Compound microbial agent for preventing and treating tomato fusarium wilt and preparation method of compound microbial agent

Abstract

The invention discloses a compound microbial agent for preventing and treating tomato fusarium wilt and a preparation method thereof, and belongs to the technical field of microbial agent preparation. The compound microbial agent for preventing and treating tomato fusarium wilt is prepared from the following raw materials in parts by mass: 11-14 parts of mixed bacterial liquid, 33-36 parts of a carrier, 1-2 parts of L-tyrosine, 1-2 parts of sodium lignin sulfonate and 1-2 parts of soluble starch, the mixed bacterial liquid is prepared from bacillus vallismortis bacterial liquid, bacillus subtilis bacterial liquid and serratia marcescens bacterial liquid according to the mass ratio of 3: 1: 1. The compound microbial agent disclosed by the invention is environment-friendly and safe to use, can remarkably improve the stress resistance of tomatoes, and has a relatively good prevention and treatment effect on tomato fusarium wilt.

Description

Technical Field

[0001] The invention relates to the technical field of microbial agent preparation, in particular to a composite microbial agent for preventing and treating tomato wilt and a preparation method thereof. Background Art

[0002] Tomato wilt is one of the most serious soil-borne diseases in greenhouse tomato production. In recent years, with the continuous increase in the planting area of ​​greenhouse tomatoes, soil-borne tomato wilt has also become serious, becoming a bottleneck problem in tomato facility production. Tomato wilt is caused by infection with the tomato-specific type of Fusarium oxysporum, which belongs to the subphylum Ascomycota. Studies have shown that the incidence of wilt in fields with continuous cropping for many years is as high as 20-40%, and there is a trend of increasing year by year. Tomato wilt mainly harms the roots and stem base of tomatoes, and the disease often begins to occur during the flowering and fruiting period, invading the root vascular tissue of the plant and causing the death of the plant. According to statistics, more than 40% of tomatoes are reduced in yield each year due to wilt, and even a total crop failure in severe cases. Therefore, prevention and control is difficult.

[0003] The prevention and control of tomato wilt in the prior art mainly include the following methods: 1. Use disease-resistant varieties to plant plants, but this method is not enough to resist a variety of pathogenic bacteria, and resistance is easy to lose; 2. Chemical control, chemical control is effective quickly, but large-scale use will cause pathogens to develop drug resistance, and excessive drug residues will cause environmental pollution and endanger human health; 3. Biocontrol agents, which are safe for crops and humans, green and environmentally friendly, and have a simple production process and can promote crop growth. However, there are still many problems in the application of existing microorganisms in biological control, such as easy inactivation of microorganisms, low release rate, unstable efficacy, short storage time, and easy contamination and degradation of strains. Summary of the invention

[0004] The purpose of the present invention is to provide a composite microbial agent for preventing and treating tomato wilt and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a composite microbial agent for preventing and treating tomato wilt, the raw materials of which include: Bacillus valerianus, Bacillus subtilis and Serratia marcescens.

[0007] Furthermore, the composite microbial agent comprises, by weight, 11 to 14 parts of mixed bacterial solution, 33 to 36 parts of carrier, 1 to 2 parts of L-tyrosine, 1 to 2 parts of sodium lignin sulfonate and 1 to 2 parts of soluble starch;

[0008] The mixed bacterial liquid is a mixed bacterial liquid of Bacillus valerius, Bacillus subtilis and Serratia marcescens.

[0009] Furthermore, the mixed bacterial solution consists of a bacterial solution of Bacillus vallis, a bacterial solution of Bacillus subtilis and a bacterial solution of Serratia marcescens in a mass ratio of 3:1:1.

[0010] Furthermore, the OD values ​​of the Bacillus subtilis solution, the Bacillus subtilis solution and the Serratia marcescens solution are 600 Both are 2.0.

[0011] Furthermore, the method for preparing the mixed bacterial solution comprises the following steps:

[0012] The Bacillus thuringiensis, Bacillus subtilis and Serratia marcescens were inoculated into solid LB medium respectively, and cultured at 37°C for 48 hours to obtain activated Bacillus thuringiensis, Bacillus subtilis and Serratia marcescens; the activated Bacillus thuringiensis, Bacillus subtilis and Serratia marcescens were inoculated into liquid LB medium respectively, and cultured at 37°C and 220 r / min for 12 hours in a shaking incubator, and the bacterial solution concentration was adjusted to OD600=2.0 to obtain Bacillus thuringiensis, Bacillus subtilis and Serratia marcescens bacterial solutions;

[0013] The Bacillus vallis bacterial solution, the Bacillus subtilis bacterial solution and the Serratia marcescens bacterial solution were uniformly mixed in a mass ratio of 3:1:1 to obtain the mixed bacterial solution.

[0014] The solid LB medium is composed of: 10.0 g peptone, 10.0 g NaCl, 5 g yeast powder, 20 g agar, and distilled water to 1 L, with a pH value of 7.0;

[0015] The composition of the liquid LB medium is: 10.0 g of peptone, 3.0 g of NaCl, 3 g of yeast powder, and the volume is adjusted to 1 L with distilled water, and the pH value is 7.0.

[0016] Furthermore, the carrier comprises bentonite and biochar in a mass ratio of 2:1.

[0017] The composite microbial agent of the invention uses Bacillus vallis, Bacillus subtilis and Serratia marcescens as effective bacterial flora, bentonite and biochar as carriers, soluble starch as a binder, L-tyrosine as a stabilizer, and sodium lignin sulfonate as a wetting agent, and has a good tomato wilt prevention and control effect.

[0018] The second technical solution of the present invention is a method for preparing the composite microbial agent for preventing and treating tomato wilt, comprising the following steps:

[0019] Firstly, soluble starch is added into water for pre-dissolution, and when cooled to below 40°C, mixed bacterial solution is added, and then carrier, L-tyrosine and sodium lignin sulfonate are added and mixed evenly, and the composite microbial agent is obtained after granulation.

[0020] The third technical solution of the present invention: an application of the above-mentioned composite microbial agent in the preparation of an agent for preventing and treating tomato wilt.

[0021] The present invention discloses the following technical effects:

[0022] (1) The present invention adopts a combination of Bacillus vallis, Bacillus subtilis and Serratia marcescens, which is reasonably matched and highly complementary. After use, the beneficial bacteria can rapidly reproduce and form a dominant population, which has a synergistic and synergistic effect on the inhibition of tomato wilt pathogens. And the composite microbial agent prepared by using the bacterial combination has an excellent control effect on tomato wilt.

[0023] (2) The bentonite and biochar in the composite microbial agent of the present invention have good biocompatibility with microorganisms. The mixed bacterial solution can be adsorbed and fixed by bentonite and biochar, thereby improving the stability and activity of the microorganisms and enhancing the prevention and control effect of tomato wilt. In addition, bentonite and biochar have strong adsorption and hydrophilicity. After being applied to the soil, they can slow down the loss of soil nutrients and water, enhance the soil's ability to retain water and fertilizer, and promote the development of tomato roots.

[0024] (3) The preparation method of the composite microbial agent of the present invention is simple and easy to operate, easy to store and transport, and the materials used to prepare the composite microbial agent have good biocompatibility with microorganisms, high safety, low cost, are easy to obtain on the market, easy to industrialize and produce, and have good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The antibacterial effect of the mixed bacterial solution prepared in Example 1 of the present invention on tomato wilt pathogenic bacteria;

[0027] Figure 2 The antibacterial effect of the mixed bacterial solution prepared in Comparative Example 1 of the present invention on tomato wilt pathogenic bacteria;

[0028] Figure 3 The antibacterial effect of the mixed bacterial solution prepared in Comparative Example 2 of the present invention on tomato wilt pathogenic bacteria;

[0029] Figure 4 The antibacterial effect of the mixed bacterial solution prepared in Comparative Example 3 of the present invention on tomato wilt pathogenic bacteria is shown. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The “parts” described in the following examples are all “parts by mass”.

[0036] In a specific embodiment of the present invention, the composition of the solid LB medium is: 10.0 g of peptone, 10.0 g of NaCl, 5 g of yeast powder, 20 g of agar, and the volume is adjusted to 1 L with distilled water, and the pH value is 7.0;

[0037] In a specific embodiment of the present invention, the composition of the liquid LB culture medium is: 10.0 g of peptone, 3.0 g of NaCl, 3 g of yeast powder, and the volume is fixed to 1 L with distilled water, and the pH value is 7.0.

[0038] In a specific embodiment of the present invention, Bacillus vallismortis is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with an address in Beijing, China, a deposit number of CGMCC NO.21871, and a deposit date of March 5, 2021; Serratia sp. is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with an address in Beijing, China, a deposit number of CGMCC NO.7340, and a deposit date of March 20, 2013; Bacillus subtilis is purchased from China Industrial Microorganism Culture Collection Administration Center, product number: CICC 24713; Bacillus amyloliquefaciens is purchased from China Industrial Microorganism Culture Collection Administration Center, product number: CICC 20229.

[0039] In a specific embodiment of the present invention, the activation method of Bacillus vallis, Bacillus subtilis, Serratia marcescens and Bacillus amyloliquefaciens is as follows: Bacillus vallis, Bacillus subtilis, Serratia marcescens and Bacillus amyloliquefaciens are inoculated into solid LB culture medium respectively, and cultured at 37° C. for 48 hours to obtain activated Bacillus vallis, Bacillus subtilis and Serratia marcescens.

[0040] Example 1

[0041] Antagonistic effect test of composite microbial flora on tomato wilt pathogen on plate:

[0042] The plate confrontation method was used to test the antibacterial effect of the composite microbial flora on tomato wilt, specifically:

[0043] (1) The activated Bacillus subtilis, Bacillus subtilis and Serratia marcescens were inoculated into fresh LB liquid medium, respectively, and cultured in a shaking incubator at 37°C and 220 r / min for 12 h. The concentrations of the three bacterial cultures were adjusted to OD 600 =2.0, to obtain Bacillus vallis fermentation liquid, Bacillus subtilis fermentation liquid and Serratia marcescens fermentation liquid, and to uniformly mix the Bacillus vallis fermentation liquid, Bacillus subtilis fermentation liquid and Serratia marcescens fermentation liquid in a mass ratio of 3:1:1 to obtain a mixed bacterial liquid.

[0044] (2) After the tomato wilt pathogen was beaten with a 5 mm pipette tip, the bacterial cake was inoculated in the center of the PDA culture medium. Then, 2 μL of the prepared mixed bacterial solution was dripped on the four sides 2.5 cm away from the center of the culture medium. An equal amount of sterile water was dripped as a control. Each treatment was repeated three times and cultured in a 28°C incubator. When the control grew to 3 / 4 of the culture dish, the antibacterial rate was measured and calculated.

[0045] Comparative Example 1

[0046] Same as Example 1, except that Bacillus valerianus was replaced with an equal amount of Bacillus amyloliquefaciens.

[0047] Comparative Example 2

[0048] Same as Example 1, except that Bacillus subtilis was replaced by an equal amount of Bacillus amyloliquefaciens.

[0049] Comparative Example 3

[0050] Same as Example 1, except that Serratia marcescens was replaced by an equal amount of Bacillus amyloliquefaciens.

[0051] Effect Example 1

[0052] The antibacterial rates of Example 1 and Comparative Examples 1 to 3 against tomato wilt pathogens are shown in Tables 1 and Figures 1 to 4 .

[0053] Table 1 Antibacterial rate

[0054] project Antibacterial rate / % Example 1 72.12±0.79 Comparative Example 1 63.07±0.64 Comparative Example 2 58.44±1.21 Comparative Example 3 54.05±0.42

[0055] It can be seen from Table 1 that the antibacterial rate of the mixed bacterial solution prepared in Example 1 of the present invention against tomato wilt pathogen is 72.12%. Compared with Comparative Examples 1 to 3, the antibacterial rate of the combination of beneficial bacteria of the present invention against tomato wilt pathogen is the highest. Figures 1 to 4 It can be seen that when an equal amount of composite microbial liquid is added to the plate, the beneficial bacteria in the mixed liquid prepared in Example 1 of the present invention can grow rapidly, the colony growth is the best, and the inhibition of tomato wilt pathogens has a synergistic and synergistic effect.

[0056] Example 2

[0057] A preparation method of a composite microbial agent for preventing and controlling tomato wilt:

[0058] (1) A composite microbial agent for preventing and controlling tomato wilt is prepared from the following raw materials in parts by weight: 14 parts of a mixed bacterial solution (prepared in Example 1), 33 parts of a carrier, 1 part of L-tyrosine, 1 part of sodium lignin sulfonate, and 1 part of soluble starch.

[0059] The carrier is composed of bentonite and biochar (commercially available) in a mass ratio of 2:1.

[0060] (2) Preparation method: First, add soluble starch into water for pre-dissolution, and when cooled to below 40°C, add the mixed bacterial solution to fully blend, then add the carrier, L-tyrosine and sodium lignin sulfonate and mix evenly, put into a wet granulator with a pore size of 2 mm and extrude to obtain a composite microbial agent.

[0061] Example 3

[0062] The same as Example 2, except that the composite microbial agent for controlling tomato wilt is prepared from the following raw materials in parts by weight: 12 parts of mixed bacterial solution (prepared in Example 1), 35 parts of carrier, 1 part of L-tyrosine, 1 part of sodium lignin sulfonate and 1 part of soluble starch.

[0063] Example 4

[0064] The same as Example 2, except that the composite microbial agent for controlling tomato wilt is prepared from the following raw materials in parts by weight: 11 parts of mixed bacterial solution (prepared in Example 1), 36 parts of carrier, 1 part of L-tyrosine, 1 part of sodium lignin sulfonate and 1 part of soluble starch.

[0065] Comparative Example 4

[0066] The same as Example 2, except that the composite microbial agent for controlling tomato wilt is prepared from the following raw materials in parts by weight: 16 parts of mixed bacterial solution (prepared in Example 1), 30 parts of carrier, 1 part of L-tyrosine, 1 part of sodium lignin sulfonate and 1 part of soluble starch.

[0067] Comparative Example 5

[0068] The same as Example 2, except that the composite microbial agent for controlling tomato wilt is prepared from the following raw materials in parts by weight: 10 parts of mixed bacterial solution (prepared in Example 1), 37 parts of carrier, 1 part of L-tyrosine, 1 part of sodium lignin sulfonate and 1 part of soluble starch.

[0069] Effect Example 2

[0070] The preparation states of the composite microbial agents prepared in Examples 2 to 4 and Comparative Examples 4 to 5 are shown in Table 2.

[0071] Table 2 Preparation status

[0072] project Preparation status Example 2 The preparation is solid, easy to granulate, and has good disintegration Example 3 The preparation is solid, easy to granulate, and has good disintegration Example 4 The preparation is solid, easy to granulate, and has good disintegration Comparative Example 4 The preparation is viscous, difficult to granulate, and cannot be formed Comparative Example 5 The preparation is semi-solid, brittle and difficult to shape

[0073] As can be seen from Table 2, the composite microbial agents prepared according to the ratios of Examples 2, 3, and 4 are solid in preparation state, easy to granulate, and have good disintegration. However, the composite microbial agents prepared according to the ratios of Comparative Examples 4 and 5 cannot be formed and do not meet the requirements.

[0074] Example 5

[0075] Field experiment on the efficacy of composite microbial agents on tomato wilt:

[0076] The experiment was conducted in the greenhouse of the vegetable base of Daguanting Village, Daguanting Town, Raoyang County, Hengshui City, Hebei Province from August 2024 to October 2024. The test tomato variety was Kaide, and the tomato wilt in this greenhouse has been more serious over the years. This embodiment has a total of 3 treatments, namely T1 treatment (4 kg / mu of composite microbial agent prepared in Example 3), T2 treatment (5 kg / mu of composite microbial agent prepared in Example 3) and CK treatment (blank treatment), each treatment was repeated 3 times, and each repetition was set to a 6m×2.5m community. The compound microbial agent was applied once on August 28, 2024, September 8, 2024, and September 18, 2024, respectively. The compound microbial agent was applied by broadcasting, and the same water and fertilizer management method was adopted. On the 7th and 14th days after the last application of the drug, each community investigated the incidence of 10 consecutive tomato plants.

[0077] Disease investigation methods:

[0078] Tomato wilt disease classification standard:

[0079] Level 0, the plant is healthy and symptom-free;

[0080] Level 1: 1 or 2 true leaves wilt and droop or turn yellow obviously, and even fall off;

[0081] Level 2: 3 or 4 true leaves turn yellow or the whole plant turns yellow, and the leaves wilt and droop;

[0082] Level 3: 5 or 6 true leaves obviously wilt and droop or turn seriously yellow, and the plant growth is inhibited and dwarfed;

[0083] Level 4: The whole plant wilts severely and even dies.

[0084] Disease index = ∑ (number of diseased plants at each level × corresponding level) / (total number of plants surveyed × maximum level) × 100;

[0085] Control effect (%) = (blank control disease index - treatment disease index) / blank control disease index × 100%.

[0086] Comparative Example 6

[0087] The same as Example 5, except that the application amount of the composite microbial agent for controlling tomato wilt (prepared in Example 3) is changed to 1 kg / mu.

[0088] Comparative Example 7

[0089] The same as Example 5, except that the application amount of the composite microbial agent for controlling tomato wilt (prepared in Example 3) is changed to 2 kg / mu.

[0090] Comparative Example 8

[0091] The same as Example 5, except that the application amount of the composite microbial agent for controlling tomato wilt (prepared in Example 3) is changed to 3 kg / mu.

[0092] Comparative Example 9

[0093] Same as Example 5, except that the three applications of the composite microbial agent were replaced by three sprayings of 75% thiophanate-methyl wettable powder at the recommended dosage (150 g / mu each time, 30 L / mu each time) as a positive control.

[0094] Comparative Example 10

[0095] The preparation method of the composite microbial agent is the same as that of Example 3, except that the carrier is replaced by bentonite alone. The test on the protective effect of the composite microbial agent on tomato wilt is the same as that of Example 5.

[0096] Comparative Example 11

[0097] The preparation method of the composite microbial agent is the same as that of Example 3, except that L-tyrosine is replaced by sodium carboxymethyl cellulose. The test on the protective effect of the composite microbial agent on tomato wilt is the same as that of Example 5.

[0098] Comparative Example 12

[0099] The preparation method of the composite microbial agent is the same as that of Example 3, except that sodium lignin sulfonate is replaced by sodium dodecylbenzene sulfate. The test on the protective effect of the composite microbial agent on tomato wilt is the same as that of Example 5.

[0100] Effect Example 3

[0101] The control effects of the composite microbial agent on tomato wilt in Example 5 and Comparative Examples 6 to 12 are shown in Table 3.

[0102] Table 3 Control effect of composite microbial agent on tomato wilt

[0103]

[0104] As can be seen from Table 3, the composite microbial agent prepared by the present invention has a disease index of 21.94 and 20.00 for tomato wilt in T1 treatment and T2 treatment on the 7th day after the last application, and the prevention effect is 57.98% and 61.70%. On the 14th day after application, the disease index of 22.50 and 22.22 for tomato wilt in T1 treatment and T2 treatment is 59.50% and 60.00%. Compared with Comparative Examples 6 to 8, the two dosages of T1 and T2 treatments in Example 5 have better control effects on tomato wilt, and there is no significant difference with the positive control (Comparative Example 9). Compared with Comparative Examples 10 to 12, the T1 and T2 treatments in Example 5 have better control effects on tomato wilt, indicating that the components used in the composite microbial agent of the present invention have good synergistic effects.

[0105] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite microbial agent for preventing and treating tomato wilt, characterized in that: Ingredients include: Bacillus valerianus, Bacillus subtilis, and Serratia marcescens.

2. The composite microbial agent according to claim 1, characterized in that: The raw materials include, by weight: 11 to 14 parts of mixed bacterial liquid, 33 to 36 parts of carrier, 1 to 2 parts of L-tyrosine, 1 to 2 parts of sodium lignin sulfonate and 1 to 2 parts of soluble starch; The mixed bacterial liquid is a mixed bacterial liquid of Bacillus valerius, Bacillus subtilis and Serratia marcescens.

3. The composite microbial agent according to claim 2, characterized in that: The mixed bacterial liquid consists of a bacterial liquid of Bacillus vallis, a bacterial liquid of Bacillus subtilis and a bacterial liquid of Serratia marcescens in a mass ratio of 3:1:

1.

4. The composite microbial agent according to claim 3, characterized in that: The OD values ​​of the Bacillus subtilis solution, the Bacillus subtilis solution and the Serratia marcescens solution are as follows: 600 Both are 2.

0.

5. The composite microbial agent according to claim 2, characterized in that: The carrier includes bentonite and biochar in a mass ratio of 2:

1.

6. A method for preparing the composite microbial agent for preventing and controlling tomato wilt according to any one of claims 2 to 5, characterized in that: The following steps are involved: Firstly, soluble starch is added into water for pre-dissolution, and when cooled to below 40°C, mixed bacterial solution is added, and then carrier, L-tyrosine and sodium lignin sulfonate are added and mixed evenly, and the composite microbial agent is obtained after granulation.

7. Use of the composite microbial agent according to any one of claims 1 to 5 in the preparation of an agent for preventing and treating tomato wilt.

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