Biocontrol complex microbial inoculant for preventing and treating curvularia maize leaf spot disease and application of biocontrol complex microbial inoculant

Abstract

The invention discloses a biocontrol complex microbial inoculant for preventing and treating maize curvularia leaf spot and application thereof, and belongs to the field of crop disease prevention and treatment. The biocontrol complex microbial inoculant comprises the following active ingredients: bacillus cereus, pseudomonas chlororaphis and streptomyces microflavus, and / or metabolites thereof. The beneficial microorganisms with different action mechanisms are combined, and the prevention and control effect on the curvularia maize leaf spot disease is enhanced through the synergistic effect. Natural microbial resources are adopted to replace traditional chemical pesticides, harmful substance residues are reduced, and the ecological environment is protected. Through reasonable matching of different types of beneficial microorganisms, effective prevention and control of the curvularia maize leaf spot disease are realized, and healthy growth of plants can be promoted. Compared with a single strain preparation, the complex microbial inoculant provided by the invention has stronger stability and durability, and can significantly reduce the incidence rate of diseases.

Description

Technical Field

[0001] The invention relates to the field of crop disease prevention and control, and in particular to a biocontrol composite bacterial agent for preventing and controlling corn Curvularia leaf spot disease and application thereof. Background Art

[0002] As one of the most important food crops in the world, corn is widely planted in my country and even around the world. However, due to its long growth cycle and strong environmental adaptability, corn is susceptible to various diseases during its growth. Among them, Curvularia leaf spot is one of the main fungal diseases that endanger corn production. The disease is mainly caused by Curvularia lunata, a fungus of the genus Curvularia in the subphylum Ascomycetes. In the early stage of the disease, dot-like chlorotic spots appear on the leaves. The spots gradually expand and are circular or oval in shape, with yellow-white in the center and brown edges or chlorotic halos. The diameter of the spots is 1 to 2 mm, and can reach 4 to 7 mm in some varieties. In susceptible varieties, the spots cover the entire leaf. In severe cases, the spots are connected into pieces, causing the leaves to die.

[0003] At present, the prevention and control measures for corn Curvularia leaf spot mainly include spraying chemical pesticides and breeding disease-resistant varieties. Although these methods can control the disease to a certain extent, the long-term and large-scale use of chemical pesticides will not only increase production costs, but may also cause pathogen resistance (resistance) and environmental problems; while breeding new disease-resistant varieties requires a long period of time and resistance is easily lost as pathogens mutate. Therefore, the development of new safe, efficient and environmentally friendly biological control technologies has become one of the current research hotspots.

[0004] In recent years, with the development of microbiology and molecular biology technology, the use of beneficial microorganisms for biological control of plant diseases has gradually attracted attention. Studies have shown that certain types of bacteria, fungi and other microorganisms can inhibit the growth and reproduction of pathogens through competition, antagonism and other means, thereby achieving the purpose of preventing or alleviating diseases. Based on this principle, the present invention proposes a composite microbial agent containing multiple functional microorganisms, aiming to provide a green and sustainable solution for corn Curvularia leaf spot disease. Summary of the invention

[0005] The purpose of the present invention is to provide a biocontrol composite microbial agent for preventing and controlling corn Curvularia leaf spot and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention combines beneficial microorganisms with different action mechanisms to enhance the control effect of corn Curvularia leaf spot through synergistic effect.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a biocontrol composite bacterial agent for preventing and controlling corn Curvularia leaf spot disease. The effective ingredients include Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuiflavos, and / or their metabolites.

[0008] Optionally, the ratio of the number of live bacteria of the Bacillus cereus, the Pseudomonas chlororaphis and the Streptomyces tenuifolius is (2-4):(2-3):(1-2).

[0009] Optionally, the formulation of the biocontrol composite bacterial agent includes powder, suspension, granule and liquid fermentation agent.

[0010] Bacillus cereus can produce lipopeptides and polypeptide antibiotics, and Pseudomonas chlororaphis can secrete a variety of antimicrobial compounds such as phenazines and 2,4-diacetylphloroglucinol, which can inhibit the growth and reproduction of pathogens. In addition, while secreting antimicrobial substances, Bacillus cereus and Pseudomonas chlororaphis can also competitively occupy the ecological niches of the plant rhizosphere and leaves, preventing the invasion of pathogens; Streptomyces tenuifolius, as an actinomycete, can produce a variety of antibiotics and secondary metabolites, and can also promote the growth of plant roots and improve their ability to absorb nutrients.

[0011] The present invention also provides a method for preparing the biocontrol composite bacterial agent, comprising the following steps:

[0012] Mixing the seed liquids of the Bacillus cereus, the Pseudomonas chlororaphis and the Streptomyces tenuiflavos to obtain a mixed seed liquid;

[0013] inoculating the mixed seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid;

[0014] The fermentation liquid is prepared into the biocontrol composite bacterial agent.

[0015] Optionally, the concentrations of the seed solution of Bacillus cereus, the seed solution of Pseudomonas chlororaphis and the seed solution of Streptomyces tenuifolius are all 1.0×10 7～9 cfu / mL.

[0016] Optionally, the inoculation amount of the mixed seed liquid is 10% to 15% of the volume of the fermentation medium.

[0017] Optionally, diatomaceous earth is added to the fermentation broth, mixed and dried to obtain a composite fermentation broth preformulation, and the composite fermentation broth preformulation is prepared into the biocontrol composite bacterial agent; the added mass of the diatomaceous earth accounts for 15% to 20% of the volume of the fermentation broth.

[0018] Optionally, the composite fermentation liquid preformulation is mixed with auxiliary materials and then subjected to air flow pulverization to obtain the biocontrol composite bacterial agent.

[0019] Optionally, the auxiliary materials include organosilicon, sodium carboxymethyl cellulose, chitosan, sodium lignin sulfonate and coenzyme A.

[0020] The biocontrol composite bacterial agent product prepared by the present invention has various forms, and the application method can be flexibly selected according to actual needs.

[0021] The invention also provides application of the biocontrol composite bacterial agent in preventing and controlling corn Curvularia leaf spot disease.

[0022] The present invention discloses the following technical effects:

[0023] In the biocontrol composite microbial agent provided by the present invention, Bacillus cereus and Pseudomonas chlororaphis belong to bacteria, and Streptomyces tenuifolius belongs to actinomycetes. The beneficial microorganisms with different action mechanisms are combined to enhance the control effect of corn Curvularia leaf spot disease through synergistic effect. The use of natural microbial resources to replace traditional chemical pesticides reduces the residual harmful substances and is conducive to protecting the ecological environment.

[0024] The present invention achieves effective prevention and control of corn Curvularia leaf spot disease by rationally combining different types of beneficial microorganisms, and can promote the healthy growth of plants. Compared with single strain preparations, the composite bacterial agent provided by the present invention has stronger stability and durability, and can significantly reduce the incidence of diseases. DETAILED DESCRIPTION

[0025] 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.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment 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. The intermediate value in any stated value or stated range, and each smaller range between 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.

[0027] 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.

[0028] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The invention provides a microbial composition of bacteria and actinomycetes, which is prepared into a biocontrol composite bacterial agent for preventing and controlling corn Curvularia leaf spot disease.

[0031] The bacteria are Bacillus cereus and Pseudomonas chlororaphis, and the actinomycetes are Streptomyces tenuiflavos.

[0032] In a specific embodiment, the ratio of the number of live bacteria of the Bacillus cereus, the Pseudomonas chlororaphis and the Streptomyces tenuifolius in the biocontrol composite bacterial agent is (2-4):(2-3):(1-2).

[0033] Optionally, the Bacillus cereus, the Pseudomonas chlororaphis and the Streptomyces tenuiflavos are all conventional strains used for agricultural production in the art, and there is no particular requirement for the specific strains.

[0034] The Bacillus cereus used in the specific embodiment of the present invention is Bacillus cereus CICC 21687, the Pseudomonas chlororaphis is Pseudomonas chlororaphis CICC 20676, and the Streptomyces microflavus is Streptomyces microflavus CICC 23627. The above strains are all from the China Industrial Microbiological Culture Collection Center, and the public can obtain them from the above institutions through conventional channels.

[0035] The present invention can prepare the above-mentioned microorganism combination into a variety of different dosage forms of biocontrol composite bacterial agents, including powder, suspension, granule and liquid fermentation agent.

[0036] In a specific embodiment, the above-mentioned microbial combination is activated and fermented to prepare a powdered biocontrol composite bacterial agent.

[0037] In a specific embodiment, the preparation method of the biocontrol composite bacterial agent of the present invention comprises the following steps:

[0038] Mixing the seed liquids of the Bacillus cereus, the Pseudomonas chlororaphis and the Streptomyces tenuiflavos to obtain a mixed seed liquid;

[0039] inoculating the mixed seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid;

[0040] The fermentation liquid is prepared into the biocontrol composite bacterial agent.

[0041] Optionally, the concentrations of the seed solution of Bacillus cereus, the seed solution of Pseudomonas chlororaphis and the seed solution of Streptomyces tenuifolius are all 1.0×10 7～9 cfu / mL.

[0042] Optionally, the inoculation amount of the mixed seed liquid is 10% to 15% of the volume of the fermentation medium.

[0043] Optionally, diatomaceous earth is added to the fermentation broth, mixed and dried to obtain a composite fermentation broth preformulation, and the composite fermentation broth preformulation is prepared into the biocontrol composite bacterial agent; the added mass of the diatomaceous earth accounts for 15% to 20% of the volume of the fermentation broth.

[0044] Optionally, the composite fermentation liquid preformulation is mixed with auxiliary materials and then subjected to air flow pulverization to obtain the biocontrol composite bacterial agent.

[0045] Optionally, the auxiliary materials include organosilicon, sodium carboxymethyl cellulose, chitosan, sodium lignin sulfonate and coenzyme A.

[0046] Unless otherwise specified, the methods mentioned in the embodiments of the present invention are all conventional methods in the art, and the materials and reagents used can be purchased through conventional channels.

[0047] The present invention is described in detail below with reference to specific embodiments.

[0048] Example 1

[0049] A biocontrol composite bacterial agent for preventing and controlling corn Curvularia leaf spot disease, the preparation method of which is as follows:

[0050] (1) The preserved Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius were inoculated into liquid LB medium respectively and cultured at 30°C until the logarithmic growth phase. The seed liquid concentration was not less than 1.0×10 8 cfu / mL, to obtain seed solution for inoculation;

[0051] (2) mixing the seed solutions of Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius obtained in step (1) to obtain a mixed seed solution, wherein the ratio of the number of live bacteria of Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius in the mixed seed solution is 3:2:1;

[0052] (3) The mixed seed solution obtained in step (2) is inoculated into the fermentation medium at an inoculation rate of 10% (volume percentage) so that the initial concentration of the bacteria in the fermentation culture system is not less than 107 cfu / mL, the fermentation medium was: starch 0.5%, yeast extract 0.04%, soybean meal 0.5%, urea 0.2%, dipotassium hydrogen phosphate 0.5%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.1%, manganese sulfate 0.009%; the culture conditions were: at 30°C, aeration pressure of 0.05MPa, dissolved oxygen 20%, rotation speed 220r / min, culture for 36h, fermentation liquid pH 7.0-7.2;

[0053] (4) adding 20% ​​(mass to volume ratio) diatomaceous earth to the fermentation broth obtained in step (3), mixing thoroughly, and spray drying to obtain a composite fermentation broth preformulation of Bacillus cereus, Pseudomonas chlororaphis, and Streptomyces tenuifolius;

[0054] (5) 91 parts of the composite fermentation broth preformulation obtained in step (4), 1 part of organosilicon BD-3077, 1.5 parts of sodium carboxymethyl cellulose, 2 parts of chitosan, 3.5 parts of sodium lignin sulfonate and 1 part of coenzyme A were mixed thoroughly, and the mixture was crushed to Φ = 45 μm or less by a jet mill to obtain a powdered composite bacterial agent, the number of viable bacteria in the preparation being not less than 1.0 × 10 11 cfu / g.

[0055] Example 2

[0056] A biocontrol composite bacterial agent for preventing and controlling corn Curvularia leaf spot disease, the preparation method of which is as follows:

[0057] (1) The preserved Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius were inoculated into liquid LB medium respectively and cultured at 30°C until the logarithmic growth phase. The seed liquid concentration was not less than 1.0×10 9 cfu / mL, to obtain seed solution for inoculation;

[0058] (2) mixing the seed solutions of Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius obtained in step (1) to obtain a mixed seed solution, wherein the ratio of the number of live bacteria of Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius in the mixed seed solution is 2:3:2;

[0059] (3) The mixed seed solution obtained in step (2) was inoculated into the fermentation medium at an inoculation rate of 15% (volume percentage) so that the initial concentration of the bacteria in the fermentation culture system was not less than 10 7 cfu / mL, the fermentation medium was: starch 0.5%, yeast extract 0.04%, soybean meal 0.5%, urea 0.2%, dipotassium hydrogen phosphate 0.5%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.1%, manganese sulfate 0.009%; the culture conditions were: at 30°C, aeration pressure of 0.05MPa, dissolved oxygen 20%, rotation speed 220r / min, culture for 36h, fermentation liquid pH 7.0-7.2;

[0060] (4) adding diatomaceous earth at a mass volume ratio of 15% to the fermentation broth obtained in step (3), mixing well, and spray drying to obtain a composite fermentation broth preformulation of Bacillus cereus, Pseudomonas chlororaphis, and Streptomyces tenuifolius;

[0061] (5) 91 parts of the composite fermentation broth preformulation obtained in step (4), 1 part of organosilicon BD-3077, 1.5 parts of sodium carboxymethyl cellulose, 2 parts of chitosan, 3.5 parts of sodium lignin sulfonate and 1 part of coenzyme A were mixed thoroughly, and the mixture was crushed to Φ = 45 μm or less by a jet mill to obtain a powdered composite bacterial agent, the number of viable bacteria in the preparation being not less than 1.0 × 10 10 cfu / g.

[0062] Example 3

[0063] A biocontrol composite bacterial agent for preventing and controlling corn Curvularia leaf spot disease, the preparation method of which is as follows:

[0064] (1) The preserved Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius were inoculated into liquid LB medium respectively and cultured at 30°C until the logarithmic growth phase. The seed liquid concentration was not less than 1.0×10 7 cfu / mL, to obtain seed solution for inoculation;

[0065] (2) mixing the seed solutions of Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius obtained in step (1) to obtain a mixed seed solution, wherein the ratio of the number of live bacteria of Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius in the mixed seed solution is 4:2:1;

[0066] (3) The mixed seed solution obtained in step (2) was inoculated into the fermentation medium at an inoculation rate of 12% (volume percentage) so that the initial concentration of the bacteria in the fermentation culture system was not less than 10 6 cfu / mL, the fermentation medium was: starch 0.5%, yeast extract 0.04%, soybean meal 0.5%, urea 0.2%, dipotassium hydrogen phosphate 0.5%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.1%, manganese sulfate 0.009%; the culture conditions were: at 30°C, aeration pressure of 0.05MPa, dissolved oxygen 20%, rotation speed 220r / min, culture for 36h, fermentation liquid pH 7.0-7.2;

[0067] (4) adding 20% ​​(mass to volume ratio) diatomaceous earth to the fermentation broth obtained in step (3), mixing thoroughly, and spray drying to obtain a composite fermentation broth preformulation of Bacillus cereus, Pseudomonas chlororaphis, and Streptomyces tenuifolius;

[0068] (5) 91 parts of the composite fermentation broth preformulation obtained in step (4), 1 part of organosilicon BD-3077, 1.5 parts of sodium carboxymethyl cellulose, 2 parts of chitosan, 3.5 parts of sodium lignin sulfonate and 1 part of coenzyme A were mixed thoroughly, and the mixture was crushed to Φ = 45 μm or less by a jet mill to obtain a powdered composite bacterial agent, the number of viable bacteria in the preparation being not less than 1.0 × 10 12 cfu / g.

[0069] Comparative Example 1

[0070] The same as Example 3, except that Paenibacillus polymyxa (CICC10494) was used to replace Bacillus cereus.

[0071] Comparative Example 2

[0072] The method is the same as Example 3, except that in step (2), the ratio of the number of live bacteria of Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius in the mixed seed liquid is 1:2:3.

[0073] Comparative Example 3

[0074] Same as Example 3, except that only Bacillus cereus is retained.

[0075] Comparative Example 4

[0076] The same as Example 3, except that only Pseudomonas chlororaphis is retained.

[0077] Comparative Example 5

[0078] The same as Example 3, except that only Streptomyces tenuiflavus is retained.

[0079] Verification Example

[0080] Experimental groups: divided into treatment groups 1-8 and a control group.

[0081] Treatment groups 1-8: 10 g of the composite bacterial agent (prepared in Examples 1-3 and Comparative Examples 1-5) was added to each liter of nutrient soil (the nutrient soil was purchased from Piedman Company, and the formula of the nutrient soil was: organic matter content ≥45%, humic acid content ≥15%, pH value: 5.5-6.8, and free water content ≤20%), and the culture medium I was obtained after sufficient mixing. The corn was sown and managed according to the conventional method;

[0082] Control group: using nutrient soil as cultivation medium II, sowing and managing corn according to conventional methods;

[0083] The corn leaf spot pathogen Curvularia lunata used in this experiment was isolated and identified by the Institute of Plant Protection, Liaoning Academy of Agricultural Sciences. The pathogen was cultured on PDA medium for 7 days. A large number of conidia were produced on the colonies. The conidia were washed with sterile water containing 0.01% Tween 80. The spore concentration was measured with a hemocytometer or microscope and adjusted to 1-2×10 5 / mL, prepared into a spore suspension of corn Curvularia leaf spot pathogen. When the corn seedlings grow to the 3-6 leaf stage, the spore suspension is evenly sprayed on the leaves of the treatment group and the control group to ensure that the leaf surface is fully moistened. After inoculation, the treatment groups 1-6 are sprayed with the solution prepared by the composite bacterial agent obtained by Examples 1 to 3 and Comparative Examples 1 to 5, respectively, and evenly sprayed on the leaves, sprayed once a week, and sprayed continuously for 3 times. The control group is sprayed with an equal amount of clean water. Each group treats 30 plants, repeated 3 times, and the disease level is investigated after 28 days, and the disease index and relative prevention effect are calculated.

[0084] The disease grading standards for corn Curvularia leaf spot are shown in Table 1.

[0085] Table 1 Grading standards for corn Curvularia leaf spot disease

[0086]

[0087]

[0088] The disease index and relative prevention efficacy calculation formula are as follows:

[0089] Disease index = 100 × ∑ (number of diseased leaves at each level × relative disease level) / (total number of leaves investigated × 9),

[0090] Relative protective effect (%) = [(disease index in control area - disease index in treated area) / disease index in control area] × 100%.

[0091] Data statistical analysis: Statistical analysis was performed using EXCEL2007 and SPSS19.0 software, and multiple comparisons were performed using Duncan's new multiple range test. The results are shown in Table 2.

[0092] Table 2 The control effect of biocontrol compound fungi on corn Curvularia leaf spot

[0093]

[0094] It can be seen from the data in Table 2 that compared with the control group, the disease index of the treatment group using the composite bacterial agent decreased. Among them, the disease index of the composite bacterial agent prepared by Examples 1-3 of the present invention was controlled at about 20, and the relative prevention effect could reach more than 69%, indicating that the composite bacterial agent prepared by the embodiment of the present invention has the best control effect on corn Curvularia leaf spot.

[0095] 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 biocontrol composite bacterial agent for preventing and controlling corn Curvularia leaf spot disease, characterized in that: The active ingredients include Bacillus cereus, Pseudomonas chlororaphis and Streptomyces tenuifolius, and / or their metabolites.

2. The biocontrol composite bacterial agent according to claim 1, characterized in that: The ratio of the number of live bacteria of the Bacillus cereus, the Pseudomonas chlororaphis and the Streptomyces tenuiflavos is (2-4):(2-3):(1-2).

3. The biocontrol composite bacterial agent according to claim 1, characterized in that: The dosage forms of the biocontrol composite fungus agent include powder, suspension, granule and liquid fermentation agent.

4. The method for preparing the biocontrol composite bacterial agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: Mixing the seed liquids of the Bacillus cereus, the Pseudomonas chlororaphis and the Streptomyces tenuiflavos to obtain a mixed seed liquid; inoculating the mixed seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid; The fermentation liquid is prepared into the biocontrol composite bacterial agent.

5. The preparation method according to claim 4, characterized in that: The concentrations of the seed solution of Bacillus cereus, the seed solution of Pseudomonas chlororaphis and the seed solution of Streptomyces tenuifolius were all 1.0×10 7～9 cfu / mL.

6. The preparation method according to claim 4, characterized in that: The inoculation amount of the mixed seed liquid is 10% to 15% of the volume of the fermentation medium.

7. The preparation method according to claim 4, characterized in that: Diatomaceous earth is added to the fermentation liquid, mixed and dried to obtain a composite fermentation liquid preformulation, and the composite fermentation liquid preformulation is used to prepare the biocontrol composite bacterial agent; the added mass of the diatomaceous earth accounts for 15% to 20% of the volume of the fermentation liquid.

8. The preparation method according to claim 7, characterized in that: The composite fermentation liquid preformulation is mixed with auxiliary materials and then subjected to air flow pulverization to obtain the biocontrol composite bacterial agent.

9. The preparation method according to claim 8, characterized in that: The auxiliary materials include organic silicon, sodium carboxymethyl cellulose, chitosan, sodium lignin sulfonate and coenzyme A.

10. Use of the biocontrol composite bacterial agent according to any one of claims 1 to 3 in controlling corn Curvularia leaf spot disease.

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