Composition for preventing and controlling clubroot of rape, its preparation method and application

Abstract

The present application relates to the field of agricultural biotechnology and plant disease control technology, and specifically discloses a composition for preventing and controlling rape root rot, a preparation method and application thereof. The composition is prepared by fermentation of the following components in parts by weight: tea seed cake 10-30 parts, oyster shell powder 20-30 parts, waste molasses 10-20 parts, brewer's yeast powder 1-5 parts, xylo-oligosaccharide industrial residue 1-3 parts, and a composite fermentation agent containing lactobacillus plantarum, bacillus coagulans and propionic acid-producing propionibacterium 1-3 parts. The fermentation process adopts a two-stage pH control timing inoculation process: in the first stage, brewer's yeast is inoculated; in the second stage, the composite fermentation agent is inoculated under acidic conditions, and through synergistic metabolism, the sugar source is efficiently converted into a short-chain fatty acid composite system mainly composed of lactic acid and propionic acid. The prevention and control effect on rape root rot is significantly improved, and the composition also has the functions of improving soil and promoting crop growth, and has the advantages of high efficiency, environmental protection and multi-effect combination.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and plant disease control technology, and to a biological agent prepared using microbial fermentation technology, particularly a composition for the prevention and control of clubroot disease in rapeseed, its specific preparation method, and its application in agricultural production. Background Technology

[0002] Rapeseed is an important oilseed and cash crop in my country, ranking among the world's top in terms of planting area and total yield. However, clubroot disease is a devastating soil-borne disease that seriously threatens the healthy development of the rapeseed industry. This disease is caused by an obligate parasite, *Plasmodiophora brassicae*. After infecting the rapeseed roots, the pathogen stimulates abnormal cell division and swelling, forming tumor-like clubroots of varying sizes. This damages the normal structure and function of the root system, hindering the plant's absorption of water and nutrients. As a result, the above-ground parts of the plant exhibit slow growth, stunted growth, yellowing and wilting of leaves, ultimately leading to a significant reduction in yield or even crop failure.

[0003] Currently, the main methods for controlling clubroot disease in rapeseed include breeding highly resistant varieties, chemical control, agricultural control, biological control, and soil improvement.

[0004] The breeding of disease-resistant varieties is the foundation of clubroot disease control. There are extremely significant differences in the incidence of clubroot disease among different cruciferous plant varieties. At the same time, different varieties of rapeseed also have significant differences in their resistance to different physiological races of clubroot fungus. Different varieties should be used to reduce the incidence of disease in plants with different physiological races of clubroot pathogens. Rapeseed varieties that are artificially bred are much more resistant than natural varieties. In different disease-affected areas, locally resistant crops should be selected as the main varieties for cultivation in the local clubroot-affected areas.

[0005] Chemical control mainly relies on the application of fungicides, such as fluazinam and cyazofamid. Although chemical agents can play a certain role in inhibiting pathogens in the short term, their drawbacks are becoming increasingly apparent. First, long-term use of chemical agents alone can easily induce pathogens to develop drug resistance, leading to a year-by-year decline in control effectiveness. Second, chemical pesticide residues may pose a potential threat to the soil environment, water bodies, and agricultural product safety, disrupting the soil microecological balance and killing beneficial microorganisms.

[0006] Agricultural control measures include selecting disease-resistant varieties, implementing crop rotation, and adjusting soil pH. Breeding and promoting disease-resistant varieties is an economical and effective approach, but the physiological races of pathogens differentiate rapidly, and a single resistance gene can easily be overcome by a new dominant race, leading to the loss of varietal resistance. Although crop rotation can reduce the number of dormant spores in the soil, dormant spores of clubroot can survive in the soil for several years or even more than ten years. For severely diseased fields, a very long rotation period is required to see results. Applying alkaline substances such as lime to increase the soil pH can inhibit the germination and infection of dormant spores, but excessive application of lime may lead to problems such as soil compaction and reduced nutrient availability.

[0007] Biological control, as an environmentally friendly strategy, has received widespread attention in recent years. It mainly utilizes antagonistic microorganisms (such as Trichoderma and Bacillus) or plant-derived active substances (such as plant extracts) to inhibit pathogens. However, existing biological control products also have certain limitations. For example, single antagonistic microbial agents often have unstable colonization and antagonistic effects in complex and variable field soil environments, and are easily affected by soil physicochemical properties, temperature, humidity, and competition from native microbial communities. On the other hand, single plant extracts have relatively singular targets, making it difficult to achieve comprehensive inhibition of multiple stages of pathogen growth, reproduction, and infection, and they usually do not have the comprehensive function of improving soil microecology and enhancing soil fertility.

[0008] Soil provides the substrate with the water and nutrients necessary for plant growth and development. The main condition affecting soil-borne diseases is the soil micro-ecological environment. In addition to soil nutrients, pH, and water content, the soil micro-ecological environment also includes soil microbiota. There are significant differences in the soil micro-ecology between rapeseed clubroot outbreaks and healthy production plots. The regulation of soil micro-ecology to reduce or control the occurrence of clubroot disease is a current research hotspot.

[0009] In summary, besides breeding resistant varieties, the control of clubroot disease in rapeseed should adopt a prevention-oriented, integrated approach. Existing methods or materials for controlling clubroot disease in rapeseed often have limited functions, failing to simultaneously address multiple needs such as efficient antibacterial activity, soil microecological regulation, and crop nutrient supplementation. They also lack synergistic effects among their functional components, resulting in unsatisfactory and unstable integrated control effects. Therefore, there is an urgent need to develop a novel, multifunctional, synergistic, and environmentally friendly biological agent and its preparation technology that targets and regulates the soil microecology to effectively solve the challenge of controlling clubroot disease in rapeseed.

[0010] Recent studies have also shown that functional xylooligosaccharides and their industrial byproducts can serve as highly efficient prebiotics, selectively promoting the proliferation of beneficial microorganisms such as lactic acid bacteria and Bacillus in the soil, optimizing the rhizosphere microbial community structure, and enhancing crop system resistance. This also provides new ideas for developing compound microbial preparations containing prebiotics. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition, its preparation method and application that can synergistically enhance and effectively control clubroot disease in rapeseed.

[0012] The first technical solution of the present invention is made by fermentation of the following components in parts by weight: 10-30 parts tea seed cake, 20-30 parts oyster shell powder, 10-20 parts waste molasses, 1-5 parts brewing yeast powder, 1-3 parts xylooligosaccharide industrial residue, and 1-3 parts a compound fermentation agent containing *Lactobacillus plantarum*, *Bacillus coagulans*, and *Propionibacterium propionate*. The parts by weight are based on a total raw material mass of 100 parts. The waste molasses has a total sugar content of not less than 40% on a dry basis.

[0013] Preferably, the product is made by fermentation from the following components in parts by weight: 15-25 parts tea seed cake, 22-28 parts oyster shell powder, 12-18 parts waste molasses, 2-4 parts brewing yeast powder, 1.5-2.5 parts xylooligosaccharide industrial residues, and 1.5-2.5 parts a compound fermentation agent containing Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate. The parts by weight are based on a total raw material mass of 100 parts.

[0014] Preferably, the product is made by fermentation from the following components in parts by weight: 20 parts tea seed cake, 25 parts oyster shell powder, 15 parts waste molasses, 3 parts brewing yeast powder, 2 parts xylooligosaccharide industrial residue, and 2 parts of a compound fermentation agent containing Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionitum. The parts by weight are based on a total raw material mass of 100 parts.

[0015] Preferably, in the compound fermentation agent, the ratio of viable bacteria of *Lactobacillus plantarum* strain, *Bacillus coagulans* strain, and *Propionibacterium propionate* strain is configured as 1–5:1–3:1. More preferably, the ratio of viable bacteria of *Lactobacillus plantarum* strain, *Bacillus coagulans* strain, and *Propionibacterium propionate* strain is configured as 2–4:1.5–2.5:1. Even more preferably, the ratio of viable bacteria of *Lactobacillus plantarum* strain, *Bacillus coagulans* strain, and *Propionibacterium propionate* strain is configured as 3:2:1.

[0016] Preferably, the *Lactobacillus plantarum* is *Lactobacillus plantarum* with accession number CCTCC No. M2021135, the *Bacillus coagulans* is *Bacillus coagulans* with accession number CCTCC No. M2021497, and the *Propionibacterium propionatum* is *Propionibacterium propionatum* with accession number CCTCC No. M2021136. This compound formulation aims to efficiently convert the sugar source in waste molasses into lactic acid through synergistic metabolism between microorganisms, and further convert lactic acid as a precursor into propionic acid, thereby forming a short-chain fatty acid complex system with broad-spectrum inhibitory activity against pathogens.

[0017] Preferably, after fermentation, the key functional indicators of the composition are defined as follows: the total short-chain fatty acid concentration in the fermentation broth reaches 150–250 mmol / L, of which the propionic acid concentration is not less than 50 mmol / L; the concentration of tea saponin reaches 3–5 g / L; and the total number of beneficial live bacteria is not less than 1.0 × 10⁻⁶. 8 The total number of beneficial live bacteria was CFU / mL, of which Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate accounted for more than 80%.

[0018] The second technical solution of the present invention: a method for preparing a combined extract for controlling clubroot disease in rapeseed, comprising the following steps:

[0019] a. Prepare raw materials according to the following weight parts: 10-30 parts of tea seed cake, 20-30 parts of oyster shell powder, 10-20 parts of waste molasses, 1-5 parts of brewing yeast powder, 1-3 parts of xylooligosaccharide industrial residue, and 1-3 parts of compound fermentation agent containing Lactobacillus plantarum, Bacillus coagulans and Propionibacterium propionate.

[0020] b. A two-stage pH-controlled sequential inoculation fermentation process is used to ferment the mixture of raw materials and water described in step a to obtain the combined extract.

[0021] Preferably, the two-stage pH-controlled sequential inoculation fermentation process specifically includes:

[0022] In the first fermentation stage, brewer's yeast is first inoculated into the mixture of raw materials and water for anaerobic fermentation. The yeast metabolizes sugars to produce ethanol and carbon dioxide, and at the same time, it initially decomposes macromolecular organic matter to provide nutrient precursors for subsequent bacterial growth.

[0023] In the second fermentation stage, after the pH of the system naturally drops to an acidic environment due to the first stage of fermentation, a compound fermentation agent composed of Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate is inoculated. This acidic environment is used to inhibit the growth of other bacteria and promote the efficient conversion of organic matter into short-chain fatty acids, mainly lactic acid and propionic acid, by lactic acid bacteria and propionic acid bacteria.

[0024] Preferably, the control parameters of the two-stage fermentation process are precisely defined as follows: the raw materials and water are mixed at a material-to-liquid ratio of 1:3 to 1:5, and the initial pH is adjusted to 6.5 to 7.0; in the first fermentation stage, after inoculation with brewer's yeast, fermentation is carried out at 28 to 32°C for 48 to 72 hours until the pH of the system drops to 4.0 to 4.5; then the second fermentation stage begins, inoculated with the compound fermentation agent, and fermentation continues at 28 to 32°C for 5 to 10 days, during which anaerobic conditions are maintained until the pH of the system stabilizes at 3.8 to 4.2 and the content of the main short-chain fatty acids reaches its peak. More preferably, the control parameters of the two-stage fermentation process are precisely defined as follows: the raw materials and water are mixed at a material-to-liquid ratio of 1:4, and the initial pH is adjusted to 6.7-6.8; in the first fermentation stage, after inoculation with brewer's yeast, fermentation is carried out at 29-31°C for 54-66 hours until the pH of the system drops to 4.2-4.3; then the second fermentation stage begins, inoculated with the compound fermentation agent, and fermentation continues at 29-31°C for 7-8 days, during which anaerobic conditions are maintained until the pH of the system stabilizes at 3.9-4.1 and the content of the main short-chain fatty acids reaches its peak.

[0025] Preferably, the temperature during the fermentation process is maintained at 25–35°C throughout, the total fermentation period is 7–14 days, and the fermentation endpoint is defined as the system pH stabilizing at 3.8–4.2 and the short-chain fatty acid concentration, monitored by gas chromatography, showing a change rate of less than 5% over 24 consecutive hours. More preferably, the temperature during the fermentation process is maintained at 28–32°C throughout, the total fermentation period is 9–12 days, and the fermentation endpoint is defined as the system pH stabilizing at 3.9–4.1 and the short-chain fatty acid content showing a change rate of less than 5% over 24 consecutive hours.

[0026] The third technical solution of the present invention provides an application, which includes diluting the combined extract obtained by the aforementioned preparation method by 500 to 650 times before use, and applying it to rapeseed by seed dressing, root irrigation or root dipping.

[0027] The present invention has the following beneficial effects:

[0028] (1) Multiple mechanisms, synergistic effect: By combining various functional raw materials such as tea seed cake, oyster shell powder, and waste molasses with specific compound microbial agents, a multifunctional prevention and control system is constructed; the tea saponins and alkaloids in the fermentation products can directly inhibit clubroot pathogens; oyster shell powder regulates soil pH and supplements calcium; while the broad-spectrum short-chain fatty acids produced by the compound microbial agents, especially propionic acid, have a strong chemical inhibitory effect on pathogens; the mechanisms of action of these components are complementary and synergistic, which significantly improves the comprehensive prevention and control effect;

[0029] (2) Technological innovation and stable quality: The unique two-stage pH-controlled sequential inoculation fermentation process cleverly utilizes the different microorganisms' preferences for environmental pH; firstly, yeast fermentation creates an acidic environment suitable for the growth of lactic acid bacteria and propionic acid bacteria, while initially degrading the raw materials. This process solves the contradiction between the extraction of active substances and the efficient proliferation of beneficial bacteria, which increases the extraction rate of active substances such as tea saponins by 20-30% and increases the number of viable beneficial microorganisms by 1-2 orders of magnitude, ensuring the stability and efficiency of product quality between batches;

[0030] (3) Regulate ecology and improve soil: The prepared extract can not only inhibit harmful pathogens, but also contains a high density of beneficial microorganisms, such as Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate. After being applied to the soil, these microbial agents can quickly colonize the rhizosphere of rapeseed and form a dominant population. This type of compound microbial agent can drive the soil microbial community to evolve towards a healthy direction, significantly increase the relative abundance of beneficial bacteria in the rhizosphere, inhibit the ecological niche of pathogens, and further inhibit the growth of clubroot pathogens through nutrient competition and spatial occupation effect, thus reshaping a healthy soil microecological balance.

[0031] (4) Rich in nutrients and promotes growth: The fermentation process decomposes the large organic molecules in the raw materials into small molecule nutrients that are more easily absorbed by plants, such as amino acids, polypeptides, vitamins and minerals. Therefore, the combined extract is also a high-quality biological organic fertilizer. A healthy soil microecology can effectively enhance the activity of soil enzymes such as catalase and urease, promote the circulation and release of soil nutrients, thereby enhancing soil fertility, promoting the robust growth of rapeseed plants, and improving their own disease resistance. Experiments have shown that the efficacy of using the product of this invention is at least 25% higher than that of traditional methods. Attached Figure Description

[0032] Figure 1 This is a flowchart of a combined extract preparation method provided in an embodiment of the present invention.

[0033] Figure 2 This is a detailed flowchart of the two-stage pH-controlled sequential inoculation fermentation process in an embodiment of the present invention.

[0034] Figure 3 This is a schematic diagram illustrating the mechanism of synergistic effect of the components in the composition provided in the embodiments of the present invention.

[0035] Figure 4 This is a schematic diagram illustrating the application of the combined extract provided in this embodiment of the invention in rapeseed cultivation.

[0036] Figure 5 This is a graph showing the comparative experimental results of the combined extract of the present invention and different control groups on the prevention of clubroot disease in rapeseed;

[0037] Figure 6This is a diagram of the physical and chemical properties of sample soils from different plots of rapeseed at different growth stages in Experiment Example 2 of this invention. In the diagram, A is soil moisture, B is soil pH, C is soil temperature, D is soil electrical conductivity, E is soil phosphorus (P) content, and F is soil potassium (K) content.

[0038] Figure 7 This is a diagram showing the difference in bacterial composition between the soil in the comparative example group and the soil in the example group in Experiment 2 of this invention;

[0039] Figure 8 This is a diagram showing the differences in fungal composition between the soil in the comparative example group and the soil in the example group in Experiment 2 of this invention. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0041] The composition for controlling clubroot disease in rapeseed is prepared by fermentation from the following components in parts by weight: 10-30 parts tea seed cake, 20-30 parts oyster shell powder, 10-20 parts waste molasses, 1-5 parts brewer's yeast powder, 1-3 parts xylooligosaccharide industrial residue, and 1-3 parts a compound fermentation agent containing *Lactobacillus plantarum*, *Bacillus coagulans*, and *Propionibacterium propionitum*. In the compound fermentation agent, the ratio of viable *Lactobacillus plantarum* strain, *Bacillus coagulans* strain, and *Propionibacterium propionitum* strain is 1-5:1-3:1; the *Lactobacillus plantarum* is the strain with accession number CCTCC No. M2021135, the *Bacillus coagulans* is the strain with accession number CCTCC No. M2021497, and the *Propionibacterium propionitum* is the strain with accession number CCTCC No. M2021136. After fermentation, the total short-chain fatty acid concentration in the fermentation broth reaches 150–250 mmol / L, with propionic acid concentration not less than 50 mmol / L; the concentration of tea saponins is 3–5 g / L; and the total number of beneficial viable bacteria is not less than 1.0 × 10⁻⁶. 8 The total number of beneficial live bacteria was CFU / mL, of which Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate accounted for more than 80%.

[0042] The preparation method of the combined extract for controlling clubroot disease in rapeseed includes the following steps:

[0043] a. Prepare raw materials according to the following weight parts: 10-30 parts of tea seed cake, 20-30 parts of oyster shell powder, 10-20 parts of waste molasses, 1-5 parts of brewing yeast powder, 1-3 parts of xylooligosaccharide industrial residue, and 1-3 parts of compound fermentation agent containing Lactobacillus plantarum, Bacillus coagulans and Propionibacterium propionate.

[0044] b. A two-stage pH-controlled sequential inoculation fermentation process is used to ferment the mixture of raw materials and water in step a to obtain a combined extract.

[0045] The two-stage pH-controlled sequential inoculation fermentation process specifically includes,

[0046] In the first fermentation stage, brewer's yeast is first inoculated into the mixture of raw materials and water for anaerobic fermentation. The yeast metabolizes sugars to produce ethanol and carbon dioxide, and at the same time, it initially decomposes macromolecular organic matter to provide nutrient precursors for subsequent bacterial growth.

[0047] In the second fermentation stage, after the first stage of fermentation has naturally lowered the pH of the system to an acidic environment, a compound fermentation agent composed of *Lactobacillus plantarum*, *Bacillus coagulans*, and *Propionibacterium propionate* is inoculated. This acidic environment inhibits the growth of other microorganisms and promotes the efficient conversion of organic matter into short-chain fatty acids, primarily lactic and propionic acids, by lactic and propionic bacteria. The control parameters for the two-stage fermentation process are precisely defined as follows: the raw materials and water are mixed at a ratio of 1:3 to 1:5, and the initial pH is adjusted to 6.5–7.0; in the first fermentation stage, after inoculation with *Saccharomyces cerevisiae*, fermentation is carried out at 28–32°C for 48–72 hours until the pH of the system drops to 4.0–4.5; then, the second fermentation stage begins, with inoculation of the compound fermentation agent and continued fermentation at 28–32°C for 5–10 days, maintaining anaerobic conditions until the pH of the system stabilizes at 3.8–4.2 and the content of the main short-chain fatty acids reaches its peak. The temperature is maintained at 25–35°C throughout the fermentation process. The total fermentation cycle is 7 to 14 days. The fermentation endpoint is defined as the system pH stabilizing at 3.8 to 4.2 and the short-chain fatty acid content changing by less than 5% over 24 consecutive hours.

[0048] A method for preparing a solid-state sustained-release composition for controlling clubroot disease in rapeseed includes the following steps:

[0049] a. Prepare tea seed cake, oyster shell powder, waste molasses, brewer's yeast powder, xylooligosaccharide industrial residue and compound fermentation agent according to the component ratio of claim 1, and add crop straw powder passing through a 35-45 mesh sieve as a solid fermentation substrate, which is equivalent to 50-100% of the weight of tea seed cake.

[0050] b. Mix the raw materials with water, adjust the initial moisture content of the mixture to 50-60% and the initial pH value to 6.5-7.0, and then use a two-stage pH-controlled sequential inoculation fermentation process to ferment the solid mixture.

[0051] The specific control parameters for the two-stage pH-controlled sequential inoculation fermentation process are as follows:

[0052] In the first fermentation stage, after inoculating with brewing yeast, the pile is fermented for 24-48 hours, during which the temperature at the center of the pile is strictly controlled not to exceed 35°C. The pH value of the pile is naturally reduced to below 4.5 by the acid produced by yeast metabolism, so as to initially soften the cellulose structure. In the second fermentation stage, the pile is spread out and evenly sprayed with compound fermentation agent, then turned over and compacted and sealed. Anaerobic fermentation is carried out at 25-35°C for 7-14 days until the pH value of the system stabilizes at 3.8-4.2 and a distinct sour aroma is produced.

[0053] The method also includes a post-processing granulation step designed to maintain the activity and shape of the strain.

[0054] The fermented solid material is dried in a low-temperature airflow below 45℃ to reduce its moisture content to 15-20% and induce a dormant state. Then, 3-5% of a binder, selected from pregelatinized starch, sodium alginate, or bentonite, is added to the dried material. The mixture is then granulated into cylindrical solid slow-release granules with a particle size of 2-5 mm using a flat die extrusion granulator. The granules have a porous microstructure composed of straw and tea seed cake residue. This structure adsorbs high concentrations of short-chain fatty acids and tea saponins. After application to the soil, the active ingredients are slowly released as soil moisture penetrates. The effective antibacterial concentration in the rhizosphere soil is maintained for 30-45 days, significantly longer than that of liquid formulations.

[0055] The application of compositions or solid slow-release granules in the control of clubroot disease in rapeseed includes differentiated application methods based on the product formulation: when the product is a liquid extract, it should be diluted 500-650 times before use for seed dressing, root drenching, or root dipping; when the product is a solid slow-release granule, it should be applied as a base dressing, hole application, or trench application at a rate of 20-40 kg per acre, in the soil layer 5-10 cm away from the rapeseed roots, thereby constructing a dual protective barrier of microecology and chemicals in the rhizosphere. When using liquid extracts for root drenching, the specific timing is as follows: the first drenching should be carried out immediately after rapeseed sowing and covering with soil, followed by second drenching on the 5th, 10th, and 15th days after rapeseed emergence. Through high-frequency early intervention, a dominant beneficial microbial community is established during the most sensitive window period of rapeseed root development, blocking the initial infection of dormant spores of the pathogen.

[0056] Example 1: This example provides a composition for controlling clubroot disease in rapeseed. The composition is a liquid biological agent prepared by a specific microbial fermentation process. It is made by fermentation of the following components in parts by weight: 10-30 parts tea seed cake, 20-30 parts oyster shell powder, 10-20 parts waste molasses, 1-5 parts brewing yeast powder, 1-3 parts xylooligosaccharide industrial residue, and 1-3 parts a special compound fermentation agent.

[0057] The core of this composition design lies in the scientific compatibility and synergistic effect of its components, aiming to generate a complex system containing a variety of bioactive substances and a high density of beneficial microorganisms through a single fermentation process.

[0058] Specifically, the functions of each raw material component and the basis for their selection are as follows:

[0059] Tea seed cake, with a weight percentage of 10-30 parts, preferably 15-25 parts, is the residue after pressing and extracting oil from the seeds of Camellia plants. It is rich in a key bioactive substance—tea saponin. Tea saponin is a pentacyclic triterpenoid saponin with good surface activity. Its molecular structure allows it to bind to sterols on the cell membranes of pathogenic microorganisms, disrupting cell membrane integrity and causing leakage of cell contents, thereby directly inhibiting or killing pathogens. It has an inhibitory effect on both dormant and zoospores of Plasmodiophora abrassicae, the pathogen of clubroot disease. In addition, tea seed cake also contains... Tannins, alkaloids, and other secondary metabolites also possess certain antibacterial activities and can complement tea saponins. In this composition, a ratio of 10 to 30 parts is selected to ensure that a sufficient concentration of tea saponins can be extracted from the fermentation broth to exert a disease-inhibiting effect. If the ratio is too low, the concentration of active substances will be insufficient; if the ratio is too high, the high concentration of tea saponins may inhibit some microorganisms in the early stage of fermentation and increase the cost of raw materials. The preferred range of 15 to 25 parts is an optimized result that comprehensively considers antibacterial effect, fermentation compatibility, and economy.

[0060] Oyster shell powder, in the form of 20-30 parts by weight, preferably 22-28 parts, is used. The main component of oyster shell powder is calcium carbonate, which plays multiple roles in this composition. Firstly, as a soil conditioner, after being applied to the soil, the oyster shell powder slowly decomposes and releases calcium carbonate. 2+ and CO3 2- This process gradually increases the soil pH. Clubroot pathogens thrive in acidic environments; raising the soil pH to neutral or slightly alkaline effectively inhibits the germination and infection of their dormant spores. Secondly, calcium is a crucial component of plant cell walls; supplementing with calcium strengthens the cell walls of rapeseed roots, enhancing the plant's physical resistance to pathogen infection. Finally, during fermentation, oyster shell powder, as a slow-release alkaline substance, acts as a pH buffer, neutralizing excess organic acids produced in the early stages of fermentation and preventing a rapid drop in pH, thus maintaining a relatively stable environment for microbial growth and metabolism. A ratio of 20-30 parts provides sufficient pH regulation and calcium, with an optimal range of 22-28 parts better balancing the dynamic pH changes during fermentation.

[0061] Waste molasses, comprising 10-20 parts by weight, contains a total sugar content of not less than 40% on a dry basis. Waste molasses is a byproduct of the sugar industry, containing a large amount of fermentable sugars such as sucrose, fructose, and glucose. It is an ideal and inexpensive carbon source for microbial fermentation, providing ample energy and material basis for the rapid growth and reproduction of *Saccharomyces cerevisiae*, *Lactobacillus plantarum*, and *Propionibacterium propionitum* in this composition. Besides sugars, waste molasses is also rich in various trace elements, vitamins, and amino acids. These substances not only promote the metabolism of microorganisms during fermentation but also ultimately enter the extract, becoming biological nutrients for rapeseed growth and enhancing soil fertility.

[0062] The brewing yeast powder accounts for 1 to 5 parts by weight. In the specific fermentation process of this invention, the brewing yeast plays a pioneering role. In the early stage of fermentation, it can quickly utilize the sugar in molasses to carry out anaerobic respiration, producing ethanol and a large amount of carbon dioxide. The production of carbon dioxide can quickly form an anaerobic environment in the fermentation system, effectively inhibiting the growth and reproduction of various aerobic bacteria. At the same time, the yeast's own metabolic activities can initially decompose some of the large molecular organic matter in the tea seed cake, releasing small molecular nutrients, providing more easily utilized nutrient precursors for the bacteria that are subsequently inoculated.

[0063] The xylooligosaccharide industrial residue comprises 1-3 parts by weight. This residue is a byproduct of xylooligosaccharide production, rich in xylooligosaccharides, xylooligosaccharides, and lignocellulose. In this invention, it acts as a highly efficient prebiotic, specifically promoting the colonization and proliferation of beneficial bacteria such as *Lactobacillus plantarum* and *Bacillus coagulans* in the fermentation system and soil rhizosphere. The lignocellulose it contains can serve as a physical carrier in solid-state fermentation, forming a porous structure that facilitates microbial attachment and slow release. This further enriches the organic matter and active sugar components in the fermentation products, enhancing soil fertility and microbial diversity.

[0064] The compound fermentation agent, with a weight ratio of 1 to 3 parts, is one of the core technical features of this invention. The agent is composed of three specific functional strains: Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium freudenreichii.

[0065] The preservation information of the *Lactobacillus plantarum* is as follows: it is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on March 15, 2021, with accession number CCTCCNo.M2021135.

[0066] The preservation information of the Bacillus coagulans is as follows: it is deposited at the China Center for Type Culture Collection (CCTCC), at Wuhan University, Wuhan, China, on May 14, 2021, with accession number CCTCCNO.M2021497.

[0067] The preservation information of the Propionibacterium propionitum is as follows: it is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 15, 2021, with accession number CCTCCNo.M2021136.

[0068] There is a precise synergistic metabolic relationship among these three strains. *Lactobacillus plantarum* is a highly efficient homofermenting lactic acid bacterium that can rapidly convert sugars into lactic acid, causing a rapid drop in the pH of the fermentation system and further creating an acidic environment unfavorable to the growth of contaminating and pathogenic bacteria. Subsequently, in this acidic environment, *Propionibacterium propionatum* can utilize the lactic acid produced by *Lactobacillus plantarum* as its main substrate and, through its unique metabolic pathway, generate short-chain fatty acids (SCFAs) such as propionic acid and acetic acid.

[0069] The significance of this synergistic effect lies in the fact that the final product not only contains lactic acid, but also propionic acid, which has stronger antibacterial activity and a broader antibacterial spectrum. Short-chain fatty acids such as propionic acid can penetrate the cell membrane of pathogens and dissociate into H+ in the cytoplasm. + This leads to intracellular acidification, interfering with the activity of key enzymes and energy metabolism, thereby achieving a strong antibacterial effect. This broad-spectrum short-chain fatty acid complex system composed of lactic acid and propionic acid is the key chemical weapon for this composition to effectively inhibit clubroot bacteria. In order to achieve the optimal synergistic metabolic efficiency, the ratio of viable bacteria of the three strains is precisely configured as 1-5:1-3:1, that is, the number of Lactobacillus plantarum is 1 to 5 times that of Propionibacterium propionate, and Bacillus coagulans is 1 to 3 times that of Propionibacterium propionate, ensuring that sufficient lactic acid is produced for the use of Propionibacterium propionate.

[0070] After fermentation of the above raw materials, the final product composition has clear functional indicators. The total short-chain fatty acid concentration in the fermentation broth reaches 150–250 mmol / L. This concentration range has been experimentally verified to have a significant inhibitory effect on clubroot pathogens and to be safe for rapeseed roots. Crucially, the propionic acid concentration is not less than 50 mmol / L, ensuring the product's core antibacterial efficacy. Simultaneously, thanks to the cell wall disruption effect of microbial fermentation on tea seed cake, the extraction rate of tea saponins is significantly improved, reaching a concentration of 3–5 g / L. Furthermore, the product contains a high density of beneficial live bacteria, with a total count of not less than 1.0 × 10⁻⁶. 8The concentration of CFU / mL, with core functional strains such as Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate accounting for over 80%, ensures its strong colonization and microecological regulation capabilities in the soil.

[0071] Example 2: This example provides a method for preparing a combined extract for controlling clubroot disease in rapeseed and its application. This method ensures that the various functional indicators of the composition described in Example 1 are achieved.

[0072] Reference Figure 1 The preparation method includes a raw material preparation step S101, a mixing and pH adjustment step S102, a two-stage fermentation step S103, and a product application step S104. Among them, the core is the two-stage fermentation step S103.

[0073] Step a, Raw material preparation (corresponding to) Figure 1 S101) The following raw materials shall be prepared in the following proportions by weight: 10-30 parts of tea seed cake (e.g., 20 parts), 20-30 parts of oyster shell powder (e.g., 25 parts), 10-20 parts of waste molasses (e.g., 15 parts), 1-5 parts of brewing yeast powder (e.g., 2 parts), 1-3 parts of xylooligosaccharide industrial residue (e.g., 2 parts), and 1-3 parts (e.g., 2 parts) of a compound fermentation agent containing Lactobacillus plantarum (CCTCC No. M2021135), Bacillus coagulans (CCTCC No. M2021497) and Propionibacterium propionate (CCTCC No. M2021136). All raw materials shall meet the requirements of agricultural production, and the microbial preparation shall ensure its activity.

[0074] Step b, two-stage pH-controlled sequential inoculation fermentation (corresponding to...) Figure 1 S102 and S103) are the core processes of this invention, and their detailed flow is as follows: Figure 2 As shown.

[0075] First, perform mixing and initial condition setting (corresponding to...) Figure 1 (S102) Place the prepared solid and semi-solid raw materials (tea seed cake, oyster shell powder, waste molasses, xylooligosaccharide industrial residues) into a clean fermentation tank, add sterile water at a material-to-liquid ratio of 1:3 to 1:5 (e.g., 1:4), stir thoroughly to form a uniform slurry, monitor the pH value of the slurry with a pH meter, and precisely adjust its initial pH value to 6.5-7.0 using food-grade acid or alkali (such as citric acid or sodium hydroxide). This neutral pH range is the ideal condition for starting the first stage of yeast fermentation.

[0076] Then, it enters the first fermentation stage (corresponding to...) Figure 2 (S201~S203).

[0077] In step S201, pre-activated brewing yeast powder is inoculated into the pH-adjusted slurry. After inoculation, the fermentation tank is sealed. A one-way exhaust valve can be installed to ensure that the carbon dioxide produced during fermentation can be discharged, while preventing outside air from entering, so as to maintain a strict anaerobic environment.

[0078] In step S202, the fermentation tank is placed in a constant temperature environment, and the fermentation temperature is controlled at 28-32℃, which is the suitable range for the growth and metabolism of brewing yeast.

[0079] In step S203, the fermentation in this stage continues for 48 to 72 hours. During this period, the pH value of the fermentation broth is monitored in real time. As yeast metabolism produces a small amount of organic acid and the generated CO2 dissolves in water to form carbonic acid, the pH value of the system will naturally and slowly decrease. When the pH value is monitored to decrease to the target range of 4.0 to 4.5, the first fermentation stage ends.

[0080] At this point, the fermentation system has completed the initial transformation: sugars have been consumed, creating an anaerobic environment; macromolecular organic matter has begun to decompose; and an acidic environment has been formed in the system. Then, the second fermentation stage begins (corresponding to...). Figure 2 (S204~S206).

[0081] In step S204, without altering the existing environment, a pre-activated compound fermentation agent, a mixture of Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate, is directly inoculated into the fermenter. This operation of inoculating subsequent strains under specific pH conditions is called sequential inoculation. The pH environment of 4.0 to 4.5 is the growth advantage zone for lactic acid bacteria and propionibacteria, while it constitutes an effective growth barrier for many putrefactive bacteria and miscellaneous bacteria. This demonstrates the key role of pH control in ensuring fermentation success rate and product purity.

[0082] In step S205, fermentation continues at a temperature of 28–32°C, while maintaining anaerobic conditions.

[0083] In step S206, fermentation lasts for 5–10 days. Samples are taken daily during this period, and the concentration of short-chain fatty acids is monitored by gas chromatography. The fermentation process is considered complete when the pH of the system stabilizes at 3.8–4.2 and the total short-chain fatty acid concentration changes by less than 5% over 24 consecutive hours.

[0084] In general, the temperature of the entire fermentation process can be broadly controlled between 25 and 35°C, and the total fermentation cycle is about 7 to 14 days. The fermentation endpoint is defined as the system pH stabilizing at 3.8 to 4.2 and the short-chain fatty acid content changing by less than 5% over 24 consecutive hours. This covers the optimized parameter range mentioned above.

[0085] After fermentation, the resulting brownish-red liquid with a sour, fermented aroma is the original extract of the present invention. After filtering or settling to remove large particles of residue, the original extract can be packaged and stored for later use.

[0086] Application steps (corresponding) Figure 1 (S104) Reference Figure 3 and Figure 4 The combined extract prepared in this invention acts synergistically on the soil-plant system through multiple mechanisms. Figure 3 Its application is flexible ( Figure 4 Before use, the extract stock solution needs to be diluted with water 500 to 650 times. Too high a concentration may stress the seedling roots, while too low a concentration will not be effective.

[0087] For live-streaming rapeseed:

[0088] 1. Seed dressing: Before sowing, mix rapeseed with a 500-fold diluted solution in an amount that can evenly coat the seeds. After slightly drying, the seeds can be sown. This ensures that the seeds are protected by beneficial bacteria and active substances from the time they germinate.

[0089] 2. Root irrigation: Immediately after sowing and covering with soil, irrigate once with a 500-650 times diluted solution to allow the solution to penetrate into the soil around the seeds. Repeat the root irrigation treatment on the 5th, 10th and 15th day after the rapeseed emerges. This series of operations aims to build a stable microbial and chemical protective barrier in the rhizosphere soil during the critical period of rapeseed root development.

[0090] For transplanted rapeseed:

[0091] 1. Root dipping: Before transplanting rapeseed seedlings, immerse their roots in a 500-650 times diluted solution for a few seconds to one minute to ensure that the roots are in full contact with and carry the extract.

[0092] 2. Root irrigation: On the day of transplanting, irrigate the roots with the same concentration of diluted solution while watering the roots. Afterward, irrigate the roots again every 5 to 7 days, for a total of 3 times. This is to help the seedlings recover and quickly establish a dominant rhizosphere microbial community in the new growth environment.

[0093] Example 3

[0094] This embodiment provides a sustained-release granular dosage form composition based on solid-state fermentation technology and its preparation method. This embodiment can solve the problems of high transportation costs and short duration of effect of liquid fermentation.

[0095] Based on the raw materials described in Example 1, crop straw powder (such as rapeseed straw and corn straw, crushed through a 40-mesh sieve) is additionally introduced as a solid fermentation substrate; the specific ratio is as follows: the amount of tea seed cake, oyster shell powder, waste molasses, brewer's yeast powder, xylooligosaccharide industrial residue and compound fermentation agent is the same as in Example 1, and crop straw powder equivalent to 50-100% of the weight of tea seed cake is added.

[0096] 2. Solid-state fermentation process:

[0097] Step a: Mix tea seed cake, oyster shell powder, and crop straw powder evenly. Dissolve waste molasses and xylooligosaccharide industrial residues in water, spray them onto the mixture, adjust the initial moisture content of the mixture to 50-60%, and adjust the initial pH to 6.5-7.0.

[0098] Step b: Two-stage solid-state fermentation.

[0099] First stage: Inoculate with brewer's yeast powder, mix evenly and then pile up for fermentation; let it pile up naturally for the first 24 hours, using the yeast to consume the oxygen between the particles; then compact and cover it, controlling the temperature at the center of the pile to not exceed 35℃, ferment for 36 to 48 hours, until the pH value of the substrate naturally drops below 4.5.

[0100] The second stage involves spreading out the pile, evenly spraying in a compound bacterial solution of Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate, mixing it thoroughly again, packing it into a fermentation tank or fermentation bag, compacting it tightly, and sealing it for anaerobic fermentation. Fermentation takes place at 25–35°C for 7–14 days, until the material has a distinct sour aroma and the pH stabilizes at around 4.0.

[0101] After fermentation, the moisture content of the material is reduced to 15-20% using low-temperature airflow drying technology, and the temperature is controlled below 45℃ to protect the activity of the microorganisms. Then, a binder, such as pregelatinized starch or sodium alginate, accounting for 3-5% of the total weight of the material is added, and solid slow-release granules with a particle size of 2-5mm are prepared by a disc granulator.

[0102] This embodiment transforms liquid-phase fermentation into solid-state fermentation (SSF), significantly reducing production water consumption and achieving clean production with zero wastewater discharge. The resulting slow-release granules are easy to store and transport. After being applied to the soil, the active ingredients are slowly released with soil moisture. According to measurements, the effective antibacterial concentration in the rhizosphere soil can last for 30 to 45 days, which is significantly better than liquid root irrigation treatment.

[0103] Experimental Example 1: In order to verify the actual control effect of the combined extract of the present invention on clubroot disease of rapeseed, the following potted plant comparative experiment was designed.

[0104] Experimental materials: The susceptible rapeseed variety "Huashuang No. 5" and the diseased soil was taken from a field with severe clubroot disease in rapeseed.

[0105] Experimental treatment:

[0106] Comparative Example 1: Water Control (CK).

[0107] Comparative Example 2: Single tea seed cake water extract treatment. The same amount of tea seed cake as in Example 1 was soaked in the same amount of water for 24 hours. The supernatant was then diluted by the same multiple before use.

[0108] Comparative Example 3: Incomplete fermentation broth treatment, using the same raw materials and ratios as Example 1, but using only Saccharomyces cerevisiae and Lactobacillus plantarum as fermentation strains, and employing a one-time mixed fermentation without sequential inoculation or pH adjustment.

[0109] Embodiment of the present invention: A combined extract prepared using the method of Example 2.

[0110] All treatment groups were applied according to the application method for transplanted rapeseed. Sixty days after transplanting, the incidence of clubroot disease in rapeseed was investigated in each treatment group, the disease index was calculated, and the control efficacy was calculated. Disease grading criteria: Grade 0, no clubroot; Grade 1, a few small tumors on fibrous roots; Grade 2, several tumors on fibrous roots and lateral roots; Grade 3, tumors on the main root, and many tumors on lateral roots and fibrous roots; Grade 4, swollen main root and rotten fibrous roots.

[0111] Disease index = [Σ(Number of diseased plants at each level × Relative grade value) / (Total number of plants surveyed × Highest relative grade value)] × 100

[0112] Prevention efficacy (%) = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100

[0113] The test results are shown in Table 1 below. Figure 5 As shown.

[0114] Table 1. Control effects of different treatments on clubroot disease in rapeseed.

[0115] Processing group Disease index Prevention efficacy (%) Comparative Example 1 (Water Control) 82.5 - Comparative Example 2 (Tea Seed Cake Aqueous Extract) 55.3 33.0 Comparative Example 3 (Incomplete Fermentation Broth) 37.1 55.0 Embodiments of the present invention 12.4 85.0

[0116] From Table 1 and Figure 5The experimental results show that, compared with the water control, all treatment groups have a certain control effect on clubroot disease of rapeseed. The control effect of Comparative Example 2 (single tea seed cake water extract) is 33.0%, indicating that tea saponins have a direct antibacterial effect, but the effect is limited. The control effect of Comparative Example 3 (incomplete fermentation liquid) is increased to 55.0%, indicating that the addition of microbial fermentation products (such as lactic acid) and beneficial bacteria enhances the control effect. However, the treatment group of the present invention has a disease index that is significantly lower than all comparative examples, and the control effect is as high as 85.0%, which is far superior to other treatment groups. This result fully demonstrates the synergistic effect of each component in the composition of the present invention, as well as the decisive role of the innovative two-stage pH-controlled sequential inoculation fermentation process in improving the final efficacy of the product. In particular, the broad-spectrum short-chain fatty acid system produced by the compound microbial agent greatly enhances the inhibitory ability against clubroot pathogens.

[0117] Experimental Example 2: In order to verify the actual control effect of the combined extract of the present invention on clubroot disease of rapeseed, the following potted comparative experiment was designed to analyze the soil physicochemical properties and microbial community structure before and after planting.

[0118] Experimental materials: The susceptible rapeseed variety "Huashuang No. 5" and the diseased soil was taken from a field with severe clubroot disease in rapeseed.

[0119] Experimental treatment:

[0120] Comparative Example 1: Water control (GZ group).

[0121] Example of the present invention: Combined extract (JK group) prepared using the method of Example 2.

[0122] In 2024-2025, the rapeseed planting area in Kaihua County was 89,300 mu (approximately 5,953 hectares). The core rapeseed planting areas were Lijiangfan in Chihuai Town, Yanglinfan in Yanglin Town, and Zhongcun Township, with areas reaching 11,600 mu (approximately 7,800 hectares), 7,800 mu (approximately 5,500 hectares), and 5,600 mu (approximately 3,000 hectares) respectively. In recent years, clubroot disease has frequently occurred in these main production areas. Soil samples were collected from three rapeseed planting areas during their growing season, showing different growth characteristics: normal plants, robust plants, and dwarf plants. The dwarf plant group was identified as exhibiting clubroot disease and bacterial wilt. Soil physicochemical properties and nutrient content were tested, and the differences were compared and analyzed. The results are as follows: Figure 6 As shown, Figure 2This study compares the differences in soil physicochemical properties under different physiological states in three rapeseed growing areas. Regarding soil moisture content, the healthy, robust rapeseed group in all three regions had lower moisture content than the diseased, dwarfed group, indicating that clubroot pathogens prefer moist soil for growth and reproduction. Regarding soil pH, the robust rapeseed group had a slightly higher pH than the dwarfed group, suggesting that clubroot pathogens reproduce more easily in slightly acidic soil environments. Regarding electrical conductivity, the robust rapeseed group had a slightly lower electrical conductivity than the dwarfed group, indicating that the soil samples from which clubroot disease occurred had higher salinity. Regarding soil phosphorus and potassium content, the robust rapeseed group had slightly lower phosphorus and potassium content than the dwarfed group, which is related to the slower plant growth and poorer nutrient absorption after clubroot disease, leading to poorer soil fertility absorption.

[0123] Soil samples were collected from the roots of the diseased rapeseed plants and placed in pots with an inner diameter of 30 cm and a height of 25 cm (approximately 15 L). Each pot contained 12 kg of diseased soil. The pots were randomly divided into two groups, with at least three replicates per group. Healthy rapeseed seedlings of the variety "Huashuang No. 5" at the 4-5 leaf stage were transplanted into the pots. Every 7 days, the two groups of pots were treated with 350 ml of water (control group, GZ group) and 350 ml of the extract of this invention diluted 500 times (example group, JK group), respectively. The treatment was carried out for 60 consecutive days. This application rate simulated the recommended concentration for field root irrigation. The microecology of the rapeseed root soil in each pot was investigated. Total bacterial DNA was extracted from cryopreserved soil samples using a commercially available DNA extraction kit, strictly following its standard operating procedures. The samples were then stored at -80°C. Subsequently, bacterial 16S rDNA genes and fungal internal transcribed spacer sequences were selected as specific molecular markers. Using a high-throughput sequencing platform, a systematic sequencing and data analysis of soil bacterial and fungal community diversity was conducted. The results are as follows: Figure 7 and Figure 8 As shown.

[0124] Figure 7 This demonstrates the differences in bacterial composition of the root soil between the clubroot disease control group GZ and the normal healthy rapeseed plant sample group JK. Figure 7 The bacterial diversity analysis results presented by AB show that there are certain differences in the species diversity of bacterial communities in the soil samples of plant roots after the occurrence of clubroot disease. The diversity in the soil samples of the example group is slightly higher, but due to the influence of soil composition in different plots and the small sampling volume, no significant difference was found. Figure 7CE demonstrated the differences in bacterial composition and genera between two groups of soil samples. The results showed that the example group was enriched in Bacillota, while the control group was enriched in Patescibacteria, with a decreased proportion of Bacillota. Bacillota bacteria are one of the core beneficial functional bacterial groups, playing a role in multiple aspects such as optimizing soil physicochemical properties, inhibiting clubroot disease, and enhancing plant resistance. Among them, the representative group of Bacillota, the genus Bacillus, such as Bacillus subtilis and Bacillus amyloliquefaciens, can produce lipopeptide antibiotics and bacteriocins, directly inhibiting the germination of dormant spores and hyphal growth of *Plasmodiophora brassicae*. They also secrete extracellular enzymes such as chitinase and glucanase, degrading key components of the *Plasmodiophora brassicae* cell wall and disrupting the pathogen's infection structure. Patescibacteria reproduce rapidly and can quickly colonize the rhizosphere of rapeseed, competing for adsorption sites of clubroot bacteria and soil nutrients such as carbon, nitrogen, and iron. This forms a biological barrier, reducing the probability of contact between pathogens and rapeseed roots. Patescibacteria also play a crucial role in inducing systemic resistance in rapeseed plants, enhancing stress resistance, regulating soil microbial community structure, and maintaining microecological balance. Furthermore, Patescibacteria in the rapeseed rhizosphere are predominantly low-abundance but functionally unique dark matter bacteria. Their effects on clubroot are mainly through community balance regulation, microenvironment optimization, and indirect antagonism, and are influenced by soil pH, organic matter, and tillage patterns. The proliferation of Patescibacteria in soil samples from clubroot outbreaks is related to their resistance to and slowing of clubroot spore germination. After application of the extract from the surface example, it can effectively regulate the microbial community structure of diseased soil towards a healthy soil state, increasing the abundance of beneficial bacteria and optimizing soil pH and nutrient availability.

[0125] Figure 8 This study demonstrates the differences in fungal composition from ITS high-throughput sequencing of soil samples from the roots of rapeseed with different growth traits; from Figure 8 The results shown in AB indicate that at the fungal level, there are no significant differences in diversity composition, but there are large differences between the two groups. Figure 8CE demonstrated the differences in fungal composition and genera between the two groups of soil samples. The results showed that the example group was enriched with Chytridiomycota, while the comparative group was enriched with Basidiomycota and Mortierellomycota. Chytridiomycota is a group of lower fungi characterized by the production of zoospores and is widely distributed in soil and aquatic environments. In the rhizosphere soil of rapeseed, chytrid fungi play a dual role. Some groups can indirectly increase the risk of clubroot disease, while certain strains can inhibit Plasmodiophora brassicae through parasitism and microecological regulation. Their function ultimately depends on the synergistic effect of soil physicochemical properties and microbial community structure. Basidiomycota and Mortierellomycota are important components of the soil fungal community. In the rhizosphere microecology of rapeseed, they play a dominant role in disease suppression. They regulate the survival and infection of Plasmodiophora brassicae through parasitic antagonism, soil microenvironment improvement, and plant resistance induction. They are also regulated by soil physicochemical properties and cultivation patterns, and are the core fungal groups for constructing a disease-suppressive soil microecology. The enrichment of Basidiomycota and Mortierellomycota fungi in the comparative group is due to antagonism against or mitigation of the outbreak of Plasmodiophora brassicae.

[0126] It is evident that there are significant differences between the example group and the comparative group in terms of physicochemical properties and microecology. The combined extract in the example group has a good effect on improving soil, increasing the relative abundance of beneficial rhizosphere bacteria, and inhibiting the ecological niche of pathogens.

[0127] In summary, this invention provides a composition and its preparation method for controlling clubroot disease in rapeseed, characterized by scientific component design, unique preparation process, and clearly defined application. This technical solution, through the synergistic effect of multiple mechanisms of action, not only effectively inhibits pathogens but also improves soil microecology and supplements crop nutrition, providing an innovative and effective solution for the green and sustainable control of clubroot disease in rapeseed.

Claims

1. A composition for controlling clubroot disease in rapeseed, characterized in that: It is made by fermentation of the following components in parts by weight: 10-30 parts tea seed cake, 20-30 parts oyster shell powder, 10-20 parts waste molasses, 1-5 parts brewing yeast powder, 1-3 parts xylooligosaccharide industrial residue, and 1-3 parts compound fermentation agent containing Lactobacillus plantarum, Bacillus coagulans and Propionibacterium propionate.

2. The composition for controlling clubroot disease in rapeseed according to claim 1, characterized in that: In the compound fermentation agent, the ratio of viable bacteria of *Lactobacillus plantarum* strain, *Bacillus coagulans* strain, and *Propionibacterium propionitum* strain is configured as 1–5:1–3:1; the *Lactobacillus plantarum* strain is *Lactobacillus plantarum* with accession number CCTCC No. M2021135, the *Bacillus coagulans* strain is *Bacillus coagulans* with accession number CCTCC No. M2021497, and the *Propionibacterium propionitum* strain is *Propionibacterium propionitum* with accession number CCTCC No. M2021136.

3. The composition for controlling clubroot disease in rapeseed according to claim 2, characterized in that: After fermentation, the total short-chain fatty acid concentration in the fermentation broth reaches 150–250 mmol / L, with propionic acid concentration not less than 50 mmol / L; the concentration of tea saponin is 3–5 g / L; and the total number of beneficial live bacteria is not less than 1.0 × 10⁻⁶. 8 The total number of beneficial live bacteria was CFU / mL, of which Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate accounted for more than 80%.

4. A method for preparing a combined extract for controlling clubroot disease in rapeseed, characterized by: Including the following step, a. Prepare raw materials according to the following weight parts: 10-30 parts of tea seed cake, 20-30 parts of oyster shell powder, 10-20 parts of waste molasses, 1-5 parts of brewing yeast powder, 1-3 parts of xylooligosaccharide industrial residue, and 1-3 parts of compound fermentation agent containing Lactobacillus plantarum, Bacillus coagulans and Propionibacterium propionate. b. A two-stage pH-controlled sequential inoculation fermentation process is used to ferment the mixture of raw materials and water described in step a to obtain the combined extract.

5. The method for preparing the combined extract for controlling clubroot disease in rapeseed according to claim 4, characterized in that: The two-stage pH-controlled sequential inoculation fermentation process specifically includes: In the first fermentation stage, brewer's yeast is first inoculated into the mixture of raw materials and water for anaerobic fermentation. The yeast metabolizes sugars to produce ethanol and carbon dioxide, and at the same time, it initially decomposes macromolecular organic matter to provide nutrient precursors for subsequent bacterial growth. In the second fermentation stage, after the pH of the system naturally drops to an acidic environment due to the first stage of fermentation, a compound fermentation agent composed of Lactobacillus plantarum, Bacillus coagulans, and Propionibacterium propionate is inoculated. This acidic environment is used to inhibit the growth of other bacteria and promote the efficient conversion of organic matter into short-chain fatty acids, mainly lactic acid and propionic acid, by lactic acid bacteria and propionic acid bacteria.

6. A method for preparing a solid-state sustained-release composition for controlling clubroot disease in rapeseed, characterized in that: Includes the following steps: a. Prepare tea seed cake, oyster shell powder, waste molasses, brewer's yeast powder, xylooligosaccharide industrial residue and compound fermentation agent according to the component ratio described in claim 1, and add crop straw powder passing through a 35-45 mesh sieve as an additional 50-100% of the weight of tea seed cake as a solid fermentation substrate. b. Mix the raw materials with water, adjust the initial moisture content of the mixture to 50-60% and the initial pH value to 6.5-7.0, and then use a two-stage pH-controlled sequential inoculation fermentation process to ferment the solid mixture.

7. The method for preparing the combined extract for controlling clubroot disease in rapeseed according to claim 6, characterized in that: The specific control parameters for the two-stage pH-controlled sequential inoculation fermentation process are as follows: In the first fermentation stage, after inoculating with brewing yeast, the pile is fermented for 24-48 hours, during which the temperature at the center of the pile is strictly controlled not to exceed 35°C. The pH value of the pile is naturally reduced to below 4.5 by the acid produced by yeast metabolism, so as to initially soften the cellulose structure. In the second fermentation stage, the pile is spread out and the compound fermentation agent is sprayed evenly, then it is turned over again, compacted and sealed. Anaerobic fermentation is carried out at 25-35°C for 7-14 days until the pH value of the system stabilizes at 3.8-4.2 and a distinct sour aroma is produced.

8. The method for preparing the combined extract for controlling clubroot disease in rapeseed according to claim 6 or 7, characterized in that: The method also includes a post-processing granulation step designed to maintain the activity of the strain and to shape it. The fermented solid material is placed in a low-temperature airflow below 45°C for drying, reducing the moisture content to 15-20% to induce a dormant state. Subsequently, 3-5% of a binder selected from pregelatinized starch, sodium alginate, or bentonite is added to the dried material. The mixture is then processed into cylindrical solid slow-release granules with a particle size of 2-5 mm using a flat die extrusion granulator.

9. A solid slow-release granule for controlling clubroot disease in rapeseed, prepared by the method of claim 8, characterized in that: The granules have a porous microstructure composed of straw and tea seed cake residue. This structure adsorbs high concentrations of short-chain fatty acids and tea saponins. After being applied to the soil, the active ingredients can be slowly released as soil moisture penetrates. The effective antibacterial concentration in the rhizosphere soil is released continuously for 30 to 45 days, which is significantly longer than that of liquid formulations.

10. The application of the composition according to any one of claims 1 to 3 or the solid slow-release granules according to claim 9 in the control of clubroot disease in rapeseed, characterized in that: The application includes selecting differentiated application methods based on the product formulation: when the product is a liquid extract, it should be diluted 500 to 650 times before use for seed dressing, root irrigation, or root dipping; when the product is a solid slow-release granule, it should be applied as a base application, hole application, or trench application at a rate of 20 to 40 kg per acre in the soil layer 5 to 10 cm away from the rapeseed roots, thereby constructing a dual protective barrier of microecology and chemical properties in the rhizosphere.

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