Composite microbial agent for preventing and treating cotton verticillium wilt and preparation method thereof

Abstract

The invention discloses a composite microbial agent for preventing and treating cotton Verticillium wilt and a preparation method thereof. The composite microbial agent comprises the following raw materials in parts by weight: 20 to 60 parts of microbial powder, 40 to 70 parts of growth auxiliary materials, and 2 to 8 parts of desiccant. The microbial powder comprises Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez, and the Fusarium is deposited as CGMCC NO.11314. Fusarium and Trichoderma harzianum in the composite microbial agent have antagonistic effects on cotton Verticillium wilt, lactic acid bacteria, Bacillus cereus, and Bacillus velez are beneficial soil bacteria, and their metabolites contain regulating substances necessary for crop growth such as auxin, antibiotics, and cytokinins, and also have the functions of inhibiting the reproduction of Verticillium wilt, colonization and growth of functional bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of crop disease prevention and control, and specifically relates to a composite microbial agent for preventing and controlling cotton verticillium wilt and a preparation method and application thereof. Background Art

[0002] Cotton Verticillium wilt is a soil-borne vascular disease of cotton caused by Verticillium dahliae. The pathogen usually infects the roots of cotton, causing diseases in the roots of cotton and even the whole plant, causing significant economic losses. It is commonly known as a "cancer" disease, and there are no ideal prevention and control measures at home and abroad. Cotton is an important economic crop in Xinjiang. Due to continuous cropping and returning cotton stalks to the fields, the accumulation of diseased residues in the soil is increasing. As a soil-borne disease, the occurrence of cotton Verticillium wilt is getting worse year by year. The survey shows that the proportion of cotton fields with Verticillium wilt in Xinjiang exceeds 50%, and Verticillium wilt has become an important factor restricting cotton production in Xinjiang. Scholars at home and abroad have carried out a lot of research, such as breeding resistant varieties, crop rotation, developing soil fumigants and chemical fungicides, and the recent rise of microbial preparations. Unfortunately, due to the lack of host-resistant germplasm resources, there are no highly resistant cotton varieties; the biological control bacteria with Bacillus subtilis as the core are affected by the soil environment and climate, and the control effect is unstable; chemical fungicides are forced to be selected as the main measure for the prevention and control of cotton Verticillium wilt. However, the long-term use of chemical fungicides not only poses a threat to the ecological environment and human health, but also increases the resistance of cotton Verticillium wilt. Therefore, it is urgent to develop environmentally friendly measures to effectively control cotton Verticillium wilt. Biological control has the advantages of green environmental protection and great development potential, and has attracted people's attention and been applied in practice. Bacillus subtilis, Bacillus velez, Bacillus amyloliquefaciens, Bacillus cereus, Trichoderma harzianum, and Paecilomyces lilacinus are the most studied and applied.

[0003] In the early stage, the team used microbiome methods and combined with metagenomic sequencing technology to systematically analyze the microbial community structure of healthy soil and healthy cotton plants, the interaction between microorganisms and pathogens, and the relationship with the occurrence of diseases. Through the theory of ecological control, the endophytic antagonistic fungi and rhizosphere beneficial bacteria are combined to form a composite microbial group, which is released into the soil, integrating growth promotion, occupation, antagonism, and induced immunity. It is of great significance for preventing and controlling cotton wilt and improving the economic benefits of cotton. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to provide a composite microbial agent for preventing and treating cotton Verticillium wilt and a preparation method thereof.

[0005] The technical scheme of the present invention is: a composite microbial agent, comprising the following raw materials in parts by weight: 20 to 60 parts of microbial powder, 40 to 70 parts of growth auxiliary materials, and 2 to 8 parts of desiccant, wherein the microbial powder comprises Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez. The preservation number of Fusarium is CGMCC NO.11314, and the strain has been disclosed in a patent with application number CN201610425079.1. The present invention has no special limitation on the sources of Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez.

[0006] Furthermore, the ratio of the number of live bacteria of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus and Bacillus velez is 2-4:3-5:1-2:1-2:1-2, preferably 2:4:1:1:1.

[0007] Furthermore, the total viable count of the composite microbial agent is not less than 5×10 9 CFU / g.

[0008] Furthermore, the growth auxiliary material is chitosan or fulvic acid or any mixture thereof.

[0009] Furthermore, the desiccant is bentonite or attapulgite, or any mixture thereof.

[0010] The present invention also discloses a method for preparing a composite microbial agent, comprising the following steps:

[0011] (1) Prepare seed solutions of Fusarium spp., Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velezii, respectively.

[0012] (2) wheat bran or corn grits or any mixture thereof is used as a matrix, the moisture content of which is controlled to be 30-50%, and the microbial seed solution prepared in step (1) is added, wherein the inoculation amount of the microbial seed solution is 20-50 ml seed solution / kg matrix.

[0013] (3) fermenting the substrate to which the microbial seed solution is added in step (2) at a temperature of 25 to 35° C. for 5 to 7 days, and then drying the fermented culture at 35 to 40° C. and crushing the culture to obtain microbial powder.

[0014] (4) The microbial powder in step (3) is mixed with growth aids and a desiccant to obtain a composite microbial agent.

[0015] The composite microbial agent of the present invention is used for preventing and controlling Verticillium wilt of cotton. Applying the composite microbial agent to the roots of cotton by drip irrigation can effectively prevent and control Verticillium wilt of cotton, increase cotton yield and improve fiber quality. The specific method is as follows:

[0016] The composite microbial agent of the present invention is applied to the roots of cotton by drip irrigation in the early stage of cotton Verticillium wilt from mid-to-late June to early July, with a dosage of 0.25-0.75 kg / mu each time and applied 1-2 times, which can effectively prevent and control cotton Verticillium wilt, increase cotton yield and improve fiber quality.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The Fusarium and Trichoderma harzianum in the composite microbial agent of the present invention have antagonistic effects on cotton Verticillium wilt pathogen, while lactic acid bacteria, Bacillus cereus and Bacillus velez are beneficial soil bacteria, and their metabolites contain regulatory substances necessary for crop growth such as auxin, antibiotics and cytokinins, and also have the function of inhibiting the reproduction of Verticillium wilt pathogen and the colonization and growth of functional bacteria.

[0019] The synergistic effect of antagonistic fungi such as Fusarium sphaeroides and Trichoderma harzianum and beneficial bacteria such as lactic acid bacteria, Bacillus cereus and Bacillus velezii enhanced the inhibitory effect on Verticillium wilt, while increasing the diversity of microorganisms in the soil, achieving a balance in the soil microecology, thereby enhancing the absorption and utilization efficiency of nutrients by the cotton roots, improving root vitality, further increasing cotton yield and improving fiber quality.

[0020] The preparation method of the invention has simple process and is easy to operate; the prepared composite microbial agent has no pollution to the environment and has low cost. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0022] The present invention provides a composite microbial agent, comprising the following raw materials in parts by weight: 20 to 60 parts of microbial powder, 40 to 70 parts of growth auxiliary materials, and 2 to 8 parts of desiccant, wherein the microbial powder comprises Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez. The Fusarium is deposited with CGMCC NO.11314, and the strain has been disclosed in the patent with application number CN201610425079.1. The present invention has no special limitation on the sources of Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez.

[0023] In the present invention, preferably, the ratio of the number of live bacteria of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez is 2-4:3-5:1-2:1-2:1-2, and more preferably 2:4:1:1:1. The total number of live bacteria of the microbial composite agent is not less than 5×10 9 CFU / g.

[0024] In the present invention, the method for preparing the microbial seed liquid is as follows: the strains of Fusarium and Trichoderma harzianum are respectively activated and cultured in liquid for 2 days, the activation culture medium is calculated as 1 L, including 2g NaNO3, 1g K2HPO4, 0.5g KCI, 0.5g MgSO4·7H2O, 0.01g FeSO4, and 30g sucrose, and the activation temperature is 25°C, so as to obtain the seed liquid of Fusarium and Trichoderma harzianum. The lactic acid bacteria, Bacillus cereus, and Bacillus velez are respectively activated and cultured in liquid for 1 day, the activation culture medium is calculated as 1 L, including 10g peptone, 5g yeast extract, and 10g NaCl, and the activation temperature is 28°C, so as to obtain the seed liquid of lactic acid bacteria, Bacillus cereus, and Bacillus velez.

[0025] In the present invention, the method for preparing a microbial fermentation matrix comprises: using wheat bran, corn grits or any mixture thereof as a matrix, preferably a mixture of wheat bran and corn grits; controlling the moisture content to be 30-50%, preferably 35-40%; adding microbial seed liquid of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus and Bacillus velez. The inoculation amount of the microbial seed liquid is 20-50 ml seed liquid / kg matrix.

[0026] In the present invention, the substrate with the added microbial seed liquid of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus and Bacillus velez is fermented and cultured at a temperature of 25 to 35°C, preferably 30°C; the culture time is 5 to 7 days. The total viable count of the microbial composite agent is continuously monitored. When the total viable count of the microbial composite agent is 5×10 9 CFU / g, the fermentation culture is dried at 35-40°C, preferably at 38°C; crushed, and passed through a 200-mesh sieve to obtain microbial powder.

[0027] In the present invention, the microbial powder is mixed with a growth auxiliary material and a desiccant to obtain a composite microbial agent. Preferably, the growth auxiliary material is chitosan, fulvic acid or any mixture thereof. Preferably, the desiccant is bentonite, attapulgite or any mixture thereof.

[0028] Example 1 Preparation of composite microbial agent

[0029] The microbial composite agent includes the following raw materials in parts by weight: 40 parts of microbial powder, 60 parts of growth auxiliary materials, and 5 parts of desiccant. The microbial powder includes Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez. The Fusarium uses the Fusarium with a preservation number of CGMCC NO.11314.

[0030] In this embodiment, the ratio of the number of live bacteria of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez is 2:4:1:1:1, and the total number of live bacteria of the microbial composite bacterial agent is not less than 5×10 9 CFU / g.

[0031] In this embodiment, the method for preparing the microbial seed solution is as follows: the strains of Fusarium and Trichoderma harzianum are respectively activated and cultured in liquid for 2 days, and the activation culture medium, in terms of 1 L, includes 2g NaNO3, 1g K2HPO4, 0.5g KCI, 0.5g MgSO4·7H2O, 0.01g FeSO4, and 30g sucrose, and the activation temperature is 30°C, to obtain the seed solution of Fusarium and Trichoderma harzianum. The lactic acid bacteria, Bacillus cereus, and Bacillus velez are respectively activated and cultured in liquid for 1 day, and the activation culture medium, in terms of 1 L, includes 10g peptone, 5g yeast extract, and 10g NaCl, and the activation temperature is 28°C, to obtain the seed solution of lactic acid bacteria, Bacillus cereus, and Bacillus velez.

[0032] In this embodiment, the method for preparing a microbial fermentation matrix is ​​as follows: wheat bran is used as a matrix, the water content is controlled to be 35%, and microbial seed liquids of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velezii are added, and the inoculation amount of the microbial seed liquid is 10 ml seed liquid / kg matrix.

[0033] In this embodiment, the substrate with the added microbial seed solution of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez was fermented and cultured at a temperature of 30°C for 5 to 7 days. The total viable count of the microbial composite inoculant was continuously monitored. When the total viable count of the microbial composite inoculant was greater than 5×10 9 CFU / g, the fermentation culture was dried at 38°C, crushed, and passed through a 200-mesh sieve to obtain microbial powder.

[0034] In the present invention, the microbial powder is mixed with a growth auxiliary material and a desiccant to obtain a composite microbial agent. The growth auxiliary material is chitosan, fulvic acid or any mixture thereof. Preferably, the desiccant is bentonite.

[0035] Example 2 Preparation of composite microbial agent

[0036] The microbial composite agent comprises the following raw materials in parts by weight: 20 parts of microbial powder, 40 parts of growth auxiliary materials, and 3 parts of desiccant. The microbial powder comprises Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez.

[0037] In this embodiment, the ratio of the number of live bacteria of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez is 2:3:1:1:1, and the total number of live bacteria of the microbial composite agent is 5×10 9 CFU / g.

[0038] The method for preparing the microbial seed solution is the same as that in Example 1.

[0039] In this embodiment, the method for preparing the microbial fermentation matrix is ​​as follows: the matrix is ​​used, the water content is controlled to be 40%, and Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez are added. The inoculation amount of the microbial seed liquid is 50 ml seed liquid / kg matrix.

[0040] In this embodiment, the substrate with the added microbial seed solution of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez was fermented and cultured at a temperature of 35°C for 5 to 7 days. The total viable count of the microbial composite inoculant was continuously monitored. When the total viable count of the microbial composite inoculant was 5×10 9 CFU / g, the fermentation culture was dried at 40°C, crushed, and passed through a 200-mesh sieve to obtain microbial powder.

[0041] In the present invention, microbial powder is mixed with growth auxiliary materials and desiccant to obtain a composite microbial agent, wherein the growth auxiliary material is fulvic acid and the desiccant is attapulgite.

[0042] Example 3 Preparation of composite microbial agent

[0043] The microbial composite agent includes the following raw materials in parts by weight: 60 parts of microbial powder, 40 parts of growth auxiliary materials, and 8 parts of desiccant. The microbial powder includes Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez.

[0044] In this embodiment, the ratio of the number of live bacteria of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez is 4:5:2:2:2, and the total number of live bacteria of the microbial composite agent is 5×10 9 CFU / g.

[0045] In this example, the method for preparing the microbial seed solution is the same as that in Example 1.

[0046] In this embodiment, the method for preparing a microbial fermentation matrix is ​​as follows: wheat bran is used as a matrix, the water content is controlled to be 40%, and microbial seed liquids of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velezii are added, and the inoculation amount of the microbial seed liquid is 30 ml seed liquid / kg matrix.

[0047] In this embodiment, the substrate with the added microbial seed solution of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez was fermented and cultured at a temperature of 33°C for 5 to 7 days. The total viable count of the microbial composite inoculant was continuously monitored. When the total viable count of the microbial composite inoculant was 5×10 9 CFU / g, the fermentation culture was dried at 40°C, crushed, and passed through a 200-mesh sieve to obtain microbial powder.

[0048] In the present invention, microbial powder is mixed with growth auxiliary materials and desiccant to obtain a composite microbial agent, wherein the growth auxiliary material is chitosan and the desiccant is bentonite.

[0049] Experimental example: Disease prevention effect of compound microbial agent in the field

[0050] The composite microbial agents obtained in Example 1, Example 2, and Example 3 were subjected to field tests to identify the disease prevention effect of the composite microbial agents on cotton Verticillium wilt. The field test was conducted in Jinghe County, Bortala Prefecture, Xinjiang, and plots with relatively uniform fertility, flat terrain, and a high incidence of Verticillium wilt in the past were selected as the test fields. The test set up 4 treatments, arranged in random blocks, repeated 3 times, and the plot area was 200m 2 . Planting density 9,000 to 10,000 plants / 666.67m 2 , one-film three-row planting mode, and field management are carried out in accordance with high-quality cotton cultivation technology.

[0051] Treatment group 1: The composite microbial agent obtained in Example 1 was drip-irrigated to the roots of cotton at the early stage of cotton bud formation, with a dosage of 0.75 kg / mu each time, once every 10 days, for a total of 2 times;

[0052] Treatment group 2: The composite microbial agent obtained in Example 2 was drip-irrigated to the roots of cotton at the early stage of cotton bud formation, with a dosage of 0.75 kg / mu each time, once every 10 days, for a total of 2 times;

[0053] Treatment group 3: The composite microbial agent obtained in Example 3 was drip-irrigated to the roots of cotton at the early stage of cotton bud formation, with a dosage of 0.75 kg / mu each time, once every 10 days, for a total of 2 times;

[0054] Control group: Bacillus subtilis microbial agent (commercially available) was applied in the same manner and dosage as the treatment group.

[0055] Drip irrigation of composite microbial agents and Bacillus cereus microbial agents was carried out on June 25 and July 5, respectively. The incidence of cotton Verticillium wilt was investigated on July 30 and August 25. Field investigation and seed testing were carried out on September 30 (two points were randomly selected in each plot, and 20 plants were continuously taken from each point for field investigation and seed testing. The number of bolls and the number of fallen bolls were investigated, and 30 bolls were picked and weighed indoors. Cotton picking began on October 8 and was completed on October 25. During the harvest period, each plot was collected and weighed and accumulated. The results are shown in Tables 1 and 2. The treatment of cotton with composite microbial agents can significantly reduce the harm of cotton Verticillium wilt. The disease index of cotton Verticillium wilt on July 30 was 14.2-22.8, and the disease prevention effect was 53.28%-59.54%. The disease index on August 25 was 21.6-25.3, and the disease prevention effect was 53.06%-59.93%. The compound microbial agent also had a significant effect on increasing cotton yield. The number of bolls per mu of cotton treated with the compound microbial agent was 77025-79861, an increase of 10.7%-14.8% over the control group. The yield per mu of cotton treated with the compound microbial agent was 429.6-445.4 kg, an increase of 8.8%-12.8% over the control group.

[0056] Table 1 Control effect of composite microbial agent on cotton Verticillium wilt in the field

[0057]

[0058] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0059] Table 2 Results of cotton yield measurement by composite microbial agent treatment in the field

[0060]

[0061] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0062] In summary, the composite microbial agent in the present invention has a good preventive and therapeutic effect on cotton Verticillium wilt, with a preventive and therapeutic effect of more than 50%, and can significantly increase cotton yield by about 10%. In addition, it can also improve fiber quality, improve the local cotton planting technology level, help local cotton farmers increase their income and become rich, and provide strong support for promoting rural revitalization.

Claims

1. A composite microbial agent, characterized in that: The invention comprises the following raw materials in parts by weight: 20 to 60 parts of microbial powder, 40 to 70 parts of growth auxiliary materials, and 2 to 8 parts of desiccant. The microbial powder comprises Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez. The preservation number of Fusarium is CGMCC NO.11314. The ratio of the number of live bacteria of Fusarium, Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velez is 2:4:1:1:

1. The total number of live bacteria of the composite microbial agent is not less than 5×10 9 CFU / g.

2. A composite microbial agent according to claim 1, characterized in that: The growth auxiliary material is chitosan or fulvic acid or any mixture thereof.

3. A composite microbial agent according to claim 1, characterized in that: The desiccant is bentonite or attapulgite or any mixture thereof.

4. The method for preparing a composite microbial agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) preparing seed solutions of Fusarium spp., Trichoderma harzianum, lactic acid bacteria, Bacillus cereus, and Bacillus velezensis, respectively; (2) wheat bran or corn grits or any mixture thereof is used as a matrix, the moisture content is controlled to be 30-50%, and the microbial seed solution prepared in step (1) is added, and the inoculation amount of the microbial seed solution is 20-50 ml seed solution / kg matrix; (3) fermenting the substrate to which the microbial seed solution was added in step (2) at a temperature of 25 to 35° C. for 5 to 7 days, and then drying the fermented culture at 35 to 40° C. and crushing it to obtain microbial powder; (4) The microbial powder in step (3) is mixed with growth aids and a desiccant to obtain a composite microbial agent.

5. Use of the composite microbial agent according to any one of claims 1 to 3 for preventing and controlling cotton Verticillium wilt.

6. The use according to claim 5, characterized in that The method for preventing and controlling cotton Verticillium wilt is: apply the composite microbial agent to the roots of cotton by drip irrigation in the early stage of cotton Verticillium wilt from mid-to-late June to early July, with a dosage of 0.25-0.75 kg / mu each time, and apply 1-2 times.

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