Microbial fertilizer for preventing and treating panax notoginseng root rot

Abstract

The invention provides a microbial fertilizer for preventing and treating panax notoginseng root rot, and relates to the technical field of microbial fertilizers.The microbial fertilizer comprises A-type quick-acting particles and B-type long-acting dormancy particles, the A-type particles take biochar as a carrier to bear a specific microbial complex, the B-type particles take biochar bearing the microbial complex as a core, a biodegradable film is coated outside the B-type particles, and the B-type particles are coated outside the biochar bearing the microbial complex. The microbial complex is composed of burkholderia, Savitz yeast, mortierella and streptomyces medicaginis, and the fertilizer is combined with deep ploughing preset basal application and targeted activation application methods, so that full-profile long-acting prevention and control of panax notoginseng root rot can be realized, and soil micro-ecology can be restored.

Description

Technical Field

[0001] This invention belongs to the field of microbial fertilizer technology, specifically relating to a microbial fertilizer for preventing and controlling root rot in Panax notoginseng. Background Technology

[0002] Panax notoginseng, a precious traditional Chinese medicine, has long suffered from severe constraints due to the "continuous cropping obstacle." Root rot caused by soil-borne pathogens such as Fusarium oxysporum is the primary factor leading to decreased yield and deteriorated quality. Research indicates that the outbreak of root rot in Panax notoginseng is not simply a matter of pathogen infection, but a complex process of soil microecological imbalance triggered by the plant itself. Its core pathogenic mechanism forms a vicious cycle: during the growth of Panax notoginseng roots, large amounts of saponin-like autotoxic substances are secreted and accumulated. These substances exert strong selective pressure on the soil microbial community, significantly inhibiting the original beneficial antagonistic bacteria in the soil, such as yeasts, spirochetes, and streptomyces; on the other hand, greatly promoting the growth and colonization of pathogenic Fusarium oxysporum. This ebb and flow disrupts the soil microecological balance, causing the soil to lose its natural "disease-suppressing" properties, ultimately creating conditions for a large-scale outbreak of root rot.

[0003] The main problems with currently used prevention and control measures for root rot in Panax notoginseng are: Chemical control: Soil fumigants such as dazomet and methyl bromide are used for soil disinfection. Although this method can temporarily kill pathogens, its sterilization process is non-selective and kills almost all beneficial microorganisms in the soil, creating a biological vacuum. Once external pathogens invade again, they will proliferate explosively due to the lack of antagonists, leading to disease rebound or even exacerbation. In addition, long-term use of chemical agents can also lead to serious problems such as pesticide residues, soil compaction, and environmental pollution.

[0004] Agricultural control: Crop rotation is a traditional method for controlling soil-borne diseases. However, since saponins that cause microecological imbalance and dormant Fusarium oxysporum can remain in the soil for several years, conventional crop rotation cycles are difficult to completely eliminate pathogens, and the disease is very easy to recur after replanting.

[0005] Conventional microbial fertilizers: Existing commercially available microbial fertilizer products are limited to a single form, and their application methods are mostly surface application, furrow application, or seed coating. These methods are mainly limited to the top 0-20cm of soil. However, soil profiles exhibit significant vertical heterogeneity in terms of physical, chemical, and biological properties. Panax notoginseng roots can penetrate to soil layers of 50cm or even deeper. Conventional fertilization methods cannot effectively and evenly deliver beneficial microorganisms to the entire root activity zone, especially in deeper soil layers, leaving blind spots for prevention and control.

[0006] Therefore, it is necessary to develop a new type of microbial fertilizer that can act on the entire soil profile, solve the problem of saponin autotoxicity from the root through a scientifically constructed microbial community, and combine innovative application methods to achieve a systematic and long-term control of Panax notoginseng root rot by combining prevention and treatment. Summary of the Invention

[0007] To overcome the problems in the background technology, this invention develops a microbial fertilizer for preventing and controlling root rot of Panax notoginseng. Through innovative fertilizer structure and matching application method, it achieves systematic prevention and control of root rot of Panax notoginseng in a comprehensive, full-cycle, and symptomatic way.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: A microbial fertilizer for preventing root rot in Panax notoginseng is a granular mixture containing type A and type B granules, wherein: The type A particles use biochar with a particle size of 0.25mm-2.0mm as a carrier to support microbial complexes, and are fast-acting particles; The type B particles use biochar with a particle size of 0.25 mm to 2.0 mm as a carrier to form a core, on which the microbial complex is carried. The core is coated with a biodegradable polymer membrane with a thickness of 50 μm to 150 μm. A membrane thickness of less than 50 μm will cause the membrane to degrade prematurely, resulting in long-term dormant particles with a decreased survival rate of microorganisms. A thickness of more than 150 μm will hinder the effective penetration of the activator and affect the activation efficiency. The microbial complex comprises: at least one Bacillus bacteria that degrades Panax notoginseng saponins and antagonizes root rot pathogens; at least one yeast that synergizes with the Bacillus bacteria to promote plant health; at least one Morphozoa fungus that regulates soil microbial community structure and repairs soil microecology; and at least one Streptomyces actinomycete that produces broad-spectrum antibiotics.

[0009] Furthermore, the biodegradable polymer membrane of the type B particles is made of polycaprolactone or a blend of polycaprolactone and polylactic acid.

[0010] Furthermore, the weight ratio of type A granules to type B granules in the fertilizer is 20:80-40:60.

[0011] A method for preparing the microbial fertilizer of claim 1 includes the following steps: (1) The biochar prepared by pyrolysis is pulverized and screened to obtain a carrier with a particle size of 0.25 mm-2.0 mm, and then sterilized. (2) Each strain in the microbial complex was subjected to liquid fermentation culture and the bacterial cells were collected; (3) Mix the bacterial cells obtained in step (2) with the carrier obtained in step (1) so that the bacterial cells are adsorbed onto the carrier to obtain type A particles and type B particles semi-finished products. (4) Take a portion of the B-type particle semi-finished product from step (3), and use a fluidized bed coating process to coat it with polycaprolactone or a blend of polylactic acid as the coating material to obtain B-type particles. (5) Mix the type A particles from step (3) with the type B particles obtained in step (4) in a predetermined ratio to obtain the final product.

[0012] 5. A method for preventing and controlling root rot in Panax notoginseng, characterized in that the method uses the microbial fertilizer according to any one of claims 1 to 5, and includes the following steps: S1: Base application pre-treatment step: Before planting Panax notoginseng, the microbial fertilizer is evenly spread on the soil surface and deep-plowing is carried out to a depth of 40cm-50cm. S2: Targeted activation step: During the root expansion period or the high incidence period of diseases of Panax notoginseng, apply a liquid activator to the soil at a depth of 20cm-40cm through underground drip irrigation or pre-buried pipes to activate the type B particles; the liquid activator contains at least one readily available carbon source selected from molasses, glucose, and sodium humate, and at least one fast-acting nitrogen source selected from amino acids and yeast extract.

[0013] Furthermore, the method also includes a surface replenishment step of applying type A granules to the soil surface during the Panax notoginseng growing season.

[0014] Furthermore, the method also includes a post-harvest remediation step, which utilizes the microbial complex remaining in the soil to bioremediate the soil after the harvest of Panax notoginseng.

[0015] The beneficial effects of this invention are: 1. The microbial fertilizer of the present invention combines two differentiated particle structures, A and B, with a deep-plowing pre-installed application method to construct a microbial defense line in the entire soil profile from 0 to 50 cm. Type A particles provide immediate protection on the surface, while type B particles serve as strategic reserves in the deep layers. During critical periods of high disease incidence, the dormant type B particles in the deep layers can be activated as needed to release biological control functions at the most needed time and location, thereby improving the utilization efficiency and control effect of microbial agents and solving the problem that traditional fertilization methods cannot cover the deep root system. The biochar carrier of this invention, as a long-lasting soil conditioner, combined with the colonization of beneficial microbial communities, can continuously improve the physical and chemical properties of the soil, restore the unbalanced micro-ecosystem, and achieve cyclical maintenance to break the barriers of continuous cropping. At the same time, the microbial complex carried by particles A and B can degrade saponin autotoxic substances that are the cause of diseases, and can also directly antagonize the pathogen Fusarium oxysporum, acting on both the root cause and the symptoms at the same time, thus treating both the symptoms and the root cause. Detailed Implementation

[0016] Unless otherwise stated, all materials, reagents and strains used in this invention are commercially available or can be obtained from nationally recognized strain depositories.

[0017] The core of the microbial complex used in this invention lies in the functional localization and synergistic cooperation of each strain. The following are examples of preferred strains that can be used to implement this invention: Bacillus bacteria: Bacillus amyloliquefaciens is preferred for degrading Panax notoginseng saponins and antagonizing root rot pathogens. Beijing Bio-Biotech Co., Ltd., Bio-18640. Yeast: works synergistically with the Bacillus bacteria to promote plant health, preferably Rhodotorula sphaerica, purchased from BNCC® Industrial Microbial Strains Engineering Technology Research Center, with the serial number BNCC119738; Mortierella fungi: Regulate soil microbial community structure and restore soil microecology. Mortierella alpina is preferred. Purchased from Beijing Bio-Bio Biotechnology Co., Ltd., product number: bio-68001. Streptomyces actinomycetes: produce broad-spectrum antibiotics, preferably Streptomyces griseus, purchased from BNCC® Industrial Microbial Strains Engineering Technology Research Center, with the number BNCC335914.

[0018] Example 1: Preparation of Microbial Fertilizer Preparation of biochar carrier: Disease-free corn stalks were selected as raw material, pulverized by a pulverizer, and placed in an anaerobic pyrolysis reactor for slow pyrolysis at 500°C for 2.5 hours to obtain biochar. The biochar was cooled, ground, and sieved through 2.0 mm and 0.25 mm sieves to collect particles with a diameter between 0.25 and 2.0 mm. After washing with deionized water, the pH of the suspension was adjusted to 7.0, and then sterilized by moist heat at 121°C and 0.1 MPa for 40 minutes, and cooled for later use. Culture and formulation of microbial complexes: (1) The preserved strains of Bacillus amyloliquefaciens, Rhodotorula spheroidae, Morphyra alpinea and Streptomyces griseus were inoculated into their respective suitable liquid culture media (e.g., LB medium for bacteria, PDA medium for fungi and yeasts) and cultured under suitable conditions (e.g., 28-30°C, 160-180 rpm) with shaking for 48-72 hours to obtain their respective fermentation broths; (2) Collect the bacterial cells by high-speed centrifugation (8000 rpm, 10 min) of the fermentation broth of the above four strains, resuspend them in sterile water, and mix them in a live cell ratio of 1:1:1:1 to prepare a concentrated bacterial suspension for later use.

[0019] Preparation of Type A granules: The sterilized biochar carrier was placed in a sterile rotary mixer. The concentrated bacterial suspension was evenly sprayed onto the biochar at a feed-to-liquid ratio of 10:3 (w / v), ensuring full adsorption and guaranteeing an effective viable count of no less than 2.0 × 10⁻⁶. 8 CFU / g, then, 2% sodium alginate solution was sprayed in at a ratio of 100:2 (w / w) of biochar to sodium alginate solution, granulated in a drum granulator at 30 rpm for 5 minutes, and dried at 38°C under ventilation conditions until the moisture content was less than 8%, thus obtaining type A granules.

[0020] Preparation of type B particles: Take another portion of sterilized biochar carrier and adsorb it onto the concentrated bacterial suspension using the method described above, ensuring that the effective viable count is not less than 5.0 × 10⁻⁶. 8 Using CFU / g as the core, the core particles are fed into a fluidized bed coating machine. A 10% polycaprolactone ethyl acetate solution is used as the coating liquid, and coating is performed via bottom spraying. The coating process parameters are controlled as follows: inlet air temperature 70-80°C, material temperature 40-45°C, spray rate 10-15 mL / min, and atomization pressure 0.15 MPa, to form a uniform and dense film on the particle surface, with an average film thickness of 100 µm. After coating, the particles are dried at 40°C to remove residual solvent, yielding type B particles.

[0021] Final product mixing: The prepared type A granules and type B granules are thoroughly mixed in a large sterile mixer at a weight ratio of 30:70, and then vacuum sealed and packaged to obtain the finished microbial fertilizer product of the present invention.

[0022] The application method is as follows: (1) Base application: Before planting, the fertilizer product of this invention is evenly spread on the surface of the plot at a rate of 4000 kg / hm², and then a large rotary tiller is used to carry out deep plowing to a depth of 45 cm to ensure that the fertilizer particles are evenly distributed in the 0-50 cm soil layer. (2) Targeted activation: On the 90th day after planting Panax notoginseng (the period of rapid root expansion), liquid activator is injected into the soil at a depth of 20-40 cm through the microporous pipes pre-buried in the plot. The activator formula is: 1% molasses, 0.5% yeast extract powder and 98.5% water. (3) Layer supplementation: During the rainy season (around 150 days after planting), apply type A granules at a rate of 500 kg / hm² to the soil surface.

[0023] Example 2 differs from Example 1 in that the weight ratio of type A granules to type B granules in the fertilizer is 20:80; Example 3 differs from Example 1 in that the weight ratio of type A granules to type B granules in the fertilizer is 40:60; Comparative Example 1: Commercially available green Trichoderma microbial fertilizer with an effective live bacteria count ≥200 million / g was applied by conventional trench application: when transplanting Panax notoginseng, the microbial agent was applied in the planting trench at a rate of 2500 kg / hm², and then covered with soil.

[0024] Comparative Example 2: The difference from Example 1 is that the bio-fertilizer was applied according to the method of Comparative Example 1; Comparative Example 3: The difference from Example 1 is that targeted activation was not performed; Comparative Example 4: The difference from Example 1 is that the microbial complex contains only Bacillus amyloliquefaciens; Comparative Example 5: The difference from Example 1 is that the microbial complex contains only spherical red yeast; Comparative Example 6: The difference from Example 1 is that the microbial complex contains only *Morchella alpineensis*; Comparative Example 7: The difference from Example 1 is that the microbial complex contains only Streptomyces griseus; Comparative Example 8: The difference from Example 1 is that the microbial complex contains only Bacillus amyloliquefaciens and Streptomyces griseus; Comparative Example 9: The difference from Example 1 is that the microbial complex does not contain spherical red yeast; In Comparative Examples 4-9, the total number of microorganisms, the A / B particle ratio, preparation and application methods of the microbial fertilizer were consistent with those in Example 1, with only the composition of the microbial complex differing. Experimental Example A year-long field comparison trial was conducted at a Panax notoginseng planting base in Wenshan, Yunnan: 1. Experimental Design: The soil fertility of the experimental site was moderate and relatively uniform. A randomized block design was used, with 12 treatments: Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3, plus a blank control (no microbial fertilizer applied). Each treatment was replicated three times (four blocks), resulting in a total of 36 experimental plots. Each plot was 4m × 5m in size, with 50cm buffer strips and physical partitions between plots to prevent cross-contamination of fertilizers and water. Except for the different fertilizer treatments, field management practices (such as irrigation, weeding, and routine maintenance) were kept consistent across all plots to ensure the accuracy and comparability of the experimental results. 2. Detection indicators and methods: (1) Investigation of root rot in Panax notoginseng: During the Panax notoginseng harvest season, 50 Panax notoginseng plants were randomly selected from the center of each plot. After careful digging, the roots were washed clean, and the disease status of each plant was checked. The number of diseased plants at each level was recorded according to the disease grading standard, and the incidence rate and control effect were calculated. The calculation formula is as follows: Incidence rate (%) = (Number of infected plants / Total number of plants surveyed) × 100 Prevention and control effect (%) = [(Incidence rate in blank control group - Incidence rate in treatment group) / Incidence rate in blank control group] × 100; (2) Determination of soil microbial abundance: After the harvest of Panax notoginseng, soil samples were collected from each plot using an aseptic soil auger following an "S"-shaped multi-point mixing method. To verify the effect of this invention on soil profile, soil samples were collected from the 0-20cm (top layer) and 20-40cm (deep layer) depths, respectively. After thorough mixing of the repeatedly collected soil samples, approximately 500g of each sample was placed in a sterile sampling bag using the quartering method and stored at low temperature for later use. The dilution plate count method was used to determine the abundance of target microorganisms in the soil. The procedure was performed in accordance with the method in the People's Republic of China National Standard GB20287-2006 "Agricultural Microbial Agents".

[0025] Fusarium oxysporum count: cultured on modified Komada selective medium at 28°C for 5-7 days.

[0026] Streptomyces count: Use Gao's No. 1 selective medium and incubate at 28°C for 5-7 days.

[0027] After incubation, plates with colony counts between 30 and 300 were selected for counting, with three replicates for each dilution. Results are expressed as colony forming units (CFU) and converted to colony count per gram of dry soil (CFU / g dry soil). 4. Experimental Results and Analysis: Table 1: Effects of different treatments on the occurrence and control efficacy of root rot in Panax notoginseng As can be seen from Table 1, the control effect of Example 1 (81.2%) was significantly better than all other treatment groups, and the control effect was much higher than that of the single-strain treatment groups (Comparative Examples 4-9). The single-strain treatment with the best effect, Bacillus amyloliquefaciens, had a control effect of 63.9%.

[0028] Table 2: Effects of different treatments on soil microbial abundance at harvest. As can be seen from Table 2, under the treatment of Example 1, the number of pathogenic Fusarium was suppressed to an extremely low level in the entire soil profile from 0 to 40 cm, while the number of beneficial Streptomyces was greatly increased, so that the S / F ratio reached a healthy level of nearly 700.

[0029] Therefore, combining Tables 1 and 2, this invention achieves microecological restoration and long-term disease control of the entire rhizosphere soil profile of Panax notoginseng through the combination of slow and controlled release of particles A and B, the three-dimensional distribution of deep tillage, and targeted activation during critical periods, thus having the beneficial effect of treating both the symptoms and the root cause.

[0030] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A microbial fertilizer for controlling Panax ginseng root rot, characterized by, The microbial fertilizer is a granular mixture comprising A-type granules and B-type granules, wherein: The A-type granules are biochar granules with a particle size of 0.25-2.0 mm and carry a microbial complex, and are quick-acting granules; The B-type granules are biochar granules with a particle size of 0.25-2.0 mm, and the core of the granules carries the microbial complex, and the core is coated with a biodegradable polymer film with a thickness of 50-150 μm, and are long-acting dormant granules; The microbial complex comprises Bacillus bacteria, yeast, Mortierella fungi, and Streptomyces actinomycetes.

2. The microbial fertilizer according to claim 1, characterized in that, The biodegradable polymer film of the B-type granules is made of polycaprolactone or a blend of polycaprolactone and polylactic acid.

3. The microbial fertilizer according to claim 1, characterized in that, The weight ratio of the A-type granules to the B-type granules in the fertilizer is 20:80-40:

60.

4. A method of preparing the microbial fertilizer of claim 1, characterized by, The method comprises the following steps: The pyrolysis-prepared biochar is crushed and sieved to obtain carriers with a particle size of 0.25-2.0 mm, and the carriers are sterilized; The strains in the microbial complex are separately subjected to liquid fermentation culture, and the bacterial bodies are collected; The bacterial bodies obtained in step (2) are mixed with the carriers obtained in step (1) so that the bacterial bodies are adsorbed on the carriers, and A-type granules and B-type granule semi-products are prepared; A portion of the B-type granule semi-products in step (3) is coated with polycaprolactone or a blend of polycaprolactone and polylactic acid as a coating material by using a fluidized bed coating process, and B-type granules are prepared; The A-type granules in step (3) and the B-type granules prepared in step (4) are mixed in a predetermined ratio, and the microbial fertilizer is obtained.

5. A method for controlling root rot of Panax notoginseng, characterized by, The method uses the microbial fertilizer of any one of claims 1-3, and comprises the following steps: S1: base application prepositioning step: before planting the Panax notoginseng, the microbial fertilizer is uniformly applied to the soil surface, and deep plowing with a depth of 40-50 cm is performed; S2: targeted activation step: during the root expansion period or the disease outbreak period of the Panax notoginseng, a liquid activator is applied to the deep soil with a depth of 20-40 cm by using underground drip irrigation or pre-embedded pipes to activate the B-type granules; the liquid activator comprises at least one readily available carbon source selected from molasses, glucose, and sodium humate, and at least one readily available nitrogen source selected from amino acids and yeast extract.

6. The method of claim 5, wherein, The method further comprises: surface layer supplementing step: during the growth season of the Panax notoginseng, the A-type granules are supplemented and applied to the surface layer of the soil after rainfall or irrigation.

7. The method of claim 5, wherein, The method further comprises: post-harvest repair step: after the Panax notoginseng is harvested, the biochar carriers and the microbial complex remaining in the soil are used to bioremediate the soil, continuously degrade the residual saponins, and inhibit pathogenic bacteria.

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