Compound microbial agent for preventing and treating tomato wilt and preparation method thereof

Abstract

The application discloses a kind of prevention and cure tomato wilt complex microbial inoculant and preparation method thereof, belong to the technical field of microbial inoculant preparation. Prevention and cure tomato wilt complex microbial inoculant, by mass fraction, raw material includes: mixed bacterial solution 11~14 parts, carrier 33~36 parts, L-tyrosine 1~2 parts, sodium lignosulfonate 1~2 parts and soluble starch 1~2 parts;Mixed bacterial solution is composed of dead bacillus subtilis bacterial solution, bacillus subtilis bacterial solution and Serratia marcescens bacterial solution with mass ratio of 3:1:1. The complex microbial inoculant of the application is environment-friendly, safe to use, can significantly improve the stress resistance of tomato, and has good prevention and treatment effect on tomato wilt.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial agent preparation, in particular to a compound microbial agent for preventing and treating tomato wilt and a preparation method thereof. BACKGROUND

[0002] Tomato wilt is one of the most serious soil-borne diseases in facility tomato production. In recent years, with the continuous increase of facility tomato planting area, tomato wilt transmitted by soil has also become serious, becoming a bottleneck problem in facility tomato production. Tomato wilt is caused by the infection of Fusarium oxysporum f. sp. lycopersici, belonging to the subdivision of Deuteromycotina. Studies have shown that the incidence of wilt in fields with continuous cropping for many years is as high as 20-40%, and has a tendency to increase year by year. Tomato wilt mainly damages the roots and stem base of tomato, and often begins to occur during the flowering and fruiting period, invading the vascular tissue of the roots and causing the death of the plants. According to statistics, more than 40% of the yield of tomato is reduced due to wilt each year, and even causes absolute yield loss in severe cases. Therefore, it is difficult to prevent and control.

[0003] The prevention and control of tomato wilt in the prior art mainly includes the following methods: 1. Using disease-resistant plant varieties, but this method is not enough to resist multiple pathogenic fungi, and the resistance is easy to lose; 2. Chemical control, which has a quick effect, but large-scale use will lead to drug resistance of pathogenic fungi, and excessive residues of pesticides will cause environmental pollution and harm human health; 3. Biocontrol agent, which is safe to crops and human body, green and environmentally friendly, and has a simple production process and can promote crop growth. However, the existing microbial agents still have many problems in biological control application, such as easy loss of activity, low release rate, unstable drug efficacy, short preservation time, easy contamination and degradation of strains, etc. SUMMARY

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

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions.

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

[0007] Further, the compound microbial agent, in terms of mass fraction, includes 11-14 parts of mixed bacterial solution, 33-36 parts of carrier, 1-2 parts of L-tyrosine, 1-2 parts of sodium lignosulfonate and 1-2 parts of soluble starch.

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

[0009] Further, the mixed bacteria liquid is composed of Bacillus amyloliquefaciens liquid, Bacillus subtilis liquid and Serratia marcescens liquid with a mass ratio of 3:1:1.

[0010] Further, the OD of the Bacillus amyloliquefaciens liquid, the Bacillus subtilis liquid and the Serratia marcescens liquid is 2.0. 600

[0011] Further, the preparation method of the mixed bacteria liquid comprises the following steps:

[0012] The Bacillus amyloliquefaciens, the Bacillus subtilis and the Serratia marcescens are inoculated in solid LB culture medium respectively, and cultured at 37 DEG C for 48 hours to obtain activated Bacillus amyloliquefaciens, Bacillus subtilis and Serratia marcescens; the activated Bacillus amyloliquefaciens, Bacillus subtilis and Serratia marcescens are inoculated in liquid LB culture medium respectively, and cultured at 37 DEG C with 220 r / min of shaking bed for 12 hours, and the concentration of the bacteria liquid is adjusted to OD600 = 2.0 to obtain Bacillus amyloliquefaciens liquid, Bacillus subtilis liquid and Serratia marcescens liquid;

[0013] The Bacillus amyloliquefaciens liquid, the Bacillus subtilis liquid and the Serratia marcescens liquid are mixed uniformly with a mass ratio of 3:1:1 to obtain the mixed bacteria liquid.

[0014] The solid LB culture medium comprises 10.0 g of proteose peptone, 10.0 g of NaCl, 5 g of yeast powder, 20 g of agar, and distilled water is added to 1 L, and the pH value is 7.0.

[0015] The liquid LB culture medium comprises 10.0 g of proteose peptone, 3.0 g of NaCl, 3 g of yeast powder, and distilled water is added to 1 L, and the pH value is 7.0.

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

[0017] The composite microbial inoculant of the application takes Bacillus amyloliquefaciens, Bacillus subtilis and Serratia marcescens as effective bacteria groups, bentonite and biochar as carriers, soluble starch as a binder, L-tyrosine as a stabilizer, and sodium lignosulfonate as a wetting agent, and has good tomato fusarium wilt prevention effect.

[0018] The second technical scheme of the application is a preparation method of the composite microbial inoculant for preventing and treating tomato fusarium wilt, which comprises the following steps:

[0019] First, the soluble starch is added to water for pre-dissolution, and then the mixed bacteria liquid is added when the temperature is cooled to below 40 DEG C, and then the carrier, L-tyrosine and sodium lignosulfonate are mixed uniformly, and the composite microbial inoculant is obtained after granulation. ​

[0020] The third technical solution of the present application is application of the above-mentioned composite microbial agent in preparation of a medicine for preventing and treating tomato fusarium wilt.

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

[0022] (1) The present application adopts the combination of Bacillus vallismortis, Bacillus subtilis and Serratia marcescens, which is reasonable and has strong complementarity. After use, the beneficial bacteria can rapidly reproduce to form a dominant population, and has a synergistic and synergistic effect on the inhibition of tomato fusarium wilt fungus. And the composite microbial agent prepared by using the bacterial population combination has excellent prevention and treatment effect on tomato fusarium wilt.

[0023] (2) The bentonite and biochar in the composite microbial agent of the present application have good biocompatibility with microorganisms. The bentonite and biochar can adsorb and fix the microbial liquid, thereby improving the stability and activity of the microorganisms and enhancing the prevention and treatment effect on tomato fusarium wilt. Moreover, the bentonite and biochar have strong adsorption and hydrophilicity, and can slow down the loss of soil nutrients and water after being applied to the soil, enhance the soil water and fertilizer retention capacity, and promote the development of tomato root system.

[0024] (3) The preparation method of the composite microbial agent of the present application is simple and easy to operate, easy to store and transport. Moreover, the materials used for preparing the composite microbial agent have good biocompatibility with microorganisms, high safety and low cost, are easy to obtain in the market, and are easy to industrialize production, which has good development and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0026] Figure 1 Inhibition effect of the mixed bacterial liquid prepared for the present application example 1 on tomato fusarium wilt fungus;

[0027] Figure 2 Inhibition effect of the mixed bacterial liquid prepared for the present application example 1 on tomato fusarium wilt fungus;

[0028] Figure 3 Inhibition effect of the mixed bacterial liquid prepared for the present application example 1 on tomato fusarium wilt fungus;

[0029] Figure 4 Inhibition effect of the mixed bacterial liquid prepared for the present application example 1 on tomato fusarium wilt fungus. DETAILED DESCRIPTION

[0030] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather as a description of certain aspects, features and embodiments of the application.

[0031] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of "about" in relation to a value or range of values is intended to include each individual intermediate value and each smaller range that falls within the range of values. The upper and lower limits of these smaller ranges can independently be included or excluded from the range.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to the disclosure.

[0033] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of this application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples in the specification including examples of the application solely are exemplary.

[0034] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" and the like are open-ended terms that are intended to permit but not limit the inclusion of elements or the number of elements not expressly listed by the recitation.

[0035] The "parts" in the following examples are "mass parts".

[0036] In the embodiment of the application, the solid LB culture medium is composed of 10.0 g of proteose peptone, 10.0 g of NaCl, 5 g of yeast powder, 20 g of agar, and distilled water to make up to 1 L, and the pH value is 7.0.

[0037] In the embodiment of the application, the liquid LB culture medium is composed of 10.0 g of proteose peptone, 3.0 g of NaCl, and 3 g of yeast powder, and distilled water to make up to 1 L, and the pH value is 7.0.

[0038] In the specific embodiment of the present application, Bacillus vallismortis is preserved in the China General Microbiological Culture Collection Center, Beijing, China, with a preservation number of CGMCC NO. 21871 and a preservation date of March 5, 2021; serratia sp. is preserved in the China General Microbiological Culture Collection Center, Beijing, China, with a preservation number of CGMCC NO. 7340 and a preservation date of March 20, 2013; Bacillus subtilis is purchased from the China Industrial Microbial Culture Collection Center, with a product number of CICC 24713; and Bacillus amyloliquefaciens is purchased from the China Industrial Microbial Culture Collection Center, with a product number of CICC 20229.

[0039] In the specific embodiment of the present application, the activation method of Bacillus vallismortis, Bacillus subtilis, serratia sp. and Bacillus amyloliquefaciens is as follows: Bacillus vallismortis, Bacillus subtilis, serratia sp. and Bacillus amyloliquefaciens are inoculated in solid LB medium respectively, and cultured at 37℃ for 48h to obtain activated Bacillus vallismortis, Bacillus subtilis and serratia sp.

[0040] Example 1

[0041] Plate antagonism test of the complex microbial flora on tomato Fusarium wilt fungus:

[0042] The plate confrontation method is used to test the antibacterial effect of the complex microbial flora on tomato Fusarium wilt, specifically as follows:

[0043] (1) The activated Bacillus vallismortis, Bacillus subtilis and serratia sp. are inoculated in fresh LB liquid medium, and cultured at 37℃, 220r / min for 12h, and the concentrations of the three bacterial liquids are adjusted to OD 600 =2.0 to obtain Bacillus vallismortis fermentation broth, Bacillus subtilis fermentation broth and serratia sp. fermentation broth. The Bacillus vallismortis fermentation broth, Bacillus subtilis fermentation broth and serratia sp. fermentation broth are mixed uniformly at a mass ratio of 3:1:1 to obtain a mixed bacterial liquid.

[0044] (2) After the tomato Fusarium wilt fungus is punched with a 5mm gun head, it is inoculated in the center of PDA medium, and then 2μL of the above prepared mixed bacterial liquid is added dropwise at a distance of 2.5cm from the center of the medium. An equal amount of sterile water is added as a control, and each treatment is repeated three times. Culturing is carried out in a 28℃ incubator, and when the control grows to 3 / 4 of the culture dish, the inhibition rate is measured and calculated.

[0045] Comparative Example 1

[0046] The same as example 1, the only difference is that Bacillus subtilis is replaced by an equal amount of Bacillus amyloliquefaciens.

[0047] Comparative example 2

[0048] The same as example 1, the only difference is that Bacillus subtilis is replaced by an equal amount of Bacillus amyloliquefaciens.

[0049] Comparative example 3

[0050] The same as example 1, the only difference is that Serratia marcescens is replaced by an equal amount of Bacillus amyloliquefaciens.

[0051] Example 1

[0052] The inhibition rates of examples 1 and comparative examples 1-3 on Fusarium solani f. sp. lycopersici are shown in Table 1 and Figures 1 to 4 .

[0053] Table 1 Inhibition rate

[0054] Item Bacteriostatic 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] As can be seen from Table 1, the inhibition rate of the mixed bacteria solution prepared in example 1 of the present application on Fusarium solani f. sp. lycopersici is 72.12%, compared with comparative examples 1-3, the combination of beneficial bacteria of the present application has the highest inhibition rate on Fusarium solani f. sp. lycopersici, and from Figures 1 to 4 As can be seen from Table 1, the inhibition rate of the mixed bacteria solution prepared in example 1 of the present application on Fusarium solani f. sp. lycopersici is 72.12%, compared with comparative examples 1-3, the combination of beneficial bacteria of the present application has the highest inhibition rate on Fusarium solani f. sp. lycopersici, and from

[0056] Example 2

[0057] A preparation method of a complex microbial agent for preventing and treating tomato fusarium wilt:

[0058] (1) The complex microbial agent for preventing and treating tomato fusarium wilt is made from the following raw materials in mass fraction: mixed bacteria solution (prepared in example 1) 14 parts, carrier 33 parts, L-tyrosine 1 part, sodium lignosulfonate 1 part and soluble starch 1 part.

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

[0060] (2) The preparation method is as follows: first, the soluble starch is added to water and pre-dissolved, and then the mixed bacteria solution is added when the temperature is cooled to below 40℃ for sufficient fusion, and then the carrier, L-tyrosine and sodium lignosulfonate are added and mixed uniformly, and then extruded into a shape in a wet granulator with a pore size of 2mm to obtain the complex microbial agent.

[0061] Example 3

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

[0063] Example 4

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

[0065] Comparative Example 4

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

[0067] Comparative Example 5

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

[0069] Example 2

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

[0071] Table 2 Preparation state

[0072] Item Formulation state Example 2 The formulation is solid, easy to granulate, and has good disintegrability Example 3 The formulation is solid, easy to granulate, and has good disintegrability Example 4 The formulation is solid, easy to granulate, and has good disintegrability Comparative Example 4 The formulation is mucilaginous, difficult to granulate, and cannot be shaped Comparative Example 5 The formulation is semi-solid, the formulation is crisp, and difficult to shape

[0073] As can be seen from Table 2, the complex microbial agents prepared according to the formulations of Examples 2, 3, and 4 are in solid form, are easy to granulate, and have good disintegrability. However, the complex microbial agents prepared according to the formulations of Comparative Examples 4 and 5 cannot be formed into a shape and do not meet the requirements.

[0074] Example 5

[0075] Field test of the effect of the complex microbial agent on tomato wilt:

[0076] The test was carried out in a greenhouse in Heguanting Village, Raoyang County, Hengshui City, Hebei Province from August 2024 to October 2024. The tomato variety used in the test was Kaid, and the greenhouse had been suffering from tomato wilt for many years. This example had 3 treatments, T1 treatment (4 kg / mu of the composite microbial inoculant prepared in Example 3), T2 treatment (5 kg / mu of the composite microbial inoculant prepared in Example 3) and CK treatment (blank treatment), each treatment was repeated 3 times, and each repeat was set as a 6m x 2.5m plot. Each was applied once on August 28, 2024, September 8, 2024, and September 18, 2024. The composite microbial inoculant was applied by spreading, and the same water and fertilizer management method was used. Seven days and 14 days after the last application, the disease incidence of 10 consecutive tomato plants in each plot was investigated.

[0077] Disease investigation method:

[0078] Tomato wilt disease grading standard was used:

[0079] 0 level, healthy plants without symptoms;

[0080] 1 level, 1 or 2 true leaves wilted and drooping or obviously yellowing, resulting in shedding;

[0081] 2 level, 3 or 4 true leaves yellowing or whole plant yellowing, leaf wilted and drooping;

[0082] 3 level: 5 or 6 true leaves wilted and drooping or true leaves severely yellowing, plant growth inhibited and dwarfed;

[0083] 4 level: whole plant severely wilted and dead.

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

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

[0086] Comparative Example 6

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

[0088] Comparative Example 7

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

[0090] Comparative Example 8

[0091] The same as Example 5, except that the amount of the complex microbial agent for preventing and treating tomato wilt (prepared in Example 3) was changed to 3 kg / acre.

[0092] Comparative Example 9

[0093] The same as Example 5, except that the three times of scattering the complex microbial agent was replaced by spraying three times of 75% chlorothalonil wettable powder at the recommended dose (150 g / acre each time, and 30 L / acre of water each time) as a positive control.

[0094] Comparative Example 10

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

[0096] Comparative Example 11

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

[0098] Comparative Example 12

[0099] The preparation method of the complex microbial agent was the same as Example 3, except that sodium lignosulfonate was replaced by sodium dodecyl benzene sulfate, and the test of the prevention effect of the complex microbial agent on tomato wilt was the same as Example 5.

[0100] Example 3

[0101] The prevention effects of the complex microbial agents in Examples 5 and Comparative Examples 6 to 12 on tomato wilt are shown in Table 3.

[0102] Table 3 Prevention effects of complex microbial agents on tomato wilt

[0103]

[0104] As can be seen from Table 3, the disease index of T1 treatment and T2 treatment of the composite microbial inoculant prepared in the application on the 7th day after the last application was 21.94 and 20.00, and the prevention effect was 57.98% and 61.70%. The disease index of T1 treatment and T2 treatment of the composite microbial inoculant prepared in the application on the 14th day after the last application was 22.50 and 22.22, and the prevention effect was 59.50% and 60.00%. Compared with Comparative Examples 6-8, the prevention effect of T1 and T2 treatments of Example 5 on tomato fusarium wilt was better, and there was no significant difference with the positive control (Comparative Example 9). Compared with Comparative Examples 10-12, the prevention effect of T1 and T2 treatments of Example 5 on tomato fusarium wilt was better, indicating that the components used in the composite microbial inoculant of the application had good synergistic effect.

[0105] The above examples only describe the preferred modes of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements of the technical solutions of the application made by those skilled in the art shall fall within the protection scope determined by the claims of the application.

Claims

1. A complex microbial inoculant for controlling Fusarium wilt of tomato, characterized by, The raw materials include a mixed bacterial solution composed of Bacillus vallismortis, Bacillus subtilis and Serratia sp. The Latin name of the Bacillus vallismortis is Bacillus vallismortis, the preservation number is CGMCC NO.21871, and the preservation date is March 5, 2021, which is preserved in the China General Microbiological Culture Collection Center. The Latin name of the Serratia sp. is Serratia sp., the preservation number is CGMCC NO.7340, and the preservation date is March 20, 2013, which is preserved in the China General Microbiological Culture Collection Center. The Bacillus subtilis is purchased from the China Industrial Microbial Culture Collection Center, and the product number is CICC 24713. The mixed bacterial solution is composed of Bacillus vallismortis, Bacillus subtilis and Serratia sp. with a mass ratio of 3:1:

1. The OD600 of the Bacillus vallismortis, Bacillus subtilis and Serratia sp. is 2.

0.

2. The complex microbial agent according to claim 1, characterized by, The raw materials include, in mass parts: 11-14 parts of the mixed bacterial solution, 33-36 parts of the carrier, 1-2 parts of L-tyrosine, 1-2 parts of sodium lignosulfonate and 1-2 parts of soluble starch. The carrier includes bentonite and biochar with a mass ratio of 2:

1.

3. A method for preparing the complex microbial agent for controlling tomato wilt according to any one of claims 1 to 2, characterized by, The method includes the following steps: First, the soluble starch is pre-dissolved in water, and when it is cooled to below 40℃, the mixed bacterial solution is added, then the carrier, L-tyrosine and sodium lignosulfonate are added and mixed uniformly, and the granulation is carried out to obtain the compound microbial agent.

4. Use of the compound microbial agent of any one of claims 1-2 in the preparation of a medicine for preventing and treating tomato fusarium wilt.

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