CN112501152A - Compound microbial agent for preventing and treating soil-borne diseases and preparation method thereof - Google Patents
Compound microbial agent for preventing and treating soil-borne diseases and preparation method thereof Download PDFInfo
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- CN112501152A CN112501152A CN202011409806.8A CN202011409806A CN112501152A CN 112501152 A CN112501152 A CN 112501152A CN 202011409806 A CN202011409806 A CN 202011409806A CN 112501152 A CN112501152 A CN 112501152A
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- microbial agent
- trichoderma harzianum
- bacillus amyloliquefaciens
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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Abstract
The invention provides a compound microbial agent for preventing and treating soil-borne diseases and a preparation method thereof, belonging to the technical field of agricultural microbial agents. The composite microbial agent is prepared by loading bacillus amyloliquefaciens and trichoderma harzianum on corn straw biochar-chitosan composite microspheres. The preparation method comprises the steps of respectively inoculating bacillus amyloliquefaciens and trichoderma harzianum to a solid matrix for fermentation, preparing the obtained fermentation product into a composite microorganism according to the proportion of the bacillus amyloliquefaciens (50-70%) to the trichoderma harzianum (30-50%), preparing a microsphere carrier by dripping a product formed by high-temperature carbonization of corn straws under the oxygen-limited or anaerobic condition and chitosan into spheres, mixing the microspheres and composite microorganism bacterial sludge with a cross-linking agent according to the mass ratio of 30: 1-20: 1, and curing the mixture in the cross-linking agent to obtain the composite microorganism bacterial agent. The microbial agent has strong heat resistance and acid-base stability, effectively inhibits the propagation of pathogenic bacteria, and has an antibacterial rate of more than 70%.
Description
Technical Field
The invention relates to a compound microbial agent for preventing and treating soil-borne diseases and a preparation method thereof, belonging to the technical field of agricultural microbial agents.
Background
The soil-borne diseases generally damage roots and stems of plants, and if the soil-borne diseases occur in the early growth stage of crops, roots and stems of seedlings are rotten or damping off, the seedlings die quickly, and the crop yield is seriously influenced. Diseases occur in the later growth stage of crops, and the yield is generally reduced by 20-30%. Soil-borne diseases are difficult to control, and the diseases are generally hidden in soil to live through the winter, continue to damage crops in the next year, and are repeated in a circulating way, so that the soil-borne diseases are more and more serious. The use of a large amount of chemical fertilizers, particularly nitrogen fertilizers, causes the growth of fusarium, verticillium and rhizoctonia in soil-borne diseases, thereby promoting the occurrence of soil-borne diseases. In recent years, the role of microbial agents in agricultural production has been gaining increasing attention. Many microorganisms can promote plant growth and inhibit propagation of fungi and bacteria, have important significance in the aspects of reducing the using amount of chemical fertilizers, reducing environmental pollution, improving the quality of crops and the like, and are harmless to human and livestock. Therefore, various microorganisms are prepared into biological preparations to be applied to the field in agricultural production, so that biological decontamination, plant growth promotion and biological control can be effectively combined, and the yield and quality of agricultural products are improved.
In recent years, the role of bacillus amyloliquefaciens in agricultural production is gradually emphasized. The bacillus amyloliquefaciens is prepared into a microbial agent, can effectively degrade toxic substances after being applied to soil, and creates a good soil microbial environment for the growth of crops. Meanwhile, the fertilizer has important significance in promoting plant growth, inhibiting fungi and bacteria reproduction, reducing the fertilizer dosage, reducing environmental pollution, improving the quality of crops and the like, and is harmless to human and livestock.
Trichoderma has the characteristics of resisting diseases, promoting growth and improving soil. Trichoderma harzianum is an important biocontrol strain, parasitizes in pathogenic bacteria of various plants, and the Trichoderma harzianum antagonizes the growth of the plant pathogenic bacteria through competition, heavy parasitism and other effects. The trichoderma harzianum has the advantages of strong adaptability, high propagation speed, capability of preempting surface sites of plant roots and the like, and a protective cover is formed on the plant roots to prevent the plants from being invaded by pathogenic bacteria; it can also be attached to pathogenic bacteria, and can dissolve the cell wall of the pathogenic bacteria by secreting extracellular enzyme, penetrate hypha, absorb nutrition, and further kill the pathogenic bacteria; part of antibiotics secreted by trichoderma harzianum can also inhibit the growth and colonization of pathogenic bacteria, and finally the harm of the pathogenic bacteria to plants is reduced.
The biomass charcoal is a substance which is formed by performing high-temperature and oxygen-limited pyrolysis on agricultural wastes such as straws and peanut shells, has rich carbon content and a unique porous structure, can provide good attachment space and propagation conditions for soil microorganisms, and can play a synergistic effect when being matched with a microbial fertilizer. The chitosan is easy to be protonated with H + to lose, and the stability of the chitosan in an acid solution can be enhanced or the adsorption performance can be improved through a crosslinking reaction. After the biological carbon and the chitosan are compounded, the problem that the biological carbon powder is not easy to recycle can be effectively solved, and the defect that the chitosan has low density and is difficult to contact with adsorbate is overcome.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and further provides a compound microbial agent for preventing and treating soil-borne diseases and a preparation method thereof, wherein the compound microbial agent is used for preventing and treating soil-borne diseases, promoting plant growth and improving yield and quality of agricultural products.
The purpose of the invention is realized by the following technical scheme:
a compound microbial agent for preventing and treating soil-borne diseases comprises: bacillus amyloliquefaciens, trichoderma harzianum, corn straw biochar and chitosan; the corn stalk biochar and chitosan are prepared into a microsphere carrier by adopting a dropping balling method, and the microsphere carrier and the compound microorganism are added into a cross-linking agent for solidification to prepare the compound microorganism bacterium agent.
The compound microbial agent is solid, and the mass ratio of active ingredients of the compound microbial agent is as follows: 50-70% of bacillus amyloliquefaciens, 30-50% of trichoderma harzianum and not less than 1 multiplied by 10 effective viable count of bacterial strains of microbial inoculum10CFU/g。
A preparation method of a compound microbial agent comprises the following steps:
(1) preparing a seed solution: streaking the bacillus amyloliquefaciens strain on an LB (lysogeny broth) plate, picking out a single colony after 48 hours, inoculating the single colony into 10mL of LB culture solution, and culturing at 30 ℃ for 12 hours at 200r/min to obtain a seed solution; respectively inoculating the strains into an activation culture medium at the temperature of 30-35 ℃ and at the speed of 150-200 r/min, and culturing for 48 hours to obtain seed liquid of the strains;
(2) preparing a PDA slant culture medium, inoculating Trichoderma harzianum, culturing at room temperature for 4-6 days, washing conidia of Trichoderma harzianum on the PDA slant culture medium with sterilized water, and preparing spore suspension with the concentration of 107-108 spores/mL.
(3) Respectively inoculating seed liquids of bacillus amyloliquefaciens and trichoderma harzianum strains into a solid matrix, wherein the inoculation amount of the bacillus amyloliquefaciens strains is 5-10%, the material-water ratio is 1:1, the fermentation time is 36-48 h, and the fermentation temperature is 30-35 ℃;
(4) the inoculation amount of the Trichoderma harzianum strain is 5-10%, the material-water ratio is 1:1, the fermentation time is 13-15 days, and the fermentation temperature is 25-28 ℃.
(5) The solid matrix consists of: 40% of bran, 20% of rice hull powder, 20% of corn flour, 20% of soybean meal, 10g/kg of maltose, 20g/kg of tryptone and MgSO415g/kg；
(6) Inoculating the seed liquid of the strain into a fermentation culture medium, performing amplification culture to obtain fermentation liquid of the strain of bacillus amyloliquefaciens and trichoderma harzianum, performing centrifugal concentration, and removing supernatant to prepare the compound microbial agent.
(7) Putting a certain mass of corn straws into a biochar furnace, heating to 550 ℃ at a speed of 15 ℃/min under an oxygen-limited condition, keeping the pyrolysis process for 2 hours, cooling to room temperature, and sieving with an 80-mesh sieve to obtain carbonized corn straw biomass charcoal for later use.
(8) Weighing chitosan powder, adding into 2% acetic acid solution, magnetically stirring for 1h, standing overnight to fully dissolve chitosan, and preparing into 4% chitosan solution.
(9) Adding 4% of corn straw biochar into a chitosan solution, dropwise adding the chitosan solution into a coagulation liquid (containing 2mol/L NaOH) by using an injector with a No. 5 needle head under the condition of continuous magnetic stirring, and preparing the microspheres. After being prepared, the corn straw biochar-chitosan microspheres are placed at room temperature overnight and then washed by distilled water to be neutral, so that the microspheres are prepared.
(10) The microspheres and the composite microbial bacterial sludge are soaked in a solution containing a cross-linking agent (2% glutaraldehyde) for 24 hours according to the mass ratio of 30: 1-20: 1 to prepare the composite microbial bacterial agent microspheres, and the composite microbial bacterial agent microspheres are dried in vacuum.
The compound microbial agent has the beneficial effects that:
(1) the microorganism is screened and obtained from nature, is easy to separate and culture, has rich metabolites, broad-spectrum antibacterial activity, strong stress resistance, fast growth and good stability. Prepared by a unique fermentation process, and is very suitable for mass production and application of biocontrol bacteria.
(2) The bacillus amyloliquefaciens can effectively degrade toxic substances in the environment, and a plurality of organic pollutants can be used as main carbon sources by some bacillus amyloliquefaciens strains to colonize the roots of plants through the movement of bacteria, so that the bacillus amyloliquefaciens can play a role in inhibiting and induce the in-vivo genes and the immune system of the plants to change. Trichoderma harzianum is an important biocontrol strain, can parasitize in pathogenic bacteria of various plants, and the antagonistic action of the Trichoderma harzianum on the plant pathogenic bacteria is mainly reflected in competition, heavy parasitism and antibiotic action, so that the harm of the pathogenic bacteria to the plants is reduced. The compound microbial agent is prepared, can greatly prevent soil-borne diseases when applied to soil, reduces the application of chemical fertilizers, gathers at the roots of plants, secretes stimulating hormones, promotes the growth of the plants and improves the quality of crops.
(3) The corn straw biomass charcoal can improve soil nutrients, can play a synergistic effect with microbial fertilizer in a matched manner, and can enhance the stability of the biomass charcoal and chitosan in an acid solution or improve the adsorption performance of the biomass charcoal and chitosan through cross-linking reaction.
(4) The compound microbial agent is safe and convenient to use, non-toxic, harmless, non-corrosive, harmless to human, animals, plants and plants, free of environmental pollution and ecological in safety. The bacillus amyloliquefaciens and the trichoderma harzianum have the characteristics of disease resistance, growth promotion and soil improvement, are prepared into a compound microbial agent to be applied to soil, can prevent and treat soil-borne diseases caused by continuous cropping of plants and improper application of chemical fertilizers after being colonized in the soil, and can generate a large amount of plant hormones and organic acids to stimulate the growth of root systems. The developed porous structure, rich functional groups and good acid-base buffer capacity of the corn straw biomass charcoal can provide good attachment space and propagation conditions for soil microorganisms, so that the corn straw biomass charcoal and the microbial fertilizer can play a synergistic effect in the matched application, the soil quality is improved, the physiological metabolism of crops is promoted, and a benign plant-soil-microorganism ecosystem is formed.
Detailed Description
The present invention will be described in further detail below: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation is given, but the scope of the present invention is not limited to the following embodiments.
The embodiment relates to a compound microbial agent for preventing and treating soil-borne diseases, which comprises:
example 1
Determination of the inhibition Spectrum
The bacterial inhibition spectrum of the composite microbial agent microspheres is determined by taking bacterial wilt, soybean sclerotinia rot, root mold, gray mold, rice sheath blight, cotton wilt, tomato early blight and other germs as targets, and the composite microbial agent microspheres have strong antagonistic action on the pathogens, and the antagonistic action reaches more than 70%.
TABLE 1 antibacterial spectra of complex microbial agents
Example 2
Preparation method of compound microbial agent
The invention provides a compound microbial agent. The viable count of the microbial inoculum is not less than 1 × 1010CFU/g。
The preparation process comprises the following steps:
(1) recovering the preserved strains, inoculating the bacillus amyloliquefaciens strains to an LB liquid culture medium, performing shake culture at 30 ℃ and 160r/min for 12 hours to obtain liquid strains; respectively inoculating the strains into an activation culture medium in a constant temperature shaking table at 30-35 ℃ under the shaking culture condition of 150-200 r/min, and culturing for 48h to obtain seed liquid of the strains;
(2) preparing a PDA slant culture medium, inoculating Trichoderma harzianum, culturing at room temperature for 4-6 days, washing conidia of Trichoderma harzianum on the PDA slant culture medium with sterilized water, and preparing spore suspension with the concentration of 107-108 spores/mL.
(3) Respectively inoculating the seed liquid of the strain into a solid matrix, wherein the inoculation amount of the bacillus amyloliquefaciens strain is 5-10%, the material-water ratio is 1:1, the fermentation time is 36-48 h, and the fermentation temperature is 30-35 ℃;
(4) the inoculation amount of the Trichoderma harzianum strain is 5-10%, the material-water ratio is 1:1, the fermentation time is 15 days, and the fermentation temperature is 25-28 ℃.
(5) The solid matrix consists of: 40% of bran, 20% of rice hull powder, 20% of corn flour, 20% of soybean meal, 10g/kg of maltose, 20g/kg of tryptone and MgSO415g/kg；
(6) Inoculating the seed liquid of the strain into a fermentation culture medium, performing amplification culture to obtain fermentation liquid of the strain of bacillus amyloliquefaciens and trichoderma harzianum, performing centrifugal concentration, and removing supernatant to prepare the compound microbial agent.
(7) Putting a certain mass of corn straws into a biochar furnace, heating to 550 ℃ at a speed of 15 ℃/min under an oxygen-limited condition, keeping the pyrolysis process for 2 hours, cooling to room temperature, and sieving with an 80-mesh sieve to obtain carbonized corn straw biomass charcoal for later use.
(8) Weighing chitosan powder, adding into 2% acetic acid solution, magnetically stirring for 1h, standing overnight to fully dissolve chitosan, and preparing into 4% chitosan solution.
(9) Adding 4% of corn straw biochar into a chitosan solution, dropwise adding the chitosan solution into a coagulation liquid (containing 2mol/L NaOH) by using an injector with a No. 5 needle head under the condition of continuous magnetic stirring, and preparing the microspheres. After being prepared, the corn straw biochar-chitosan microspheres are placed at room temperature overnight and then washed by distilled water to be neutral, so that the microspheres are prepared.
(10) The microspheres and the composite microbial bacterial sludge are soaked in a solution containing a cross-linking agent (2% glutaraldehyde) for 24 hours according to the mass ratio of 30: 1-20: 1 to prepare the composite microbial bacterial agent microspheres, and the composite microbial bacterial agent microspheres are dried in vacuum.
Example 3: fermentation process of microbial agent
The invention provides a composite microbial agent microsphere, which mainly comprises bacillus amyloliquefaciens and trichoderma harzianum, wherein the viable count of each strain is not less than 3 multiplied by 108CFU/g, the total viable count of the composite microbial agent is not less than 1 × 1010CFU/g. The specific fermentation process of the compound microbial agent is shown in tables 2-5.
TABLE 2
TABLE 3
TABLE 4
TABLE 5
Example 4
The proportion of the compound microbial agent
The invention provides a compound microbial agent, which mainly comprises bacillus amyloliquefaciens and trichoderma harzianum, wherein the viable count of each strain is not less than 3 multiplied by 108CFU/g, the total viable count of the composite microbial agent is not less than 1 × 1010CFU/g。
The preparation process comprises the following steps:
and (3) centrifugally concentrating the fermented strain culture, removing supernatant, and preparing the compound microbial agent, wherein the compound microbial agent comprises the following components in percentage by weight: bacillus amyloliquefaciens (50-70%) and trichoderma harzianum (30-50%).
The specific proportion of the compound microbial inoculum is shown in Table 6.
TABLE 6 weight percent of complex microbial inoculant
Example 5
Proportion ratio for preparing composite microbial agent microspheres
The invention provides a compound microbial agent microsphere, and microbial strains mainly comprise: bacillus amyloliquefaciens and trichoderma harzianum, wherein the microspheres mainly comprise: the corn straw biomass charcoal and the chitosan are prepared into the composite microbial agent microspheres by a cross-linking method.
The preparation process comprises the following steps:
putting a certain mass of corn straws into a biochar furnace, heating to 550 ℃ at a speed of 15 ℃/min under an oxygen-limited condition, keeping the pyrolysis process for 2 hours, cooling to room temperature, and sieving with an 80-mesh sieve to obtain carbonized corn straw biomass charcoal for later use.
Weighing chitosan powder, adding into 2% acetic acid solution, magnetically stirring for 1h, standing overnight to fully dissolve chitosan, and preparing into 4% chitosan solution.
Adding 4% of corn straw biochar into a chitosan solution, dropwise adding the chitosan solution into a coagulation liquid (containing 2mol/L NaOH) by using an injector with a No. 5 needle head under the condition of continuous magnetic stirring, and preparing the microspheres. After being prepared, the corn straw biochar-chitosan microspheres are placed at room temperature overnight and then washed by distilled water to be neutral, so that the microspheres are prepared.
The microspheres and the composite microbial bacterial sludge are soaked in a solution containing a cross-linking agent (2% glutaraldehyde) for 24 hours according to the mass ratio of 30: 1-20: 1 to prepare the composite microbial bacterial agent microspheres, and the composite microbial bacterial agent microspheres are dried in vacuum.
The specific ratio of microspheres to composite microbial sludge is shown in table 7.
TABLE 7 mass ratio of microspheres to microbial sludge
The above description is only a preferred embodiment of the present invention, and these embodiments are based on different implementations of the present invention, and the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. The compound microbial agent for preventing and treating soil-borne diseases is characterized by comprising the following components in parts by weight: bacillus amyloliquefaciens, trichoderma harzianum, corn straw biochar and chitosan; the corn stalk biochar and chitosan are prepared into a microsphere carrier by adopting a dropping balling method, and the microsphere carrier, bacillus amyloliquefaciens and trichoderma harzianum are added into a cross-linking agent for solidification to prepare the compound microbial agent.
2. The compound microbial agent for preventing and treating soil-borne diseases according to claim 1, wherein the compound microbial agent is prepared by mixing the raw materialsThe strains of the compound microbial agent are as follows: bacillus amyloliquefaciens and Trichoderma harzianum, the effective viable count of the strain of microbial agent is not less than 1 × 1010CFU/g。
3. The compound microbial agent for preventing and treating soil-borne diseases according to claim 1, wherein the microsphere carrier is corn stalk biochar-chitosan microspheres.
4. The compound microbial agent for controlling soil-borne diseases according to claim 1, wherein the cross-linking agent is 2% glutaraldehyde solution.
5. The preparation method of the compound microbial agent for preventing and treating soil-borne diseases based on the claim 1, 2, 3 or 4 is characterized by comprising the following steps:
the method comprises the following steps: inoculating the preserved strains of the bacillus amyloliquefaciens and the trichoderma harzianum into a solid culture medium for screening and purifying, culturing and separating to obtain single colonies of the bacillus amyloliquefaciens and the trichoderma harzianum, culturing again, and preserving the two colonies for later use;
step two: shaking and culturing the separated bacillus amyloliquefaciens in a constant-temperature shaking table at the temperature of 30-35 ℃ for 36-48 h at the speed of 200r/min to prepare liquid seeds;
step three: inoculating trichoderma harzianum to a PDA (PDA dextrose agar) slant culture medium, culturing at room temperature for 4-6 days, and washing conidia of trichoderma harzianum on the PDA slant culture medium by using sterilized water to prepare a spore suspension with the concentration of 107-108 spores/mL;
step four: inoculating the seed liquid of the strains of the bacillus amyloliquefaciens and the trichoderma harzianum to a fermentation medium, carrying out amplification culture to obtain fermentation liquid of the strains of the bacillus amyloliquefaciens and the trichoderma harzianum, carrying out centrifugal concentration, and removing supernatant to prepare a composite microbial agent;
step five: placing corn straws into a biochar furnace, heating to 550 ℃ at a speed of 15 ℃/min under an oxygen-limited condition, keeping the pyrolysis process for 2 hours, cooling to room temperature, and sieving with an 80-mesh sieve to obtain carbonized corn straw biomass charcoal for later use;
step six: weighing chitosan powder, adding the chitosan powder into 2% acetic acid solution, magnetically stirring for 1h, standing overnight to fully dissolve chitosan, and preparing chitosan solution with the final concentration of 4%;
step seven: adding 4% of corn straw biochar into a chitosan solution, dropwise adding the chitosan solution into a condensation solution by using an injector with a No. 5 needle head under the condition of continuous magnetic stirring, wherein the condensation solution is a solution containing 2mol/L NaOH to prepare microspheres, standing overnight at room temperature after the corn straw biochar-chitosan microspheres are prepared, and then washing the microspheres to be neutral by using distilled water to prepare the microspheres;
step eight: the microspheres and the composite microbial bacterial sludge are soaked in a solution containing a cross-linking agent, wherein the mass ratio of the microspheres to the composite microbial bacterial sludge is 30: 1-20: 1, the cross-linking agent is 2% glutaraldehyde solution, the composite microbial bacterial sludge microspheres are prepared by 24 hours of soaking, and the composite microbial bacterial sludge microspheres are dried in vacuum.
6. The preparation method of the compound microbial agent for preventing and treating soil-borne diseases according to claim 5, wherein in the fourth step, the bacillus amyloliquefaciens strain is inoculated into a solid fermentation culture medium, the inoculation amount is 5-10%, the material-water ratio is 1:1, the fermentation time is 36-48 h, and the fermentation temperature is 30-35 ℃.
7. The preparation method of the compound microbial agent for preventing and treating soil-borne diseases according to claim 5, wherein in the fourth step, Trichoderma harzianum strain is inoculated into a solid fermentation medium, the inoculation amount is 5-10%, the material-water ratio is 1:1, the fermentation time is 13-15 days, and the fermentation temperature is 25-28 ℃.
8. The method for preparing the compound microbial agent for controlling soil-borne diseases according to claim 7, wherein the solid substrate of the solid fermentation medium consists of the following substances: bran, rice hull powder, corn flour, bean pulp, maltose, tryptone and MgSO4。
9. The preparation method of the compound microbial agent for preventing and treating soil-borne diseases according to claim 5, wherein the content of the bacillus amyloliquefaciens is 50-70%, the content of the trichoderma harzianum is 30-50%, the two cultures are uniformly mixed according to a proportion to prepare the compound microbial agent, and the effective viable count of each strain is not less than 3 x 108 CFU/g.
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