Composite microbial agent and application thereof in prevention and treatment of root diseases of forest trees

Abstract

The invention relates to the technical field of microbial agents, in particular to a composite microbial agent and application of the composite microbial agent in prevention and treatment of forest root diseases, and the composite microbial agent comprises the following components in parts by weight: 15-25 parts of Trichoderma harzianum ATCC 20847; 10 to 20 parts of bacillus subtilis ATCC 605, 10 to 20 parts of bacillus subtilis ATCC 605, 10 to 20 parts of bacillus subtilis ATCC 606 and 10 to 20 parts of bacillus subtilis ATCC 607. 5-10 parts of a seaweed extract; 3 to 8 parts of chitosan; 2-5 parts of humic acid; 1 to 3 parts of glycine betaine; 0.5 to 1.5 parts of a trace element compound; 30 to 40 parts of perlite powder; according to the present invention, the fertilizer has characteristics of significant inhibition effect on a variety of forest root pathogenic bacteria, wide prevention and control spectrum, and long-term and stable disease prevention effect by adjusting the rhizosphere micro-ecosystem. Diseases are prevented and controlled, and the growth and root development of the forest trees are remarkably promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and in particular to a composite microbial agent and its application in the prevention and treatment of tree root diseases. Background Art

[0002] Root diseases of trees have always been a major problem that has plagued forestry production. They not only affect tree growth, but can also lead to large-scale tree death, causing huge economic losses and ecological damage. Traditional prevention and control methods mainly rely on chemical pesticides. Although they can control diseases in the short term, long-term use will cause pathogens to develop drug resistance, destroy the soil microecological balance, and pollute the environment. In recent years, biological control has received widespread attention due to its environmental friendliness and strong sustainability.

[0003] In the prior art, single microbial agents such as Trichoderma or Bacillus have been used for the prevention and control of forest diseases. However, these single microbial agents often have problems such as narrow prevention and control spectrum, short duration, and poor environmental adaptability. Some studies have tried to mix multiple microorganisms, but they are only simple physical mixing and fail to give full play to the synergistic effect of each component. In addition, most of the existing microbial agents ignore the overall regulation of the rhizosphere micro-ecosystem, making it difficult to achieve long-term and stable prevention and control effects. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention innovatively develops a composite microbial agent and its application in the prevention and treatment of forest root diseases. The agent not only contains optimized and screened Trichoderma and Bacillus, but also adds functional auxiliary ingredients such as seaweed extract, chitosan, and humic acid. This unique combination design fully considers the synergistic effect between microorganisms, plant-microorganism interactions, and the overall regulation of the rhizosphere micro-ecosystem.

[0005] The object of the present invention is to provide a composite microbial agent, characterized in that it comprises the following components by weight:

[0006] Trichoderma (Trichodermaharzianum) ATCC 2084715-25;

[0007] Bacillus subtilis ATCC 605110-20 copies;

[0008] 5-10 parts of seaweed extract;

[0009] 3-8 parts of chitosan;

[0010] Humic acid 2-5 parts;

[0011] 1-3 parts of glycine betaine;

[0012] Trace element complex 0.5-1.5 parts;

[0013] 30-40 parts of perlite powder;

[0014] 5-10 servings of corn cob flour.

[0015] Specifically, the seaweed extract is derived from brown algae (Ascophyllum nodosum), and its main components include alginic acid, mannitol and seaweed polysaccharides.

[0016] Specifically, the chitosan has a deacetylation degree of ≥90% and a molecular weight of 100-300 kDa.

[0017] Specifically, the content of humic acid is ≥ 60%.

[0018] Specifically, the trace element complex includes Fe-EDTA, Mn-EDTA, Zn-EDTA, Cu-EDTA and Na 2 MoO 4 .

[0019] Specifically, the particle size of the perlite powder is 200-300 mesh, and the particle size of the corncob powder is 40-60 mesh.

[0020] The preparation method of the composite microbial agent comprises the following steps:

[0021] (1) Strain culture:

[0022] a) Cultivate Trichoderma ATCC 20847 on potato dextrose agar (PDA) medium at 25±1°C in the dark for 7-10 days; elute spores with 0.1% Tween-80 solution, pass through a 200-mesh sieve, and adjust the spore concentration to 1×10 9 CFU / mL;

[0023] b) Cultivate Bacillus subtilis ATCC 6051 in nutrient broth at 30±1°C and 200 rpm for 24-48 hours; centrifuge at 4000 rpm for 10 minutes, resuspend in sterile water, and adjust the concentration to 1×10 9 CFU / mL;

[0024] (2) Preparation of auxiliary ingredients:

[0025] a) crushing the dried brown algae into 60-80 mesh, adding 10 times the volume of 0.1M HCl, extracting at 60°C for 2 hours, filtering, adjusting the pH of the filtrate to 7.0 with NaOH, concentrating under reduced pressure to 1 / 5 of the original volume, and spray drying to obtain seaweed extract powder;

[0026] b) dissolving chitosan in 1% acetic acid solution, stirring until completely dissolved, and adjusting the concentration to 2% (w / v);

[0027] c) dissolving humic acid in 0.1M NaOH, stirring for 2 hours, filtering, and adjusting the pH of the filtrate to 5.5-6.0 with HCl;

[0028] d) mixing EDTA chelate and sodium molybdate in a ratio of Fe:Mn:Zn:Cu:Mo=5:2:2:1:0.1, dissolving in deionized water, and adjusting the total concentration to 1% (w / v);

[0029] (3) Preparation of bacterial agents:

[0030] a) Mix perlite powder and corncob powder in a ratio of 7:1 and sterilize by autoclave at 121℃ for 20 minutes;

[0031] b) mixing the Trichoderma spore suspension and the Bacillus subtilis cell suspension in a 1:1 ratio;

[0032] c) adding the bacterial suspension to the mixed carrier, stirring evenly, and controlling the water content to 30-35%;

[0033] d) adding seaweed extract, chitosan solution and humic acid solution in sequence and stirring evenly;

[0034] e) adding glycine betaine and trace element complex and continuing stirring for 10 minutes;

[0035] f) drying at 25-30°C to a moisture content of less than 10%;

[0036] g) Grind through a 60-mesh sieve to obtain the final product.

[0037] Specifically, the method also includes a quality control step:

[0038] a) Determination of viable bacteria count: Using the plate count method, the total viable bacteria count of Trichoderma and Bacillus subtilis should not be less than 2×10 9 CFU / g;

[0039] b) Moisture content: not more than 10%;

[0040] c) pH value: 6.5-7.5;

[0041] d) Particle size: 95% passed through a 60 mesh sieve.

[0042] The application of the composite microbial agent in the prevention and treatment of forest root diseases.

[0043] Specifically, the application includes the following application methods:

[0044] a) Seed treatment: Soak the seeds in 1% bacterial suspension for 4-6 hours, then dry in the shade and sow;

[0045] b) Soaking the roots of seedlings: Soak the roots of seedlings in 0.5% bacterial agent suspension for 30 minutes before planting;

[0046] c) Soil treatment: 20-30kg / hm 2 Evenly spread the amount on the soil surface, and then shallowly till the soil;

[0047] d) Regular re-application: Re-application every 3-4 months, the dosage is 1 / 2 of the initial application amount.

[0048] The core innovation of the present invention is that: first, by optimizing the combination of microorganisms, a broad-spectrum inhibition of multiple forest root pathogens is achieved. Secondly, the addition of functional auxiliary ingredients not only provides nutritional support for microorganisms, but also directly participates in the induction of plant defense responses. Thirdly, the microbial agent can significantly improve the structure of rhizosphere microbial communities and increase the diversity and abundance of beneficial microorganisms. Finally, by promoting root development and enhancing plant stress resistance, comprehensive protection and growth promotion of forests are achieved.

[0049] From a microbiological perspective, the Trichoderma and Bacillus in the present invention complement each other in terms of metabolites and mechanisms of action. Trichoderma mainly dissolves the cell walls of pathogens by secreting hydrolases such as chitinase and β-1,3-glucanase, while Bacillus produces a variety of antibiotics and volatile organic compounds. This synergistic effect not only expands the antibacterial spectrum, but also reduces the risk of pathogens developing resistance.

[0050] From the perspective of plant pathology and forestry, the bacterial agent of the present invention enhances the disease resistance of trees through multiple mechanisms. Ingredients such as alginic acid and mannitol in seaweed extract can act as plant immune inducers to activate systemic acquired resistance (SAR) and induced systemic resistance (ISR). Chitosan, through its special molecular structure, can bind to the chitin receptors of plant cell walls and trigger a series of defense responses.

[0051] From the perspective of soil science and ecology, the present invention not only focuses on the direct inhibition of pathogens, but also pays more attention to the overall regulation of the rhizosphere micro-ecosystem. The addition of humic acid improves the soil structure, increases the effectiveness of nutrients, and creates good conditions for the colonization of beneficial microorganisms. This ecological regulation method achieves long-term improvement of the rhizosphere environment and provides continuous protection for forests.

[0052] From the perspective of biotechnology, the preparation method of the present invention fully considers the characteristics and interactions of each component. For example, by optimizing the fermentation conditions and the selection of protective agents, the activity of the microorganisms during the preparation and storage process is guaranteed. The addition of the trace element complex provides the necessary nutrients for the metabolism and reproduction of the microorganisms.

[0053] From the perspective of pesticide science, this invention breaks through the limitations of traditional chemical pesticides and provides a green and sustainable disease prevention and control solution. Compared with chemical pesticides, this biological agent not only avoids environmental pollution and drug resistance problems, but also improves the overall health of forests by improving plant growth conditions.

[0054] The beneficial effects of the present invention are mainly reflected in the following aspects:

[0055] 1. Broad-spectrum and highly effective disease prevention and control: It has a significant inhibitory effect on a variety of forest root pathogens and has a broad prevention and control spectrum.

[0056] 2. Long-lasting and stable effect: By regulating the rhizosphere micro-ecosystem, a long-term and stable disease prevention effect is achieved.

[0057] 3. Dual role in promoting growth: It not only prevents and controls diseases, but also significantly promotes the growth and root development of trees.

[0058] 4. Enhanced stress resistance: Improved the resistance of trees to abiotic stresses, such as drought resistance and salt tolerance.

[0059] 5. Environmentally friendly: It avoids the negative impact of chemical pesticides and conforms to the concept of sustainable development.

[0060] 6. Wide applicability: It can be used in multiple stages such as seed treatment, seedling transplanting and forest management.

[0061] 7. Significant economic benefits: By improving the survival rate of trees, promoting growth and effectively preventing diseases, it brings considerable economic benefits to forestry production.

[0062] In summary, the composite microbial agent of the present invention has significant innovation and practical value in the field of forest root disease prevention and control, and provides a new efficient, environmentally friendly and sustainable solution for forestry production. DETAILED DESCRIPTION

[0063] The mechanism of action of the composite microbial agent of the present invention is:

[0064] 1. Direct antagonism: Trichoderma secretes chitinase, β-1,3-glucanase and other hydrolases to dissolve the cell walls of pathogens; Bacillus produces lipopeptide antibiotics and volatile organic compounds to inhibit the growth of pathogens;

[0065] 2. Induced resistance: Seaweed extract and chitosan act as inducers to activate plant systemic acquired resistance (SAR) and induced systemic resistance (ISR);

[0066] 3. Promote growth: Humic acid improves soil structure and promotes root development; trace element complex supplements the nutrients needed by plants;

[0067] 4. Enhance stress resistance: Glycine betaine acts as an osmotic regulator to improve plant drought and salt tolerance;

[0068] 5. Improve soil: The compound bacterial agent colonizes in the rhizosphere, secretes organic acids and auxins, and improves the soil microecological environment;

[0069] By compounding the above components and optimizing the preparation method, the composite microbial agent achieves synergy, complementarity and synergy in the following aspects:

[0070] 1. The synergistic antagonism of Trichoderma and Bacillus expands the control spectrum and improves the inhibitory effect on pathogens

[0071] 2. The addition of seaweed extract and chitosan not only enhances the plant's own immunity, but also provides nutrition for microorganisms and promotes their colonization

[0072] 3. The addition of humic acid improves the soil environment, is conducive to the growth and colonization of microorganisms, and also promotes the development of plant roots

[0073] 4. The addition of glycine betaine improves the stress resistance of microorganisms and plants, making the fungicide effective under different environmental conditions

[0074] 5. The addition of trace element complexes not only meets the growth needs of plants, but also provides necessary nutrients for microorganisms, promoting their metabolism and reproduction

[0075] 6. The composite carrier of perlite and corncob powder not only ensures the stability of the product, but also provides a carbon source for the initial colonization of microorganisms

[0076] This multi-component, multi-functional composite design not only improves the disease prevention effect of the product, but also has multiple functions such as growth promotion, stress resistance, and soil improvement, which greatly enhances the application value and innovation of the product. At the same time, the detailed preparation method and quality control standards ensure the stability and repeatability of the product, laying the foundation for practical application and promotion.

[0077] Embodiment 1:

[0078] This embodiment provides a composite microbial agent and a preparation method thereof. The agent comprises the following components by weight: 15 parts of Trichoderma ATCC 2084715 parts, 10 parts of Bacillus subtilis ATCC 605110 parts, 5 parts of seaweed extract, 3 parts of chitosan, 2 parts of humic acid, 1 part of glycine betaine, 0.5 parts of trace element complex, 40 parts of perlite powder, and 5 parts of corn cob powder.

[0079] The preparation method of the composite microbial agent comprises the following steps:

[0080] (1) Strain culture:

[0081] First, Trichoderma ATCC 20847 was cultured on potato dextrose agar (PDA) medium at 24°C in the dark for 7 days. Then, the spores were eluted with 0.1% Tween-80 solution and passed through a 200-mesh sieve to adjust the spore concentration to 1×10 9 CFU / mL. Secondly, Bacillus subtilis ATCC 6051 was cultured in nutrient broth at 29°C and 200 rpm for 24 hours. Then, the mixture was centrifuged at 4000 rpm for 10 minutes and resuspended in sterile water to adjust the concentration to 1×10 9 CFU / mL.

[0082] (2) Preparation of auxiliary ingredients:

[0083] a) Seaweed extract: The dried brown algae was crushed into 60 mesh, and 10 volumes of 0.1 M HCl were added, and the mixture was extracted at 60° C. for 2 hours. The mixture was filtered, and the pH of the filtrate was adjusted to 7.0 with NaOH, and the mixture was concentrated under reduced pressure to 1 / 5 of the original volume, and spray-dried to obtain seaweed extract powder.

[0084] b) Chitosan: Chitosan with a deacetylation degree of 90% and a molecular weight of 100 kDa was dissolved in 1% acetic acid solution, stirred until completely dissolved, and the concentration was adjusted to 2% (w / v).

[0085] c) Humic acid: 60% humic acid was dissolved in 0.1 M NaOH, stirred for 2 hours, filtered, and the pH of the filtrate was adjusted to 5.5 with HCl.

[0086] d) Trace element complex: EDTA chelate and sodium molybdate were mixed in a ratio of Fe:Mn:Zn:Cu:Mo=5:2:2:1:0.1, dissolved in deionized water, and the total concentration was adjusted to 1% (w / v).

[0087] (3) Preparation of bacterial agents:

[0088] a) First, pearlite powder with a particle size of 200 mesh and corn cob powder with a particle size of 40 mesh were mixed in a ratio of 7:1 and sterilized by autoclaving at 121°C for 20 minutes.

[0089] b) Secondly, the Trichoderma spore suspension and the Bacillus subtilis cell suspension are mixed in a 1:1 ratio.

[0090] c) Then, add the bacterial suspension to the mixed carrier, stir evenly, and control the water content to 30%.

[0091] d) Again, add the seaweed extract, chitosan solution and humic acid solution in sequence and stir evenly.

[0092] e) Subsequently, glycine betaine and trace element complex were added and stirring was continued for 10 minutes.

[0093] f) Then, drying at 25°C until the moisture content is less than 10%.

[0094] g) Finally, crush and pass through a 60-mesh sieve to obtain the final product.

[0095] Preferably, in the embodiment of the present invention, Trichoderma ATCC 20847 can secrete hydrolases such as chitinase and β-1,3-glucanase to effectively dissolve the cell wall of pathogens, while Bacillus subtilis ATCC 6051 produces lipopeptide antibiotics and volatile organic compounds to inhibit the growth of pathogens. The two act synergistically to significantly improve the prevention and control effect of forest root diseases. In addition, seaweed extract and chitosan, as inducers, can activate plant systemic acquired resistance (SAR) and induced systemic resistance (ISR), further enhancing the disease resistance of plants.

[0096] Embodiment 2:

[0097] This embodiment provides another composite microbial agent and a preparation method thereof. The agent comprises the following components by weight: Trichoderma ATCC 2084725 parts, Bacillus subtilis ATCC 605120 parts, seaweed extract 10 parts, chitosan 8 parts, humic acid 5 parts, glycine betaine 3 parts, trace element complex 1.5 parts, perlite powder 30 parts, corn cob powder 10 parts.

[0098] The preparation method of the composite microbial agent comprises the following steps:

[0099] (1) Strain culture:

[0100] First, Trichoderma ATCC 20847 was cultured on potato dextrose agar (PDA) medium at 26°C in the dark for 10 days. Then, the spores were eluted with 0.1% Tween-80 solution and passed through a 200-mesh sieve to adjust the spore concentration to 1×10 9 CFU / mL. Secondly, Bacillus subtilis ATCC 6051 was cultured in nutrient broth at 31°C and 200 rpm for 48 hours. Then, the cells were centrifuged at 4000 rpm for 10 minutes and resuspended in sterile water to adjust the concentration to 1×10 9 CFU / mL.

[0101] (2) Preparation of auxiliary ingredients:

[0102] a) Seaweed extract: The dried brown algae was crushed into 80 mesh, and 10 volumes of 0.1 M HCl were added, and the mixture was extracted at 60° C. for 2 hours. The mixture was filtered, and the pH of the filtrate was adjusted to 7.0 with NaOH, and the mixture was concentrated under reduced pressure to 1 / 5 of the original volume, and spray-dried to obtain seaweed extract powder.

[0103] b) Chitosan: Chitosan with a deacetylation degree of 95% and a molecular weight of 300 kDa was dissolved in 1% acetic acid solution, stirred until completely dissolved, and the concentration was adjusted to 2% (w / v).

[0104] c) Humic acid: 70% humic acid was dissolved in 0.1 M NaOH, stirred for 2 hours, filtered, and the pH of the filtrate was adjusted to 6.0 with HCl.

[0105] d) Trace element complex: EDTA chelate and sodium molybdate were mixed in a ratio of Fe:Mn:Zn:Cu:Mo=5:2:2:1:0.1, dissolved in deionized water, and the total concentration was adjusted to 1% (w / v).

[0106] (3) Preparation of bacterial agents:

[0107] a) First, pearlite powder with a particle size of 300 mesh and corn cob powder with a particle size of 60 mesh were mixed in a ratio of 7:1 and sterilized by autoclaving at 121°C for 20 minutes.

[0108] b) Secondly, the Trichoderma spore suspension and the Bacillus subtilis cell suspension are mixed in a 1:1 ratio.

[0109] c) Then, add the bacterial suspension to the mixed carrier, stir evenly, and control the water content to 35%.

[0110] d) Again, add the seaweed extract, chitosan solution and humic acid solution in sequence and stir evenly.

[0111] e) Subsequently, glycine betaine and trace element complex were added and stirring was continued for 10 minutes.

[0112] f) Then, drying at 30°C until the moisture content is less than 10%.

[0113] g) Finally, crush and pass through a 60-mesh sieve to obtain the final product.

[0114] Preferably, in the embodiments of the present invention, the addition of humic acid not only improves the soil environment, is conducive to the growth and colonization of microorganisms, but also promotes the development of plant roots. In addition, glycine betaine, as an osmotic regulator, improves the stress resistance of microorganisms and plants, so that the bacterial agent can play a role under different environmental conditions. The addition of trace element complexes meets the growth needs of plants, and at the same time provides necessary nutrients for microorganisms to promote their metabolism and reproduction.

[0115] Embodiment 3:

[0116] This embodiment provides a third composite microbial agent and a preparation method thereof. The agent comprises the following components by weight: Trichoderma ATCC 2084720 parts, Bacillus subtilis ATCC 605115 parts, seaweed extract 7.5 parts, chitosan 5.5 parts, humic acid 3.5 parts, glycine betaine 2 parts, trace element complex 1 part, perlite powder 35 parts, corn cob powder 7.5 parts.

[0117] The preparation method of the composite microbial agent comprises the following steps:

[0118] (1) Strain culture:

[0119] First, Trichoderma ATCC 20847 was cultured on potato dextrose agar (PDA) medium at 25°C in the dark for 8.5 days. Then, the spores were eluted with 0.1% Tween-80 solution and passed through a 200-mesh sieve to adjust the spore concentration to 1×10 9 CFU / mL. Secondly, Bacillus subtilis ATCC 6051 was cultured in nutrient broth at 30°C and 200 rpm for 36 hours. Then, it was centrifuged at 4000 rpm for 10 minutes and resuspended in sterile water to adjust the concentration to 1×10 9 CFU / mL.

[0120] (2) Preparation of auxiliary ingredients:

[0121] a) Seaweed extract: The dried brown algae was crushed into 70 mesh, and 10 volumes of 0.1 M HCl were added, and the mixture was extracted at 60° C. for 2 hours. The mixture was filtered, and the pH of the filtrate was adjusted to 7.0 with NaOH, and the mixture was concentrated under reduced pressure to 1 / 5 of the original volume, and spray-dried to obtain seaweed extract powder.

[0122] b) Chitosan: Chitosan with a deacetylation degree of 92.5% and a molecular weight of 200 kDa was dissolved in 1% acetic acid solution, stirred until completely dissolved, and the concentration was adjusted to 2% (w / v).

[0123] c) Humic acid: 65% humic acid was dissolved in 0.1 M NaOH, stirred for 2 hours, filtered, and the pH of the filtrate was adjusted to 5.75 with HCl.

[0124] d) Trace element complex: EDTA chelate and sodium molybdate were mixed in a ratio of Fe:Mn:Zn:Cu:Mo=5:2:2:1:0.1, dissolved in deionized water, and the total concentration was adjusted to 1% (w / v).

[0125] (3) Preparation of bacterial agents:

[0126] a) First, pearlite powder with a particle size of 250 mesh and corn cob powder with a particle size of 50 mesh were mixed in a ratio of 7:1 and sterilized by autoclaving at 121°C for 20 minutes.

[0127] b) Secondly, the Trichoderma spore suspension and the Bacillus subtilis cell suspension are mixed in a 1:1 ratio.

[0128] c) Then, add the bacterial suspension to the mixed carrier, stir evenly, and control the water content to 32.5%.

[0129] d) Again, add the seaweed extract, chitosan solution and humic acid solution in sequence and stir evenly.

[0130] e) Subsequently, glycine betaine and trace element complex were added and stirring was continued for 10 minutes.

[0131] f) Then, drying at 27.5°C until the moisture content is less than 10%.

[0132] g) Finally, crush and pass through a 60-mesh sieve to obtain the final product.

[0133] Preferably, in the embodiment of the present invention, the composite carrier of perlite and corncob powder not only ensures the stability of the product, but also provides a carbon source for the initial colonization of microorganisms. This carrier design is conducive to maintaining the activity of microorganisms during transportation and storage, and can quickly release active ingredients after application, thereby improving the actual application effect of the product.

[0134] Embodiment 4:

[0135] This embodiment provides a fourth composite microbial agent and a preparation method thereof. The agent comprises the following components by weight: Trichoderma ATCC 2084722 parts, Bacillus subtilis ATCC 605117 parts, 8 parts of seaweed extract, 6 parts of chitosan, 4 parts of humic acid, 2.5 parts of glycine betaine, 1.2 parts of trace element complex, 32 parts of perlite powder, and 8 parts of corn cob powder.

[0136] The preparation method of the composite microbial agent comprises the following steps:

[0137] (1) Strain culture:

[0138] First, Trichoderma ATCC 20847 was cultured on potato dextrose agar (PDA) medium at 25.5°C in the dark for 9 days. Then, the spores were eluted with 0.1% Tween-80 solution and passed through a 200-mesh sieve to adjust the spore concentration to 1×10 9 CFU / mL. Secondly, Bacillus subtilis ATCC 6051 was cultured in nutrient broth at 30.5°C and 200 rpm for 42 hours. Then, the mixture was centrifuged at 4000 rpm for 10 minutes and resuspended in sterile water to adjust the concentration to 1×10 9 CFU / mL.

[0139] (2) Preparation of auxiliary ingredients:

[0140] a) Seaweed extract: The dried brown algae was crushed into 75 mesh, and 10 volumes of 0.1 M HCl were added, and the mixture was extracted at 60° C. for 2 hours. The mixture was filtered, and the pH of the filtrate was adjusted to 7.0 with NaOH, and the mixture was concentrated under reduced pressure to 1 / 5 of the original volume, and spray-dried to obtain seaweed extract powder.

[0141] b) Chitosan: Chitosan with a deacetylation degree of 93% and a molecular weight of 250 kDa was dissolved in 1% acetic acid solution, stirred until completely dissolved, and the concentration was adjusted to 2% (w / v).

[0142] c) Humic acid: 67% humic acid was dissolved in 0.1 M NaOH, stirred for 2 hours, filtered, and the pH of the filtrate was adjusted to 5.8 with HCl.

[0143] d) Trace element complex: EDTA chelate and sodium molybdate were mixed in a ratio of Fe:Mn:Zn:Cu:Mo=5:2:2:1:0.1, dissolved in deionized water, and the total concentration was adjusted to 1% (w / v).

[0144] (3) Preparation of bacterial agents:

[0145] a) First, pearlite powder with a particle size of 275 mesh and corn cob powder with a particle size of 55 mesh were mixed in a ratio of 7:1 and sterilized by autoclaving at 121°C for 20 minutes.

[0146] b) Secondly, the Trichoderma spore suspension and the Bacillus subtilis cell suspension are mixed in a 1:1 ratio.

[0147] c) Then, add the bacterial suspension to the mixed carrier, stir evenly, and control the water content to 33%.

[0148] d) Again, add the seaweed extract, chitosan solution and humic acid solution in sequence and stir evenly.

[0149] e) Subsequently, glycine betaine and trace element complex were added and stirring was continued for 10 minutes.

[0150] f) Then, drying at 28°C until the moisture content is less than 10%.

[0151] g) Finally, crush and pass through a 60-mesh sieve to obtain the final product.

[0152] Preferably, in an embodiment of the present invention, the alginic acid, mannitol and seaweed polysaccharide in the seaweed extract not only activate the defense system of the plant as an inducer, but also provide an additional nutrient source for microorganisms, promoting their rapid colonization in the rhizosphere. The high deacetylation degree and moderate molecular weight of chitosan are conducive to its interaction with the plant cell wall, enhancing the effect of induced resistance. In addition, the ratio of each element in the trace element complex is optimized, which not only meets the nutritional needs of the plant, but also provides a balanced trace element supply for microorganisms, which is conducive to maintaining the stability of the microbial community.

[0153] Through the above four embodiments, the composite microbial agent of the present invention has shown remarkable innovation and flexibility in terms of component ratio, preparation process and functionality. Each component has been carefully designed and optimized, not only playing a role in a single function, but also through the synergistic effect between each other, to achieve comprehensive prevention and control of tree root diseases. At the same time, the various parameters of the preparation method have also been carefully adjusted to ensure the quality stability and biological activity of the final product. This multi-component, multi-functional composite design not only improves the disease prevention effect of the product, but also has multiple functions such as growth promotion, stress resistance, and soil improvement, which greatly enhances the application value and innovation of the product in the prevention and control of tree root diseases.

[0154] Application Example 1: Seed Treatment

[0155] This example demonstrates the application effect of the composite microbial agent in pine seed treatment.

[0156] Experimental methods:

[0157] Fresh seeds of Pinus elliottii were selected as experimental materials. The seeds were randomly divided into a treatment group and a control group, with 100 seeds in each group. The seeds of the treatment group were soaked in a 1% suspension of the composite microbial agent prepared in Example 1 for 4 hours, while the seeds of the control group were treated with an equal amount of sterile water. The treated seeds were dried in the shade at 25°C and then sown into a pre-sterilized sand culture substrate.

[0158] After 30 days of cultivation under greenhouse conditions (temperature 25±2℃, relative humidity 70±5%), observe and record the following indicators:

[0159] 1. Germination rate (%);

[0160] 2. Seedling height (cm);

[0161] 3. Root length (cm);

[0162] 4. Root rot incidence (%);

[0163] result:

[0164] 1. Germination rate: 92% in the treatment group and 85% in the control group;

[0165] 2. Average seedling height: 8.5 cm in the treatment group and 7.2 cm in the control group;

[0166] 3. Average root length: 12.3 cm in the treatment group and 10.1 cm in the control group;

[0167] 4. Root rot incidence: 5% in the treatment group and 18% in the control group;

[0168] Result analysis:

[0169] The seeds treated with the composite microbial agent were significantly better than the control group in all indicators. The germination rate increased by 7 percentage points, the seedlings grew better, and the root system developed healthier. It is particularly noteworthy that the incidence of root rot in the treated group was greatly reduced, only 27.8% of the control group. This fully proves the significant effect of the composite microbial agent of the present invention in promoting seed germination, enhancing seedling vitality and preventing root diseases.

[0170] Application Example 2: Soaking treatment of seedling roots

[0171] This example demonstrates the application effect of the composite microbial agent in root treatment of Chinese fir seedlings before transplanting.

[0172] Experimental methods:

[0173] One-year-old Cunninghamia lanceolata seedlings were selected as experimental materials, with a seedling height of about 30 cm. The seedlings were randomly divided into a treatment group and a control group, with 50 plants in each group. The roots of the seedlings in the treatment group were soaked in a 0.5% composite microbial agent suspension prepared in Example 2 for 30 minutes, while the control group was treated with an equal amount of sterile water. The treated seedlings were immediately transplanted to a pre-prepared test plot.

[0174] The test plot is located in Sanming City, Fujian Province. The soil type is acidic red soil with a pH value of about 5.5. Conventional management measures were adopted during the test, and no additional pesticides or fertilizers were applied. Three months after transplanting, the following indicators were observed and recorded:

[0175] 1. Survival rate (%);

[0176] 2. Plant height increase (cm);

[0177] 3. Ground diameter growth (mm);

[0178] 4. Root biomass (g / plant);

[0179] 5. Incidence of wilt disease (%);

[0180] result:

[0181] 1. Survival rate: 96% in the treatment group and 88% in the control group;

[0182] 2. Average plant height increase: 18.5cm in the treatment group and 15.2cm in the control group;

[0183] 3. Average ground diameter growth: 3.2mm in the treatment group and 2.5mm in the control group;

[0184] 4. Average root biomass: 28.6 g / plant in the treatment group and 22.3 g / plant in the control group;

[0185] 5. Incidence of wilt disease: 3% in the treatment group and 12% in the control group;

[0186] Result analysis:

[0187] The Chinese fir seedlings treated with the composite microbial agent showed stronger adaptability and growth momentum after transplantation. The survival rate of the treated group was 8 percentage points higher than that of the control group, and the growth of plant height and ground diameter increased by 21.7% and 28% respectively. It is particularly noteworthy that the root biomass of the treated group was significantly higher than that of the control group, with an increase of 28.3%, which shows that the agent significantly promoted the development of the root system. At the same time, the incidence of wilt in the treated group was only 25% of that in the control group, which fully proved the excellent effect of the present invention in preventing root diseases of forest trees.

[0188] These two application examples clearly demonstrate the multiple advantages of the composite microbial agent of the present invention in actual forestry production. Whether in seed treatment or seedling transplanting stage, the agent shows significant growth promotion and disease prevention effects. This not only verifies the practical value of the present invention, but also provides strong experimental support for its application in large-scale forestry production. By improving the survival rate of trees, promoting growth and effectively preventing and controlling root diseases, the present invention is expected to bring significant economic and ecological benefits to forestry production.

[0189] Comparative Example 1: Single microbial preparation

[0190] This comparative example provides a microbial preparation containing only Trichoderma, which is used to compare the synergistic effect of the composite microorganisms in Example 1. The preparation comprises, by weight: Trichoderma harzianum ATCC 2084725 parts, perlite powder 70 parts, and corn cob powder 5 parts.

[0191] The preparation method comprises the following steps:

[0192] (1) First, Trichoderma ATCC 20847 was cultured on potato dextrose agar (PDA) medium at 24°C in the dark for 7 days. Subsequently, spores were eluted with 0.1% Tween-80 solution, passed through a 200-mesh sieve, and the spore concentration was adjusted to 1×10^9 CFU / mL.

[0193] (2) Next, pearlite powder with a particle size of 200 mesh and corn cob powder with a particle size of 40 mesh were mixed in a ratio of 14:1 and sterilized by autoclaving at 121°C for 20 minutes.

[0194] (3) Then, add the bacterial suspension to the mixed carrier, stir evenly, and control the water content to 30%.

[0195] (4) Finally, the mixture was dried at 25°C until the moisture content was less than 10%, and then crushed through a 60-mesh sieve to obtain the final product.

[0196] Comparative Example 2: Composite microbial preparation without auxiliary ingredients

[0197] This comparative example is intended to verify the importance of auxiliary components in the composite microbial preparation, and is compared with Example 2. The preparation comprises, by weight: Trichoderma (Trichodermaharzianum) ATCC 2084725 parts, Bacillus subtilis (Bacillus subtilis) ATCC 605120 parts, perlite powder 45 parts, and corn cob powder 10 parts.

[0198] The preparation method is similar to that of Example 2, but the step of adding the auxiliary components is omitted.

[0199] Comparative Example 3: Low-dose trace element complex preparation

[0200] This comparative example is intended to verify the effect of the dosage of the trace element complex on the effect of the preparation, and is compared with Example 3. The components of the preparation are the same as those of Example 3, but the content of the trace element complex is reduced to 0.2 parts.

[0201] The preparation method is basically the same as that of Example 3, but when preparing the trace element complex, the total concentration is adjusted to 0.4% (w / v).

[0202] Comparative Example 4: High pH Humic Acid Preparation

[0203] This comparative example is intended to verify the effect of the pH value of humic acid on the effect of the preparation, and is compared with Example 4. The components of the preparation are the same as those of Example 4, but the pH value of the humic acid solution is adjusted to 7.0.

[0204] The preparation method is substantially the same as that of Example 4, but in the humic acid preparation step, the pH of the filtrate is adjusted to 7.0 with NaOH.

[0205] Comparative Example 5: Chitosan-free preparation

[0206] This comparative example is intended to verify the role of chitosan in inducing plant resistance, and is compared with Example 1. The components of the preparation are the same as those of Example 1, but chitosan is removed and the content of perlite powder is increased accordingly.

[0207] The preparation method is substantially the same as that of Example 1, but the steps of preparing and adding the chitosan solution are omitted.

[0208] Comparative Example 6: High temperature drying preparation

[0209] This comparative example is intended to verify the effect of drying temperature on microbial activity and is compared with Example 2. The components of the preparation are exactly the same as those in Example 2.

[0210] The preparation method is basically the same as Example 2, but in the drying step, the temperature is increased to 40°C.

[0211] Through these six comparative examples, the effects of various key factors on the formulation effects of the present invention can be systematically evaluated. Comparative Example 1 highlights the synergistic effect of composite microorganisms; Comparative Examples 2 and 5 respectively verify the importance of auxiliary components and chitosan; Comparative Examples 3 and 4 explore the optimal parameters of trace elements and humic acid; and Comparative Example 6 focuses on the effect of the preparation process on product quality.

[0212] By comparing with the corresponding examples, the superiority of the present invention in terms of component ratio, preparation process and functionality can be clearly demonstrated. For example, it is expected that the single microbial preparation of Comparative Example 1 will be inferior to the composite preparation in terms of control spectrum and persistence; Comparative Examples 2 and 5 may perform poorly in terms of plant resistance induction and microbial colonization; Comparative Examples 3 and 4 may affect the growth of microorganisms and the stability of the product; and Comparative Example 6 may result in a significant reduction in microbial activity.

[0213] This systematic comparative design not only helps to verify the creativity of the present invention, but also provides valuable data support for further optimizing the product. Through these comparative experiments, we can have a deeper understanding of the synergistic mechanism between the components, and provide a solid scientific basis for the application of the present invention in the prevention and treatment of forest root diseases.

[0214] In order to comprehensively evaluate the effectiveness of the "composite microbial agent and its application in the prevention and treatment of forest root diseases" of the present invention, a series of experiments were designed to test its performance. These experiments covered multiple fields such as microbiology, plant pathology, forestry, soil science and ecology, aiming to fully demonstrate the core innovation and synergistic mechanism of the present invention.

[0215] Experiment 1: Analysis of the diversity of rhizosphere microbial communities

[0216] Experimental purpose: To evaluate the effect of compound microbial agents on the structure of rhizosphere microbial communities in forest trees.

[0217] Experimental methods:

[0218] Three-year-old saplings of Cunninghamia lanceolata were selected as experimental subjects. The bacterial agents of Examples 1-4 and Comparative Examples 1-6 were applied to different groups of rhizosphere soil of Cunninghamia lanceolata, with 3 replicates in each group. An untreated control group was set. 60 days after the application of the bacterial agent, rhizosphere soil samples were collected, and the 16S rRNA and ITS sequences of bacteria and fungi were analyzed using high-throughput sequencing technology.

[0219] Experimental results:

[0220] Table 1. Effects of different treatments on the diversity of rhizosphere microbial communities of Chinese fir

[0221]

[0222] Results analysis: The Shannon diversity index of Examples 1-4 was significantly higher than that of the comparative example and the control group, indicating that the composite microbial agent can effectively increase the diversity of rhizosphere microorganisms. In particular, Example 2, the Shannon index of bacteria and fungi reached 5.01 and 3.89, respectively, showing the best microbial community structure. In addition, more beneficial microbial genera appeared in the Example group, such as nitrogen-fixing bacteria Rhizobium and Azotobacter, which may be due to the addition of auxiliary ingredients that promoted the growth of these beneficial bacteria.

[0223] Experiment 2: Analysis of root morphology and physiological indicators

[0224] Experimental purpose: To evaluate the effects of complex microbial agents on the development and physiological state of tree roots.

[0225] Experimental methods:

[0226] One-year-old Masson pine (Pinus massoniana) seedlings were selected and treated with the bacterial agents of Examples 1-4 and Comparative Examples 1-6 for 30 minutes by soaking the roots, 20 plants in each group, and an untreated control group was set up. After 90 days of cultivation under greenhouse conditions (25±2°C, relative humidity 70±5%), the root morphological parameters and physiological indicators were measured.

[0227] Experimental results:

[0228] Table 2. Effects of different treatments on root development and physiological indicators of Masson pine seedlings

[0229]

[0230] Results analysis: The root morphology and physiological indicators of Masson pine seedlings treated with Examples 1-4 were significantly better than those of the comparative example and the control group. Among them, Example 2 performed best, with a root length of 172.8 cm, an increase of 42.2% over the control group; the root surface area and the number of root tips increased by 41.4% and 49.1%, respectively. More importantly, the root vitality and SOD activity of Example 2 increased by 38.6% and 32.3%, respectively, indicating that the composite microbial agent not only promoted root growth, but also significantly enhanced the physiological activity and antioxidant capacity of the root system.

[0231] Experiment 3: Evaluation of the inhibitory effect on pathogens

[0232] Experimental purpose: To evaluate the inhibitory effect of compound microbial agents on common forest tree root pathogens.

[0233] Experimental methods:

[0234] Three common forest root pathogens were selected: pine wood nematode pathogenic nematode (Bursaphelenchusxylophilus), pine root rot pathogen (Heterobasidion annosum) and fir wilt pathogen (Fusariumoxysporum). The bacterial agents of Examples 1-4 and Comparative Examples 1-6 were inoculated with pathogens on PDA culture medium by plate confrontation culture method, and the diameter of the inhibition zone was measured after culturing at 28°C for 7 days.

[0235] Experimental results:

[0236] Table 3. Inhibitory effects of different treatments on pathogens in tree roots (diameter of inhibition zone, mm)

[0237] deal with B. xylophilus H. annosum F.oxysporum Example 1 18.5 22.3 20.7 Example 2 21.6 25.8 23.9 Example 3 20.4 24.5 22.8 Example 4 19.7 23.6 21.9 Comparative Example 1 12.3 15.6 14.2 Comparative Example 2 14.8 18.2 16.7 Comparative Example 3 15.6 19.1 17.5 Comparative Example 4 14.2 17.8 16.3 Comparative Example 5 13.9 17.3 15.8 Comparative Example 6 13.5 16.9 15.4 Comparison 0 0 0

[0238] Results analysis: Examples 1-4 all showed significant inhibitory effects on the three pathogens, among which Example 2 had the best inhibitory effect. The diameter of the inhibition zone for pine wood nematode pathogenic nematodes reached 21.6 mm, which was 38.5% higher than the closest comparative example 3. This result fully demonstrated the superiority of the composite microbial agent in preventing and controlling a variety of forest root diseases, which may be due to the synergistic effect between different microorganisms and the synergistic effect of auxiliary components.

[0239] Based on the above experimental results, the following conclusions can be drawn:

[0240] 1. Microbial community optimization: The composite microbial agent of the present invention not only increases the diversity of rhizosphere microorganisms, but also promotes the colonization of beneficial microorganisms. This improved microbial community structure may be achieved through a variety of mechanisms, including mutualistic symbiosis between microorganisms, nutritional support provided by auxiliary ingredients, and improved rhizosphere microenvironment.

[0241] 2. Promotion of root development: The microbial agent significantly promoted the growth and development of the tree root system, especially increasing the root length, root surface area and number of root tips. This effect may be due to the growth hormones (such as auxin and cytokinin) produced by microorganisms and the improved soil structure and nutrient availability.

[0242] 3. Enhanced stress resistance: The increase in root vitality and SOD activity indicates that the treated trees have stronger stress resistance. This may be due to the fact that microorganisms induce plant systemic acquired resistance (SAR) and induced systemic resistance (ISR), while chitosan and other ingredients are also directly involved in the plant's defense response.

[0243] 4. Broad-spectrum disease resistance: The significant inhibitory effect on a variety of pathogens proves that the present invention has a broad-spectrum disease prevention and control ability. This effect may be due to the combined effect of multiple mechanisms such as antibiotics produced by different microorganisms, competitive effects, and inducing plants to produce defense substances.

[0244] 5. Long-term sustainability: Improvements in microbial communities and healthy root development provide long-term protection and growth promotion for trees, an advantage that is difficult to achieve with chemical pesticides.

[0245] 6. Eco-friendliness: The present invention achieves disease prevention and control by adjusting the balance of microbial communities, avoiding the negative impact of chemical pesticides on the ecological environment, and is in line with the concept of sustainable development.

[0246] These results not only verify the effectiveness of the present invention, but also reveal some unexpected technical effects. For example, the composite microbial agent not only directly inhibits pathogens, but also enhances the overall health of trees by improving the entire rhizosphere micro-ecosystem. This "ecological regulation" method provides a new idea for forest disease prevention and control, and has the potential to become an effective alternative to chemical pesticides. In addition, the comprehensive improvement effect of the microbial agent on root morphology and physiology suggests that it may also have potential in improving the drought resistance, salt resistance and other abiotic stresses of trees, which opens up new possibilities for the present invention in a wider range of forestry applications.

[0247] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Composite microbial agent, characterized by , by weight, comprising the following components: Trichoderma (Trichodermaharzianum) ATCC 2084715-25; Bacillus subtilis ATCC 605110-20 copies; 5-10 parts of seaweed extract; 3-8 parts of chitosan; Humic acid 2-5 parts; 1-3 parts of glycine betaine; Trace element complex 0.5-1.5 parts; 30-40 parts of perlite powder; 5-10 servings of corn cob flour.

2. The composite microbial agent according to claim 1, characterized in that The seaweed extract is derived from brown algae (Ascophyllum nodosum), and its main components include alginic acid, mannitol and seaweed polysaccharides.

3. The composite microbial agent according to claim 1, characterized in that The chitosan has a deacetylation degree of ≥90% and a molecular weight of 100-300 kDa.

4. The composite microbial agent according to claim 1, characterized in that , the content of humic acid is ≥60%.

5. The composite microbial agent according to claim 1, characterized in that , the trace element complex includes Fe-EDTA, Mn-EDTA, Zn-EDTA, Cu-EDTA and Na2MoO4.

6. The composite microbial agent according to claim 1, characterized in that The particle size of the perlite powder is 200-300 mesh, and the particle size of the corncob powder is 40-60 mesh.

7. The method for preparing the composite microbial agent according to any one of claims 1 to 6, characterized in that , including the following steps: (1) Strain culture: a) Cultivate Trichoderma ATCC 20847 on potato dextrose agar (PDA) medium at 25±1°C in the dark for 7-10 days; elute spores with 0.1% Tween-80 solution, pass through a 200-mesh sieve, and adjust the spore concentration to 1×10 9 CFU / mL; b) Cultivate Bacillus subtilis ATCC 6051 in nutrient broth at 30±1°C and 200 rpm for 24-48 hours; centrifuge at 4000 rpm for 10 minutes, resuspend in sterile water, and adjust the concentration to 1×10 9 CFU / mL; (2) Preparation of auxiliary ingredients: a) crushing the dried brown algae into 60-80 mesh, adding 10 times the volume of 0.1M HCl, extracting at 60°C for 2 hours, filtering, adjusting the pH of the filtrate to 7.0 with NaOH, concentrating under reduced pressure to 1 / 5 of the original volume, and spray drying to obtain seaweed extract powder; b) dissolving chitosan in 1% acetic acid solution, stirring until completely dissolved, and adjusting the concentration to 2% (w / v); c) dissolving humic acid in 0.1M NaOH, stirring for 2 hours, filtering, and adjusting the pH of the filtrate to 5.5-6.0 with HCl; d) mixing EDTA chelate and sodium molybdate in a ratio of Fe:Mn:Zn:Cu:Mo=5:2:2:1:0.1, dissolving in deionized water, and adjusting the total concentration to 1% (w / v); (3) Preparation of bacterial agents: a) Mix perlite powder and corncob powder in a ratio of 7:1 and sterilize by autoclave at 121℃ for 20 minutes; b) mixing the Trichoderma spore suspension and the Bacillus subtilis cell suspension in a 1:1 ratio; c) adding the bacterial suspension to the mixed carrier, stirring evenly, and controlling the water content to 30-35%; d) adding seaweed extract, chitosan solution and humic acid solution in sequence and stirring evenly; e) adding glycine betaine and trace element complex and continuing stirring for 10 minutes; f) drying at 25-30°C to a moisture content of less than 10%; g) Grind through a 60-mesh sieve to obtain the final product.

8. The preparation method according to claim 7, characterized in that , the method further comprises a quality control step: a) Determination of viable bacteria count: Using the plate count method, the total viable bacteria count of Trichoderma and Bacillus subtilis should not be less than 2×10 9 CFU / g; b) Moisture content: not more than 10%; c) pH value: 6.5-7.5; d) Particle size: 95% passed through a 60 mesh sieve.

9. Use of the composite microbial agent as described in any one of claims 1 to 6 in the prevention and treatment of tree root diseases.

10. The use according to claim 9, characterized in that , including the following application methods: a) Seed treatment: Soak the seeds in 1% bacterial suspension for 4-6 hours, then dry in the shade and sow; b) Soaking the roots of seedlings: Soak the roots of seedlings in 0.5% bacterial agent suspension for 30 minutes before planting; c) Soil treatment: 20-30kg / hm 2 Evenly spread the amount on the soil surface, and then shallowly till the soil; d) Regular re-application: Re-application every 3-4 months, the dosage is 1 / 2 of the initial application amount.

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