composite bacterial agent, including Bv-HR6-1 and the polymyxa bacillus XY8-23 provided in Example 1, wherein the classification name of the Bv-HR6-1 is Bacillus velezensis, the deposit number is CGMCC No.14469, the deposit unit is the General Microbiology Center of China Microbiological Culture Collection Administration; the deposit date is July 27, 2017; the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.
[0055] This embodiment also provides a method for preparing a composite bacterial agent, the steps of which include:
[0056] The strains Bv-HR6-1 and XY8-23 were streaked on a solid culture medium, cultured at 28°C for 24-96 hours, and Bv-HR6-1 and XY8-23 were obtained, respectively. The solid culture medium included 30 g / L tryptic soy broth (TSB) and 18 g / L agar.
[0057] Bv-HR6-1 and XY8-23 were inoculated into TSB medium using a bamboo stick or an inoculation loop. The culture conditions were 25-30° C. and 140-200 r / min for 16-30 h to obtain the Bv-HR6-1 seed solution and XY8-23 seed solution, respectively.
[0058] The seed liquid of Bv-HR6-1 and XY8-23 is inoculated into TSB medium, and the inoculation amount is 0.5%-2.0% of the volume of the inoculated TSB medium. The culture is fermented at 28-32°C for 36-50h to obtain Bv-HR6-1 and XY8-23 bacterial liquid (one-time propagation); the TSB liquid medium includes: 30g / L tryptic soy liquid medium (TSB).
[0059] (1) Experiment on optimizing the secondary propagation culture conditions of the two strains
[0060] Screening indicators: culture time, OD 600 (for culture time), pH value of culture medium, inoculum amount (volume ratio), culture temperature, and culture speed.
[0061] Single factor experiment: Bv-HR6-1 and XY8-23 were cultured under different environmental conditions, and the growth of the two bacteria under different treatments was investigated. Figure 4 and Figure 5 shown.
[0062] Figure 4 AF and Figure 5 AF showed that the optimal culture conditions for Bv-HR6-1 and XY8-23 were: culture time 36h, medium pH 7, inoculum 10%, culture temperature 28℃, culture speed 160rpm; among them, the optimal OD of Bv-HR6-1 and XY8-23 600 They are 2.4 and 2.8 respectively.
[0063] according to Figure 4-5 Based on the single factor experimental results shown in Table 1, the culture conditions of the above two bacteria were subjected to response surface analysis, and the response surface analysis results were verified. The results are shown in Tables 1 and 2.
[0064] Table 1. Verification of the response surface experimental results of Bv-HR6-1
[0065] Experimental conditions Response surface prediction of optimal conditions Verify the response surface prediction of the optimal conditions Time (h) 30 30 pH 7 7 Inoculation amount (%) 11 11 Temperature(℃) 28 28 Speed (rpm) 140 140 Bacterial growth (cfu / ml) <![CDATA[7.35×10 8 ]]> <![CDATA[8.10×10 8 ]]>
[0066] Table 2. Verification of response surface experimental results of XY8-23
[0067] Experimental conditions Response surface prediction of optimal conditions Verify the response surface prediction of the optimal conditions Time (h) 30 30 pH 6.75 6.75 Inoculation amount (%) 13 13 Temperature(℃) 28 28 Speed (rpm) 140 140 Bacterial growth (cfu / ml) <![CDATA[2.71×10 8 ]]> <![CDATA[3.90×10 8 ]]>
[0068] Combination Figure 4-5 From Table 1-2, we can get the optimal culture conditions for Bv-HR6-1 and XY8-23. According to the optimal culture conditions, the bacterial liquid of Bv-HR6-1 and XY8-23 (the product after one expansion) was expanded again. The bacterial liquid of Bv-HR6-1 was inoculated into the liquid culture medium at a rate of 11% (volume ratio), the rotation speed was 140r / min, the optimal fermentation temperature was 28°C, the optimal culture time was 30h, and the optimal pH of the liquid culture medium was 7; the bacterial liquid of XY8-23 was inoculated into the liquid culture medium at a rate of 13% (volume ratio), the rotation speed was 140r / min, the optimal fermentation temperature was 28°C, the optimal culture time was 30h, and the optimal pH of the liquid culture medium was 6.75. The fermentation liquid of Bv-HR6-1 and XY8-23 was obtained. Each liter of the liquid culture medium includes: 20 g of peptone, 5 g of yeast extract, 0.5 g of sodium chloride, 10 mL of 0.25 mol / L potassium chloride), 5 mL of 2 mol / L magnesium chloride, and 20 mL of 1 mol / L glucose.
[0069] (2) Screening of Bv-HR6-1 and XY8-23 bacterial agent carriers
[0070] Carrier test materials: peat (P), vermiculite (VER), coal slag (CC), chicken manure compost (CMC), sheep manure compost (SMC), peanut shell (PS), rice husk (RH), mushroom husk (MR), bean dregs (BD), tomato straw (TS), wheat straw (WS), corn straw (CS)
[0071] Experimental method: The fermentation broth of Bv-HR6-1 and XY8-23 was mixed with the 12 carriers at a mass ratio of 1:10, and fermented for 12 days. The fermented carriers were placed in a cool and ventilated place to dry. The dried fungus residue was crushed with a grinder and passed through a 20-mesh sieve. The 12 kinds of dry fungus powder were stored separately, and the growth of the two bacteria in different carriers was investigated for 30 days, 60 days, and 90 days. Figure 6 .
[0072] from Figure 6 It can be seen that Bv-HR6-1 (A) and XY8-23 (B) have the best growth on the carrier okara.
[0073] The fermented liquid of Bv-HR6-1 and XY8-23 were mixed with bean dregs at a mass ratio of 1:10, and fermented for 12 days. The fermented bean dregs were placed in a cool and ventilated place to dry. The dried composite fungus residues were crushed by a grinder and passed through a 20-mesh sieve. The two dried fungus powders were stored separately, and the viable bacterial counts were 1.86×10 12 cfu / g of Bv-HR6-1 single bacterial powder and viable count of 7.37×10 10 cfu / g of XY8-23 single bacterial powder were mixed in a 1:1 mass ratio to obtain the composite bacterial agent. When the composite bacterial agent was stored for 30 days, 60 days, and 90 days, the viable bacterial counts of Bv-HR6-1 were 1.61×10 12 cfu / g, 1.44×10 12 cfu / g, 1.21×10 12 cfu / g; the viable counts of XY8-23 were 6.34×10 10 cfu / g, 4.71×10 10 cfu / g, 3.85×10 10 cfu / g.
[0074] Example 3 Application of composite bacterial agent
[0075] (1) Comparison of the application effects of different compounding ratios of composite microbial agents
[0076] This embodiment provides a composite bacterial agent, comprising uniformly mixing Bv-HR6-1 bacterial powder and XY8-23 bacterial powder in a mass ratio of 1:1, 2:1, and 1:2, wherein the Bv-HR6-1 bacterial powder and XY8-23 bacterial powder are both prepared under optimal conditions in reference to Example 2.
[0077] Experimental raw materials: The tomato variety used in the experiment was 'Ailsa Craig' (wild type); the test substrate was Delifeng seedling substrate;
[0078] Specific formula of compound bacterial agent: The number of viable bacteria of Bv-HR6-1 bacterial powder in each formula is 1.21×10 12 cfu / g, the number of viable bacteria in XY8-23 bacterial powder was 6.34×10 10 cfu / g;
[0079] Bacterial powder 1:1: When the mass ratio of Bv-HR6-1 bacterial powder and XY8-23 bacterial powder is 1:1, 6g / L Bv-HR6-1 bacterial powder and 6g / L XY8-23 bacterial powder are uniformly mixed;
[0080] Bacterial powder 2:1: When the mass ratio of Bv-HR6-1 bacterial powder to XY8-23 bacterial powder is 2:1, 8g / L Bv-HR6-1 bacterial powder and 4g / L XY8-23 bacterial powder are uniformly mixed;
[0081] Bacterial powder 1:2: When the mass ratio of Bv-HR6-1 bacterial powder to XY8-23 bacterial powder is 1:2, 4g / L Bv-HR6-1 bacterial powder and 8g / L XY8-23 bacterial powder are evenly mixed.
[0082] Substrate treatment: Add 12g of the prepared compound bacterial agent to one liter of Delifeng seedling substrate, mix evenly, and obtain seedling substrates treated with different compounding ratios. Delifeng seedling substrate (Mock) was used as a blank test.
[0083] Experimental method: Tomato seeds were grown in plug trays in different seedling mediums, and two-leaf and one-heart tomato seedlings with consistent growth were selected and transplanted into test pots with the same seedling medium. The tomato seedlings were cultured in a climate chamber with a temperature of 28℃ / 18℃ (day / night) and a photoperiod of 14h / 10h (day / night). When the tomato seedlings grew to 3-4 true leaves, tomato seedlings with consistent growth were selected for salt and tomato blight combined stress treatment (NaCl+Pc) for 7 days, with one seedling per pot.
[0084] A total of 8 treatments were designed in the experiment, namely, clean water (CK), NaCl+Pc, bacterial powder 1:1, bacterial powder 1:1+NaCl+Pc, bacterial powder 2:1, bacterial powder 2:1+NaCl+Pc, bacterial powder 1:2, and bacterial powder 1:2+NaCl+Pc. Each treatment had 15 tomato seedlings, and the concentration of Phytophthora tomato was 1×10 4 CFU / mL, 5 mL was irrigated per plant, and the salt concentration was 200 mM NaCl, 100 mL was irrigated per plant.
[0085] The growth status of tomato plants on the 7th day after treatment is as follows Figure 7 As shown in Figure 2, tomato plant height, stem thickness, aboveground dry and fresh weight, underground dry and fresh weight, etc. Figure 8 As shown in Figure 2, the root phenotype, root length, total root length, total root surface area, average root diameter, total root volume, number of root tips, number of root forks, number of root crossings, etc. of tomato seedlings are shown in Figure 2. Figure 9-10 The test results of leaf relative conductivity and root activity under the combined stress of salt and blight are shown in Table 3.
[0086] Table 3. Effects of different compound ratios of compound microbial agents on the alleviation of combined stress
[0087]
[0088]
[0089] from Figure 7-10 It can be seen from Table 3 that the tomato seedlings added with the composite bacterial agent are larger than the blank control group added with only water in terms of plant height and stem thickness, and the tomato seedlings under stress added with the composite bacterial agent are larger than the treatment group added with only stress in terms of plant height, number of leaves, stem thickness, etc. This shows that the composite bacterial agent has an alleviating effect on the combined stress of salt and blight.
[0090] The tomato seedlings added with the compound microbial agent of 1:1 bacterial powder were greater than those of the test groups of the compound microbial agent of 2:1 bacterial powder and 1:2 bacterial powder in terms of plant height, stem diameter, number of leaves, aboveground dry and fresh weight, underground dry and fresh weight, root length, total root surface area, total root volume, and average root diameter. It can be seen that the compound microbial agent containing 1:1 bacterial powder is more effective in promoting salt tolerance and preventing and controlling tomato blight than the compound microbial agent of 2:1 bacterial powder and 1:2 bacterial powder.
[0091] (2) Comparison of the effects of different dosages of composite microbial agents
[0092] This embodiment also provides a method for applying the composite microbial agent, including mixing the seedling substrate with different amounts of the composite microbial agent when raising plant seedlings, and mixing the cultivation substrate with different amounts of the composite microbial agent when transplanting plants.
[0093] Experimental raw materials: The tomato variety used for the test was 'Ailsa Craig' (wild type) material; the test substrate was Delifeng seedling substrate; the test compound microbial agent was the best compound microbial agent in the aforementioned "(1) Comparison of the application effects of different compound microbial agent ratios", i.e., 1:1 bacterial powder; the application rates of the compound microbial agent were 6 g / L, 12 g / L, and 18 g / L.
[0094] Substrate treatment: Compound the compound bacterial agent (bacterial powder 1:1) with Delifeng seedling substrate according to different usage amounts, mix evenly to obtain seedling substrate treated with different usage amounts.
[0095] Experimental method: Tomato seeds were grown in plug trays in different seedling mediums, and two-leaf and one-heart tomato seedlings with consistent growth were selected and transplanted into test pots with the same seedling medium. The tomato seedlings were cultured in a climate chamber with a temperature of 28℃ / 18℃ (day / night) and a photoperiod of 14h / 10h (day / night). When the tomato seedlings grew to 3-4 true leaves, the tomato seedlings with consistent growth were selected for NaCl+Pc compound stress treatment, with one seedling per pot.
[0096] The experiment designed 8 treatments, namely clean water (CK), NaCl+Pc, 6g / L, 6g / L+NaCl+Pc, 12g / L, 12g / L+NaCl+Pc, 18g / L, 18g / L+NaCl+Pc. Each treatment had 15 tomato seedlings, and the concentration of Phytophthora was 1×10 4 CFU / ml, 5 ml was poured per plant, and the salt concentration was 200 mM NaCl, 100 ml was poured per plant.
[0097] The growth status of tomato plants on the 7th day after treatment is as follows Fig.11 As shown in Figure 2, tomato plant height, stem thickness, aboveground dry and fresh weight, underground dry and fresh weight, root length, total root length, and other indicators are as follows Fig.12 The test results of leaf relative conductivity and root activity under the combined stress of salt and blight are shown in Table 4.
[0098] Table 4. Effects of different dosages of compound microbial agents on alleviating compound stress
[0099]
[0100] from Figure 11-12 It can be seen from Table 4 that: 1) the tomato seedlings treated with the composite bacterial agent were greater than the blank control group treated with only water in terms of plant height, stem thickness, aboveground dry fresh weight, underground dry fresh weight, and root length, and the tomato seedlings under stress treated with the composite bacterial agent were greater than the treatment group treated with only stress in terms of plant height, leaf number, and stem thickness. This shows that the composite bacterial agent has an alleviating effect on the combined stress of salt and blight.
[0101] 2) The tomato seedlings added with 12g / L of the compound microbial agent were greater than those of the test groups with 6g / L and 18g / L of the compound microbial agent in terms of plant height, stem thickness, aboveground dry fresh weight, underground dry fresh weight, and root length, and the test groups with 6g / L and 18g / L of the compound microbial agent were also greater than those of the test groups with 6g / L and 18g / L of the compound microbial agent. It can be seen that the 12g / L compound microbial agent is more effective in promoting salt tolerance and preventing and controlling tomato blight than the 6g / L and 18g / L compound microbial agents.
[0102] (3) Comparison of the application effects of the composite microbial agent and Bacillus velezensis Bv-HR6-1 microbial agent
[0103] This embodiment also provides a method for comparing the application effects of the composite microbial agent and the Bv-HR6-1 microbial agent, including mixing the seedling substrate with different microbial agents during plant seedling cultivation, and mixing the cultivation substrate with different microbial agents during plant transplanting.
[0104] Experimental raw materials: The tomato variety used was 'Ailsa Craig' (wild type); the substrate used was Delifeng seedling substrate; the compound microbial agent used was the best compound microbial agent in the above "(1) Comparison of the application effects of different compound microbial agent ratios", i.e., 1:1 bacterial powder; the number of viable bacteria was 1.21×10 12 The dosage of Bv-HR6-1 bacterial agent for cfu / g is 12g / L.
[0105] Substrate treatment: The test compound bacterial agent was compounded with Delifeng seedling substrate at an application rate of 12 g / L and mixed evenly to obtain seedling substrates with different treatments.
[0106] Experimental method: Tomato seeds were grown in seedling trays in seedling medium with different bacterial agents added, and two-leaf and one-heart tomato seedlings with consistent growth were selected and transplanted into test pots with the same seedling medium. The tomato seedlings were cultured in a climate chamber with a temperature of 28℃ / 18℃ (day / night) and a photoperiod of 14h / 10h (day / night). When the tomato seedlings grew to 3-4 true leaves, the tomato seedlings with consistent growth were selected for salt stress treatment (NaCl), with one seedling per pot.
[0107] The experiment was designed with 6 treatments, namely clean water (CK), NaCl (salt stress treatment), 12g / L Bv-HR6-1 agent, 12g / L Bv-HR6-1 agent + NaCl, 12g / L compound agent, 12g / L compound agent + NaCl. Each treatment had 15 tomato seedlings, the salt concentration was 200mM NaCl, and each plant was irrigated with 100ml.
[0108] The results of tomato plant phenotype, plant height, stem diameter, aboveground fresh and dry weight, underground fresh and dry weight, leaf relative conductivity, root activity, stress index, relief effect and other seedling growth indicators on the 7th day after treatment are as follows: Fig.12 And as shown in Table 5.
[0109] Table 5. Effects of compound bacterial agent and Bv-HR6-1 bacterial agent on salt stress alleviation
[0110]
[0111] from Fig.12 As can be seen from Table 5: 1) The tomato seedlings with added bacterial agents were larger than the blank control group with only water added in terms of plant height and stem thickness, and the tomato seedlings with added bacterial agents under stress were larger than the experimental group with only stress treatment in terms of plant height and stem thickness. This shows that the composite bacterial agent and the Bv-HR6-1 bacterial agent have an alleviating effect on salt stress.
[0112] 2) The tomato seedlings added with 12g / L of the composite microbial agent were greater than those of the test group with 12g / L of the Bv-HR6-1 microbial agent in terms of plant height, stem diameter, aboveground fresh weight, underground fresh weight, etc. This shows that the 12g / L composite microbial agent is more effective in promoting salt tolerance and growth than the 12g / L Bv-HR6-1 microbial agent.
[0113] (4) Comparison of the application effects of composite microbial agent and XY8-23 microbial agent
[0114] This embodiment also provides a method for comparing the application effects of the composite microbial agent and the XY8-23 microbial agent, including mixing the seedling substrate with different microbial agents during plant seedling cultivation, and mixing the cultivation substrate with different microbial agents during plant transplanting.
[0115] Experimental raw materials: The tomato variety used was 'Ailsa Craig' (wild type); the substrate used was Delifeng seedling substrate; the compound microbial agent used was the best compound microbial agent in the above "(1) Comparison of the application effects of different compound microbial agent ratios", i.e., 1:1 bacterial powder; the number of viable bacteria was 6.34×10 10 The usage amount of XY8-23 bacterial agent is 12g / L cfu / g.
[0116] Substrate treatment: The test compound bacterial agent was compounded with Delifeng seedling substrate at an application rate of 12 g / L and mixed evenly to obtain seedling substrates with different treatments.
[0117] Experimental method: Tomato seeds were grown in seedling trays in seedling medium with different bacterial agents added, and two-leaf and one-heart tomato seedlings with consistent growth were selected and transplanted into test pots with the same seedling medium. The tomato seedlings were cultured in a climate chamber with a temperature of 28℃ / 18℃ (day / night) and a photoperiod of 14h / 10h (day / night). When the tomato seedlings grew to 3-4 true leaves, the tomato seedlings with consistent growth were selected for NaCl salt stress, with one seedling per pot.
[0118] The experiment designed 6 treatments, namely clean water (CK), tomato blight stress treatment (Pc), 12g / L XY8-23 agent, 12g / L XY8-23 agent + Pc, 12g / L compound agent, 12g / L compound agent + Pc. Each treatment had 15 tomato seedlings, and the concentration of Phytophthora was 1×10 4 CFU / ml, 5ml was irrigated per plant.
[0119] The results of tomato plant phenotype, plant height, stem diameter, aboveground fresh and dry weight, underground fresh and dry weight, leaf relative conductivity, root activity, stress index, relief effect and other seedling growth indicators on the 7th day after treatment are as follows: Fig.13 and as shown in Table 6.
[0120] Table 6. Effects of compound fungicide and XY8-23 fungicide on tomato blight
[0121]
[0122] from Fig.13 It can be seen from Table 6 that the tomato seedlings added with the bacterial agent are greater than the blank control group with only water added in terms of plant height and stem thickness, and the tomato seedlings under stress with the bacterial agent are greater than the experimental group with only stress treatment in terms of plant height and stem thickness. This shows that the composite bacterial agent and the XY8-23 bacterial agent have a mitigating effect on tomato blight stress. In addition, the tomato seedlings added with 12g / L of the composite bacterial agent are greater than the experimental group with 12g / L of the XY8-23 bacterial agent in terms of plant height, stem thickness, aboveground dry and fresh weight, underground dry and fresh weight, etc. This shows that the 12g / L composite bacterial agent is more effective in preventing and controlling tomato blight than the 12g / L XY8-23 bacterial agent.
[0123] Therefore, the composite bacterial agent provided by the embodiment of the present invention, which is formed by compounding the XY8-23 bacteria with the above-mentioned Bv-HR6-1 bacteria, has the effects of controlling soil secondary salinization and preventing and treating tomato blight. The seedling medium using the composite bacterial agent can also improve plant immunity, control heavy metal pollution and regulate soil microecology. In addition, the application method of the composite bacterial agent is simple, easy to operate, time-saving and labor-saving, and has wide universality.
[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.
Claims
1. A strain of Paenibacillus polymyxa, characterized in that: Paenibacillus polymyxa Paenibacillus polymyxa XY8-23, deposited on August 20, 2024 in the "General Microbiology Center of China Microorganism Culture Collection Administration", with the deposit number CGMCC No. 31696, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.
2. A composite bacterial agent, characterized in that: Comprising Bacillus Velezii Bv-HR6-1 and the polymyxa bacillus described in claim 1 Paenibacillus polymyxa XY8-23, wherein the deposit number of the Bacillus velez Bv-HR6-1 is CGMCC No. 14469, the deposit unit is the General Microbiology Center of China Microorganism Culture Collection Administration; the deposit date is July 27, 2017; the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.
3. The composite bacterial agent according to claim 2, characterized in that: The concentration of the Velez Bacillus Bv-HR6-1 is 2×10 10 -1×10 12 cfu / g, the polymyxa bacillus Paenibacillus polymyxa The concentration of XY8-23 is 2×10 9 -6×10 10 cfu / g; Preferably, the concentration of the Bacillus Velezii Bv-HR6-1 is 2×10 11 -1×10 12 / cfu / g, the polymyxa bacillus Paenibacillus polymyxa The concentration of XY8-23 is 3×10 10 -6×10 10 cfu / g.
4. The composite bacterial agent according to claim 2 or 3, characterized in that: The composite bacterial agent is a powder; Preferably, the carrier of the strain in the powder is okara; Preferably, the pH value of the carrier is 6.5-7.
5.
5. A method for preparing the composite bacterial agent according to any one of claims 2 to 4, comprising the steps of: Seed liquid culture: Bacillus velez Bv-HR6-1 and Paenibacillus polymyxa Paenibacilluspolymyxa XY8-23 were cultured separately to obtain Bv-HR6-1 seed solution and XY8-23 seed solution; Propagation culture: performing propagation culture on the Bv-HR6-1 seed solution and the XY8-23 seed solution to obtain Bv-HR6-1 fermentation solution and XY8-23 fermentation solution respectively; Preparation of composite bacterial agent: ferment the Bv-HR6-1 fermentation broth and the XY8-23 fermentation broth separately to obtain Bv-HR6-1 fermentation bacterial agent and XY8-23 fermentation bacterial agent, and evenly mix the Bv-HR6-1 fermentation bacterial agent and the XY8-23 fermentation bacterial agent to obtain the composite bacterial agent.
6. The preparation method according to claim 5, characterized in that: The seed liquid culture step includes: inoculating the Bv-HR6-1 mother strain and the XY8-23 mother strain into the seed liquid culture medium respectively, and culturing for 16-24 hours at a temperature of 25-30°C and a r / min of 140-200, to obtain the Bv-HR6-1 seed liquid and the XY8-23 seed liquid respectively.
7. The preparation method according to claim 6, characterized in that: The multiplication culture includes two multiplication cultures, specifically including: First propagation: inoculating the Bv-HR6-1 seed solution and the XY8-23 seed solution into the first liquid culture medium respectively, and the inoculation amount is 0.5%-2.0% of the volume of the liquid culture medium, and fermenting and culturing at 28-32° C. for 36-50 h to obtain Bv-HR6-1 bacterial solution and XY8-23 bacterial solution; Secondary propagation: The Bv-HR6-1 bacterial solution and the XY8-23 bacterial solution are inoculated into the second liquid culture medium respectively, and the inoculation amount is 5%-15% of the volume of the liquid culture medium, and fermented and cultured at 22-35°C and 120-200 rpm for 16-48 h to obtain Bv-HR6-1 fermentation liquid and XY8-23 fermentation liquid, wherein the pH value of the second liquid culture medium is 6-8.
8. The preparation method according to any one of claims 5 to 7, characterized in that: When the composite bacterial agent is a powder, the steps of preparing the composite bacterial agent include: fully mixing the Bv-HR6-1 fermentation liquid and the XY8-23 fermentation liquid with bean dregs, respectively, and fermenting and culturing for 7-14, drying, and crushing to obtain Bv-HR6-1 fermentation bacteria powder and XY8-23 fermentation bacteria powder; uniformly mixing the Bv-HR6-1 fermentation bacteria powder and the XY8-23 fermentation bacteria powder to obtain the composite bacterial agent; Preferably, the viable count of the Bv-HR6-1 fermentation bacteria powder is 1×10 12 -1×10 13 cfu / g, and the viable count of the XY8-23 fermentation bacteria powder is 1×10 10 -1×10 11 cfu / g; Preferably, the mass ratio of the Bv-HR6-1 fermentation bacteria powder to the XY8-23 fermentation bacteria powder is 1:2 - 2:
1.
9. A composite bacterial agent seedling medium, characterized in that: It is mainly prepared by uniformly mixing the composite bacterial agent according to any one of claims 2 to 4 with a seedling culture medium at a mass ratio of 6 to 18 g / L; Preferably, the seedling raising matrix is a seedling raising matrix for vegetables and fruits.
10. Use of the composite bacterial agent according to any one of claims 2 to 4 or the composite bacterial agent seedling medium according to claim 9 in seedling raising and cultivation of vegetables and fruits; Preferably, the composite bacterial agent or composite bacterial agent seedling matrix is used in controlling soil secondary salinization and preventing and controlling pests; Preferably, the vegetable is tomato, cucumber, pepper or eggplant, and the melon or fruit is melon or watermelon.