Method for preventing and treating rice blast by using compound microorganism fermentation liquor

Abstract

The invention discloses a method for preventing and treating rice blast by using compound microorganism fermentation liquor, and relates to the technical field of agricultural biologication.The method comprises the following steps that S1, bacillus strains with antagonistic activity on rice blast bacteria are selected for compound culture; the method comprises the following steps: respectively carrying out activation culture on selected bacillus of different species and strains to obtain respective seed solutions; and mixing the seed solutions according to a predetermined proportion, performing liquid submerged fermentation culture under controllable conditions, and controlling fermentation parameters to proliferate the compound microorganism thalli to a predetermined high concentration. According to the method for preventing and treating the rice blast through the compound microorganism fermentation liquor, the compound microorganism fermentation liquor preparation which can be directly used for spraying is prepared through compound culture of bacillus strains with antagonistic activity, so that the problems of drug resistance, pesticide residues and environmental pollution caused by chemical pesticides are effectively solved; the characteristics of greenness and environmental protection are shown, and the control effect on the rice blast is improved.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for controlling rice blast using a compound microbial fermentation broth. Background Technology

[0002] Rice blast, a significant fungal disease in rice production, can cause damage at all growth stages of rice, severely impacting yield and quality. Currently, rice blast control methods mainly include chemical pesticide application, breeding resistant varieties, and biological control. Chemical pesticide application, due to its significant efficacy, was once widely used; however, long-term use has led to increased resistance in the rice blast fungus, resulting in excessive pesticide residues in agricultural products and pollution of the farmland environment. While the application of resistant varieties has a certain degree of sustainability, resistance is often difficult to maintain due to variations in the physiological races of the rice blast fungus. Biological control, with its green and environmentally friendly characteristics, has become a research hotspot, especially the use of microorganisms and their metabolites, showing promising application prospects. However, existing single-strain biological control agents have limited colonization ability on rice leaves, insufficient resistance, and their control effects are easily affected by environmental factors, making it difficult to meet the needs of continuous and efficient rice blast control in actual production.

[0003] In the existing technology, CN120381034A-Bacillus belye TCS001 and its crude lipopeptide extracts for the control of rice blast fungus and activation of rice resistance to rice blast fungus proposes a scheme to control rice blast using a single Bacillus belye TCS001 and its fermentation broth, filtrate or crude lipopeptide extracts. Its core mechanism includes two points: first, the crude lipopeptide extracts directly inhibit the mycelial growth and appressorium formation of rice blast fungus, thereby reducing the pathogenicity of the fungus; second, it induces the expression of rice resistance immune-related genes (such as OsKS4, OsNAC4, OsPR1a, OsChitinase1) and the accumulation of reactive oxygen species, thereby activating the rice's own resistance. However, this technology still has significant limitations: at the microbial level, it relies on a single Bacillus belyssus TCS001, lacking synergistic effects between strains, resulting in limited leaf colonization and resistance, making it difficult to form a continuous and stable biological protection; at the formulation process level, the post-processing aims to "extract crude lipopeptides," using acid precipitation (adjusting pH to 2.0 with 6 mol / L hydrochloric acid) and drying, which directly kills the bacteria, making it impossible to achieve long-term control through viable colonization on the leaves; at the control mechanism level, it relies solely on "chemical inhibition (lipopeptides) + host resistance induction," lacking key dimensions such as physical shielding and nutrient competition, making the control effect susceptible to factors such as lipopeptide degradation and rice resistance attenuation; at the field application level, it does not clearly define parameters such as the key control growth period of rice (e.g., jointing-heading stage), quantitative standards for the concentration of sprayed viable bacteria, and droplet diameter control, making it difficult to guarantee the effective colonization and efficacy of the formulation on the leaves.

[0004] In summary, the environmental risks of chemical control, the limitations of resistance in resistant varieties, and the shortcomings of biological control in terms of strain synergy, live bacteria stability, and the integrity of control mechanisms all constrain the development of green control technologies for rice blast. How to screen for complex microbial strain combinations with highly efficient antagonistic activity and good compatibility, optimize fermentation processes to obtain high-concentration live bacteria preparations, and construct a multi-dimensional control system based on live bacteria colonization remain pressing technical challenges. To address these challenges, we propose a method for controlling rice blast using a complex microbial fermentation broth. Summary of the Invention

[0005] To address the aforementioned technical problems, a method for controlling rice blast using a compound microbial fermentation broth is provided. This technical solution solves the problems of resistance to chemical pesticide pathogens, pesticide residues, and environmental pollution; the difficulty in maintaining the resistance of disease-resistant varieties due to pathogen mutations; and the limited colonization ability and stress resistance of single-species biological control agents, which are greatly affected by the environment and cannot meet the needs of efficient control.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for controlling rice blast using a compound microbial fermentation broth includes the following steps:

[0008] S1. Select Bacillus species with antagonistic activity against rice blast fungus for compound culture; activate and culture different selected Bacillus species and strains separately to obtain their respective seed liquids; mix the seed liquids in a predetermined ratio and carry out liquid deep fermentation culture under controlled conditions, controlling the fermentation parameters to proliferate the compound microbial cells to a predetermined high concentration to obtain a primary fermentation broth; perform post-processing on the primary fermentation broth, including centrifugation to separate the cells, removal of the supernatant, resuspending the cells in sterile liquid, and adjusting the cell concentration to obtain a compound microbial fermentation broth preparation that can be directly used for spraying;

[0009] S2. Foliar spraying should be carried out during the time window from the jointing stage to the heading stage of rice.

[0010] S3. Using a spraying device, dilute the prepared compound microbial fermentation broth preparation and spray it evenly onto the rice plants in a way that completely and effectively covers both sides of the rice leaves. The amount of spraying should be sufficient to moisten the leaves without forming dripping liquid.

[0011] S4. Through the above spraying, the composite microorganisms colonize the surface of rice leaves and form a biological protective layer.

[0012] Preferably, in step S1, the Bacillus species with antagonistic activity against rice blast fungus include at least one antagonistic Bacillus species and Bacillus subtilis.

[0013] The antagonistic Bacillus species are selected from Bacillus amyloliquefaciens, Bacillus bellis, and Bacillus pumilus.

[0014] The Bacillus subtilis strain selected is a strain that is well compatible with the antagonistic Bacillus strain and has antagonistic activity against rice blast fungus;

[0015] The selected ratio is the volume ratio of the antagonistic Bacillus seed liquid to the Bacillus subtilis seed liquid, and this volume ratio is strictly controlled within the range of 1:0.8 to 1:1.2;

[0016] Before mixed inoculation and compound fermentation, the seed culture of each strain must be cultured to the late logarithmic growth stage, and the viable cell concentration must be no less than [missing information]. CFU / mL.

[0017] Preferably, in the liquid submerged fermentation culture of step S1, the controllable conditions specifically include:

[0018] The fermentation medium is a liquid medium containing carbon source, nitrogen source, inorganic salts and trace elements;

[0019] The initial pH value for fermentation was set between 6.8 and 7.2;

[0020] The fermentation temperature is kept constant within the range of 29℃ to 31℃;

[0021] Maintain the stirring speed at 150 rpm to 250 rpm;

[0022] Ventilation rate was maintained between 0.8 vvm and 1.2 vvm;

[0023] Fermentation culture time lasts 36 to 48 hours;

[0024] The predetermined high concentration refers to a total concentration of compound live bacteria greater than or equal to... CFU / mL.

[0025] Preferably, in the post-processing of step S1, the conditions for centrifugal separation are:

[0026] Centrifuge at a low temperature of 4°C to 10°C at a speed of 8000 rpm to 12000 rpm for 10 to 15 minutes.

[0027] The sterile liquid is 0.85% sterile physiological saline or sterile phosphate buffer with a pH of 7.0 to 7.2;

[0028] The adjustment of bacterial cell concentration refers to adjusting the concentration of the resuspended composite microbial cells to a certain level. CFU / mL to The range of CFU / mL;

[0029] The obtained compound microbial fermentation broth preparation needs to be stored at 4°C to 8°C in the dark and should be used up within 7 days after preparation.

[0030] Preferably, in step S2, the time window from the jointing stage to the heading stage is specifically defined as follows:

[0031] The process begins in the early stage of jointing, when the first internode at the base of the rice plant begins to elongate significantly, and ends in the late heading stage, when the spikelets at the top of the uppermost panicle begin to emerge from the sheath of the flag leaf.

[0032] Preferably, in step 3, the foliar spraying operation is performed 2 to 3 times within the time window from the jointing stage to the heading stage;

[0033] The specific timing of the second to third spraying applications is as follows:

[0034] The first spraying was carried out at the early stage of rice jointing, that is, when the first internode at the base of the rice has elongated by 1 to 2 cm; each spraying used the newly prepared compound microbial fermentation broth preparation with a consistent concentration of live bacteria.

[0035] Preferably, in step S3, the total concentration of live bacteria in the compound microbial fermentation broth preparation during spraying should reach [a certain level]. CFU / mL to CFU / mL;

[0036] This concentration is achieved by adjusting the prepared bacterial cell concentration to... The compound microbial fermentation broth preparation with a concentration of CFU / mL was obtained by diluting it 10 times with clean, room-temperature water.

[0037] Preferably, in step S3, the spraying amount is specifically 600 to 900 liters of the diluted compound microbial fermentation broth preparation per hectare of paddy field for each spraying;

[0038] The spraying equipment requires that the diameter of the spray droplets be controlled within the range of 100 micrometers to 200 micrometers;

[0039] Spraying operations should be carried out in sunny and windless weather conditions, with the best time being before 9:00 a.m. and after 4:00 p.m., avoiding the hottest and most sunny period of daytime.

[0040] If rain washes off the leaves within 24 hours of spraying, re-spraying should be carried out promptly after the leaves have dried.

[0041] Preferably, the composite microbial fermentation broth preparation prepared in step S1 does not contain any synthetic bactericides, antibiotics, chemically synthesized surfactants, or other chemical auxiliaries;

[0042] The mechanism by which the biological protective layer inhibits the attachment and germination of rice blast fungus spores relies entirely on the physical shielding effect produced by the colonization of the microbial cells on the leaf surface and their metabolic activities, the competitive consumption of nutrients on the leaf surface, and the secretion of antibacterial substances that inhibit the activity of rice blast fungus spores. The antibacterial substances include lipopeptide compounds, polyketide compounds, and antibacterial proteins.

[0043] Preferably, this method is applicable to rice production systems that follow green control principles or organic rice planting standards; by spraying the compound microbial fermentation liquid preparation during the rice jointing to heading stage, biological control of rice blast is achieved without the use of chemically synthesized pesticides.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention proposes a method for controlling rice blast using a compound microbial fermentation broth. By co-culturing Bacillus strains with antagonistic activity, a compound microbial fermentation broth formulation is prepared for direct spraying. This effectively solves the problems of pesticide resistance, pesticide residues, and environmental pollution caused by chemical pesticides. The formulation of this invention is a pure live bacteria system, free of any synthetic fungicides, antibiotics, or chemical adjuvants. It eliminates the need for extraction processes that kill the bacteria, such as acid precipitation or drying, thus fully preserving microbial activity. This method is more suitable for green pest control and organic rice cultivation standards, avoiding the short-term control limitations that may exist with crude lipopeptide extracts. Foliar spraying during the rice's jointing to heading stage allows the compound microorganisms to colonize the rice leaf surface and form a biological protective layer, avoiding the problems associated with single-strain microorganisms. It lacks interspecies synergy, has weak leaf colonization ability, and insufficient stress resistance. It relies entirely on the physical shielding, nutrient competitive consumption, and secreted antibacterial substances generated by the colonization of microorganisms on the leaf surface and their metabolic activities to inhibit the attachment and germination of rice blast fungus spores. In response to the lack of physical shielding and nutrient competition dimensions in the control mechanism, which is only the direct inhibition of bacteria by crude lipopeptide extracts and the induction of rice resistance genes, this product uses a triple mechanism to directly block the contact of fungal spores and deprive them of germination nutrients. At the same time, it secretes lipopeptides, polyketides, and antibacterial proteins, showing green and environmentally friendly characteristics. It is suitable for rice production systems that follow green control principles or organic rice planting standards, improves the control effect of rice blast, and promotes sustainable agricultural development. Attached Figure Description

[0046] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0047] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are merely examples, and those skilled in the art may conceive of other obvious variations.

[0048] Rice blast, a devastating fungal disease, severely threatens rice yield and quality during rice cultivation. While traditional chemical control methods offer rapid results, long-term use can lead to pathogen resistance, environmental pollution, and pesticide residues, failing to meet the requirements of modern green, ecological, and sustainable agricultural development. Therefore, developing safe, efficient, and environmentally friendly biological control technologies is crucial. This method provides a core technology based on compound microbial fermentation broth. Through careful screening of beneficial microorganisms with specific antagonistic activities, and through scientific formulation and optimized fermentation processes, a highly effective formulation is prepared and precisely applied during critical rice growth stages. This achieves effective prevention and control of rice blast, particularly suitable for green pest control systems or organic rice cultivation standards.

[0049] Reference Figure 1 As shown, a method for controlling rice blast using a compound microbial fermentation broth includes: preparing a microbial fermentation broth formulation composed of specific Bacillus species and mastering its scientific application strategy. The entire process begins with the selection and compound culture of the strains. The selected microorganisms must be members of the Bacillus genus that have clear antagonistic activity against rice blast fungus. Specifically, the strain combination must include at least two types: one is an "antagonistic Bacillus" with direct antagonistic activity, and the other is a specific "Bacillus subtilis". Antagonistic Bacillus is mainly screened from Bacillus amyloliquefaciens, Bacillus belye, and Bacillus pumilus. These strains have been verified in the laboratory to effectively inhibit the germination of rice blast fungus spores or the growth of mycelia, and their mechanism of action usually involves competition for nutrient sites and the production of antimicrobial metabolites. The selection of Bacillus subtilis is more rigorous, not only ensuring that it also has a certain antagonistic ability against rice blast fungus itself, but more importantly, that its strain characteristics have good compatibility with the selected antagonistic Bacillus. Compatibility refers to the ability of different bacterial strains to coexist harmoniously and work synergistically during mixed culture without mutual inhibition. This is a crucial foundation for the success of compound microbial agents. If antagonism exists between strains, it will not only reduce the concentration of the target bacteria but may also affect its metabolic activity, ultimately diminishing the control effect. Therefore, rigorous inter-strain compatibility tests must be conducted before determining the final Bacillus subtilis strain to be used.

[0050] After selecting specific strains and species, the seed culture preparation stage begins. This is the starting point for fermentation, and a high-quality seed culture is a prerequisite for successful large-scale fermentation. Operationally, different species or strains of Bacillus (including selected antagonistic Bacillus species and compatible Bacillus subtilis) need to be independently activated. Activation is usually carried out in shake flasks containing suitable liquid culture medium (such as LB broth, nutrient broth, or specific media optimized according to the characteristics of the strain), and cultured on a temperature-controlled shaker at a suitable temperature (usually 30-37°C) and rotation speed for a certain period (e.g., 12-24 hours). The purpose of activation is to restore the vigorous growth and metabolic activity of the preserved strains (such as slant culture or glycerol cryovials), obtaining vigorous cells in the logarithmic growth phase. The seed culture obtained after activation is the seed culture for each strain. To ensure the uniformity and high yield of subsequent combined fermentation, the seed culture of each strain must be cultured to the late logarithmic growth phase before being used for mixed inoculation. This period is characterized by rapid cell division, vigorous metabolism, and a high concentration of viable bacteria. Specifically, the viable bacteria concentration of each seed culture should not be less than 1 billion colony-forming units (1×10⁻⁶) per milliliter. 9 (CFU / mL). Seed culture that meets this standard means that the bacteria are in their optimal physiological state and are most suitable for large-scale inoculation.

[0051] The next crucial stage is the compound fermentation. The seed cultures of the different strains are mixed according to a predetermined and strictly controlled volume ratio. This ratio, optimized through extensive experimentation, is essential for the effective functioning of the compound microbial agent. Specifically, it is the volume ratio of antagonistic Bacillus seed culture to Bacillus subtilis seed culture, strictly limited to a narrow range of 1:0.8 to 1:1.2. For example, if 100 liters of antagonistic Bacillus seed culture are added, the amount of Bacillus subtilis seed culture added must be between 80 and 120 liters. This precise ratio control ensures that the compound microbial community maintains the expected population structure and balance during fermentation, preventing the overgrowth of one strain from inhibiting others, thus ensuring optimal activity and synergistic effects of various target microorganisms in the final fermentation product. The mixed seed culture is then inoculated into a large fermenter for deep liquid fermentation. This is the core step in the large-scale production of compound microbial agents, and the entire process needs to be carried out under highly controlled conditions to maximize the synergistic proliferation of target microorganisms and the accumulation of metabolites.

[0052] The conditions for liquid submerged fermentation are extremely precisely controlled, involving several key parameters: First, the fermentation medium is the nutritional basis for microbial growth. Liquid media are typically used, containing carbon sources for energy (such as glucose, sucrose, starch, or their hydrolysates), nitrogen sources (such as peptone, yeast extract, soybean meal, ammonium sulfate, urea, etc.), inorganic salts to maintain osmotic pressure and enzyme activity (such as phosphates, sulfates, sodium chloride, etc.), and essential trace elements (such as iron, manganese, zinc, copper, etc.). The formulation of the medium needs to be optimized according to the nutritional requirements and metabolic characteristics of the strain used, ensuring rapid cell growth while also promoting the synthesis of target antagonistic substances. Second, the initial pH of fermentation has a significant impact on microbial enzyme activity and metabolic pathways, and is usually set between 6.8 and 7.2, close to neutral. During fermentation, microbial metabolism produces organic acids or alkaline substances, causing pH changes; therefore, online pH monitoring and automatic acid / alkali addition devices are usually required for adjustment. Third, temperature is a key factor affecting microbial growth rate and metabolite synthesis. Fermentation temperature must be consistently controlled within a narrow range of 29°C to 31°C. This temperature range represents the optimal growth range for the selected Bacillus species and the production of antimicrobial substances. Excessively high temperatures may lead to premature spore formation or metabolic disorders, while excessively low temperatures result in slow growth. Fourth, the stirring rate is crucial for ensuring uniform distribution of the culture medium components and promoting oxygen dissolution and transfer. The stirring rate is typically maintained between 150 and 250 revolutions per minute. Sufficient stirring prevents cell sedimentation and ensures that nutrients and oxygen are evenly distributed to each cell. Fifth, aeration is the lifeline of deep aerobic fermentation because Bacillus species are strict or facultative aerobic bacteria, requiring sufficient oxygen for respiration, metabolism, and energy production. Aeration is expressed in "vvm" (volume of air per unit time per unit volume of culture) and should be maintained between 0.8 vvm and 1.2 vvm. For example, for 1 cubic meter of fermentation broth, 0.8 to 1.2 cubic meters of filtered sterile air need to be introduced per minute. Adequate oxygen supply is essential for high-density cell growth and certain aerobic metabolic pathways (such as the synthesis of lipopeptide antibiotics). Finally, the fermentation culture time needs to last 36 to 48 hours. This timeframe is determined based on the cell growth curve and the accumulation patterns of metabolites. The initial stage of culture is the logarithmic growth phase, characterized by cell adaptation and rapid proliferation, while the later stage is the stationary phase, during which cell density reaches its peak, and secondary metabolites (such as antimicrobial substances) may be synthesized and accumulated in large quantities. Monitoring the viable cell concentration is crucial for determining the fermentation endpoint. When sampling confirms that the total concentration of the combined viable cells reaches or exceeds 5 billion colony-forming units per milliliter (5 × 10⁻⁶), the fermentation endpoint is reached. 9 Fermentation can be terminated when the concentration (CFU / mL) reaches the predetermined high level, yielding the primary fermentation broth. Reaching this high concentration signifies successful fermentation and the acquisition of a culture rich in highly active complex microbial cells.

[0053] The post-fermentation processing steps are equally important, directly affecting the quality, stability, and efficacy of the final formulation. The primary fermentation broth first requires post-processing, the core of which is the isolation and formulation of the microorganisms. The first step is centrifugation. The primary fermentation broth is placed in a large centrifuge and centrifuged at a high speed of 8000 to 12000 rpm for 10 to 15 minutes at a low temperature (4°C to 10°C). The low temperature is to minimize damage to microbial activity during centrifugation. The strong centrifugal force generated by high-speed centrifugation quickly settles the microbial cells to the bottom of the centrifuge tube or drum, effectively separating them from the fermentation supernatant. After centrifugation, the supernatant is carefully removed. This supernatant may contain some soluble metabolites (including some antimicrobial substances), but it is usually removed to obtain a more stable formulation with controllable viable cell concentration and fewer impurities. After removing the supernatant, the sediment at the bottom of the centrifuge tube is a wet bacterial sludge rich in the target complex microbial cells. Next, these cells need to be resuspended in sterile liquid. There are two commonly used sterile liquid options: one is 0.85% sterile physiological saline (with an osmotic pressure similar to that of microbial cells, reducing osmotic shock), and the other is sterile phosphate buffer with a pH adjusted to 7.0 to 7.2 (providing a stable pH environment). The bacterial sludge is added to an appropriate amount of sterile liquid, and the cells are evenly dispersed by gentle stirring or agitation to form a suspension. The final step is to adjust the bacterial concentration. Depending on the specific application requirements, the concentration of the resuspended composite microbial cells is precisely adjusted to 1 billion to 10 billion colony-forming units (1×10⁻⁶) per milliliter by adding or removing sterile liquid. 9 CFU / mL to 1×10 10 The range of CFU / mL is specified. After the above steps, the final product is a compound microbial fermentation broth formulation that can be directly applied to the field. A key characteristic of this formulation is that it is completely free of any synthetic fungicides, antibiotics, chemically synthesized surfactants, or other chemical auxiliaries; its active ingredients are entirely derived from the compound microorganisms themselves and their metabolic activities. To maintain the activity of the microorganisms in the formulation, the fermentation broth must be stored in a low-temperature environment between 4°C and 8°C, protected from light. Low temperature significantly reduces the metabolic rate of microorganisms, delaying their aging and death. Protection from light is to prevent damage to microbial cells from light (especially ultraviolet light). Furthermore, considering that the activity of live bacteria formulations gradually decreases with prolonged storage, this formulation must be used within 7 days of preparation to ensure the concentration of live bacteria and the control effect at the time of application.

[0054] After preparing a high-quality compound microbial fermentation broth formulation, scientific field application is crucial to maximizing its control effect. This involves precise spraying timing, reasonable spraying frequency, correct spraying concentration, appropriate spraying amount, and suitable environmental conditions. First, the selection of the spraying timing is paramount. Rice blast can occur at different growth stages of rice, but neck blast has the most severe impact on yield, and neck blast infection mainly occurs during the booting stage of rice. Therefore, this method strictly limits the spraying time window to the jointing to heading stage, when rice is most susceptible to rice blast. This window is specifically defined as: starting from the early jointing stage when the first internode at the base of the rice plant begins to elongate significantly, and ending from the late heading stage when the spikelet (i.e., the tip of the panicle) of the uppermost panicle begins to emerge from the leaf sheath of the flag leaf (the last leaf) that encloses it. This period covers the entire process of young panicle differentiation, development, and emergence, and is the golden period for protecting future panicles from rice blast infection. To provide continuous protection throughout the entire sensitive period, a single spray is often insufficient. Typically, foliar spraying needs to be applied 2 to 3 times within the time window from the jointing stage to the heading stage. The specific spraying timing is as follows: The first spraying should be applied at the critical early jointing stage, specifically when the first internode at the base of the rice plant has elongated to 1-2 cm. Subsequent second (and possibly third) sprayings should be applied during the mid-to-late booting stage or the heading stage, depending on the rice's growth progress, disease trends, and weather conditions. Each spraying must use a freshly prepared, precisely adjusted and consistent diluted compound microbial fermentation broth to ensure a balanced and effective biological dosage for each treatment.

[0055] The spraying process itself requires precise control. Before spraying, the prepared bacterial cells need to be adjusted to a high concentration (e.g., 5 × 10⁻⁶). 9 The concentrate of the compound microbial fermentation broth preparation (CFU / mL) should be diluted. Clean, room-temperature water should be used as the diluent (avoid water containing large amounts of impurities or disinfectants). The dilution ratio must be strictly controlled to ensure that the total concentration of the compound live bacteria in the solution reaches 100 million to 500 million colony-forming units (1×10⁻⁶) per milliliter during spraying. 8 CFU / mL up to 5×10 8 The effective range of CFU / mL. Typically, a concentration of 5 × 10⁻⁶ CFU / mL is used. 9The concentration range can be achieved by diluting the CFU / mL stock solution 10 times with water (i.e., 1 part stock solution to 9 parts water). This concentration range is a balance point verified by extensive field trials: if the concentration is too low, the amount of colonizing bacteria will be insufficient, making it difficult to form an effective biological protective layer; if the concentration is too high, it may increase costs, and with limited leaf space and nutrients, competition among bacteria will intensify, which may reduce control efficiency. The spraying volume is also a key parameter, as it determines the total amount of microorganisms actually received per unit area of ​​rice plants and the coverage of the pesticide solution on the leaf surface. This method specifies that 600 to 900 liters of diluted compound microbial fermentation broth should be used per hectare of rice field for each spraying. This larger spraying volume is to ensure that the pesticide solution can fully and evenly cover the rice plants, especially the middle and lower leaves and the underside of the leaves, which are often the starting points for rice blast fungus infection. Specific requirements apply to the spraying equipment: a sprayer capable of producing good atomization and uniform droplet size must be used. The ideal droplet diameter should be controlled within the range of 100 to 200 micrometers. Droplets that are too large are prone to rolling off leaves, resulting in waste and uneven coverage; droplets that are too small (such as aerosols) are easily lost, highly susceptible to wind influence, and evaporate too quickly under strong light, hindering microbial adhesion and survival on leaf surfaces. A droplet size of 100-200 micrometers strikes a balance between leaf adhesion, uniform coverage, and resistance to drift. Environmental conditions are equally important for spraying. Spraying should be carried out in sunny, windless, or lightly windy conditions. Strong winds can cause pesticide drift, reducing effective deposition and potentially contaminating non-target areas. The optimal spraying time is before 9:00 AM or after 4:00 PM. This is primarily to avoid periods of high daytime temperatures (usually exceeding 30°C) and strong sunlight (especially intense ultraviolet radiation). High temperatures and strong ultraviolet radiation significantly reduce the survival rate of microorganisms sprayed onto leaves, weakening their colonization ability. If rainfall occurs shortly after spraying, rainwater may wash away microorganisms that have not yet firmly colonized the leaves, leading to a decrease in control effectiveness. Therefore, if effective rainfall (enough to wash away the leaves) occurs within 24 hours after spraying, a second spray should be arranged promptly after the rain stops and the leaves dry naturally in order to re-establish the biological protective layer.

[0056] The core objective of this precise foliar spraying operation is to effectively deliver a sufficient amount of active compound microorganisms to the surface of rice leaves (both sides) and create suitable colonization conditions. Once the pesticide droplets containing a high concentration of live bacteria are evenly attached to the leaves, under suitable temperature and humidity conditions, these compound Bacillus species (including Bacillus amyloliquefaciens, Bacillus belye, Bacillus pumilus, and their compatible Bacillus subtilis) begin to rapidly recover, grow, and reproduce. Utilizing the micronutrients and water in the leaf microenvironment, they form communities on the leaf surface (especially near stomata and veins), gradually building a dynamic, living "biological protective layer." This biological protective layer's inhibitory mechanism against rice blast fungus is multifaceted and comprehensive, entirely dependent on the successful colonization of these beneficial microorganisms on the leaf surface and their vigorous metabolic activity: firstly, a physical barrier effect. A large number of beneficial microorganisms and their secreted extracellular polysaccharides cover the leaf surface, forming a physical barrier that directly prevents airborne rice blast fungus conidia from directly contacting the rice leaf epidermal cells, reducing the chance of spore attachment. Secondly, there is competition for nutrients and space. After colonizing the leaf surface, beneficial microorganisms rapidly consume the limited nutrients (such as sugars and amino acids) exudated or present on the leaf surface and occupy favorable living space. This puts the rice blast fungus spores that subsequently land on the leaf surface in an unfavorable environment of nutrient scarcity and overcrowding, greatly inhibiting the energy and resources required for their germination and invasion. Most importantly, there is the antimicrobial effect. These antagonistic Bacillus species continuously secrete various secondary metabolites—antimicrobial substances—with broad-spectrum antibacterial or bactericidal activities into the surrounding environment during their growth and reproduction. These antimicrobial substances are the chemical basis for the biocontrol efficacy of this method. The main types include: lipopeptide compounds (such as surfactants, iturin, and fengycin), which can disrupt the structure and function of pathogen cell membranes; polyketide compounds (such as difficidin and macrolactin), which interfere with the metabolic pathways of pathogens; and various antibacterial proteins or peptides. When these antibacterial substances accumulate to a certain concentration in the leaf microenvironment, they can effectively inhibit or directly kill rice blast fungus spores that fall onto the leaf surface, preventing the formation of germination tubes and the penetration of invasion pins into the host cell wall, thus nipping the pathogen in the bud at the inception of infection.

[0057] In summary, this method of controlling rice blast using compound microbial fermentation broth forms a complete, efficient, and environmentally friendly biological control technology system. From the scientific screening and formulation of antagonistic strains to the precise control of the compound fermentation process, the standardization of post-treatment of the formulation, and finally the precise control of the timing, frequency, concentration, method, and environmental conditions of field spraying, it fully utilizes the natural antagonistic relationship between beneficial microorganisms and pathogens. By artificially strengthening the dominant position of beneficial microbial populations in key parts of the crop, it constructs a biological defense line of using beneficial microorganisms to control pathogens. Its mechanism of action is natural and safe, involving no chemically synthesized pesticides, and fully complies with the production principles and requirements of green plant protection and organic agriculture. Through scientific application during the high-risk period of disease infection from rice jointing to heading, it can effectively prevent and control rice blast, especially neck blast, without polluting the environment, producing pesticide residues, or inducing resistance, providing reliable technical support for the production of safe and high-quality rice products. This method embodies the advanced concepts of integrated pest management (IPM) and ecological regulation in modern agricultural development and has broad application prospects and promotional value.

[0058] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for controlling rice blast disease using a compound microbial fermentation broth, characterized in that, Includes the following steps: S1. Select Bacillus species that have antagonistic activity against rice blast fungus for compound culture; activate and culture different selected species and strains of Bacillus to obtain their respective seed solutions; After the seed liquid is mixed in a predetermined ratio, it is carried out in a liquid deep fermentation culture under controlled conditions. The fermentation parameters are controlled to make the composite microbial cells proliferate to a predetermined high concentration to obtain a primary fermentation broth. The primary fermentation broth is post-processed, including centrifugation to separate the cells, removal of the supernatant, resuspending the cells in sterile liquid, and adjusting the cell concentration to obtain a compound microbial fermentation broth preparation that can be directly used for spraying. S2. Foliar spraying should be carried out during the time window from the jointing stage to the heading stage of rice. S3. Using a spraying device, dilute the prepared compound microbial fermentation broth preparation and spray it evenly onto the rice plants in a way that completely and effectively covers both sides of the rice leaves. The amount of spraying should be sufficient to moisten the leaves without forming dripping liquid. S4. Through the above spraying, the composite microorganisms colonize the surface of rice leaves and form a biological protective layer.

2. The method for controlling rice blast disease using a compound microbial fermentation broth according to claim 1, characterized in that, In step S1, the Bacillus species with antagonistic activity against rice blast fungus include at least one antagonistic Bacillus species and Bacillus subtilis. The antagonistic Bacillus species are selected from Bacillus amyloliquefaciens, Bacillus bellis, and Bacillus pumilus. The Bacillus subtilis strain selected is a strain that is well compatible with the antagonistic Bacillus strain and has antagonistic activity against rice blast fungus; The selected ratio is the volume ratio of the antagonistic Bacillus seed liquid to the Bacillus subtilis seed liquid, and this volume ratio is strictly controlled within the range of 1:0.8 to 1:1.2; Before mixed inoculation and compound fermentation, the seed culture of each strain must be cultured to the late logarithmic growth stage, and the viable cell concentration must be no less than [missing information]. CFU / mL.

3. The method for controlling rice blast with a compound microbial fermentation broth according to claim 1, characterized in that, In the liquid submerged fermentation culture of step S1, the controllable conditions specifically include: The fermentation medium is a liquid medium containing carbon source, nitrogen source, inorganic salts and trace elements; The initial pH value for fermentation was set between 6.8 and 7.2; The fermentation temperature is kept constant within the range of 29℃ to 31℃; Maintain the stirring speed at 150 rpm to 250 rpm; Ventilation rate was maintained between 0.8 vvm and 1.2 vvm; Fermentation culture time lasts 36 to 48 hours; The predetermined high concentration refers to a total concentration of compound live bacteria greater than or equal to... CFU / mL.

4. The method for controlling rice blast with a compound microbial fermentation broth according to claim 1, characterized in that, In the post-processing described in step S1, the conditions for centrifugal separation are as follows: Centrifuge at a low temperature of 4°C to 10°C at a speed of 8000 rpm to 12000 rpm for 10 to 15 minutes. The sterile liquid is 0.85% sterile physiological saline or sterile phosphate buffer with a pH of 7.0 to 7.2; The adjustment of bacterial cell concentration refers to adjusting the concentration of the resuspended composite microbial cells to a certain level. CFU / mL to The range of CFU / mL; The obtained compound microbial fermentation broth preparation needs to be stored at 4°C to 8°C in the dark and should be used up within 7 days after preparation.

5. The method for controlling rice blast with a compound microbial fermentation broth according to claim 1, characterized in that, In step S2, the time window from the jointing stage to the heading stage is specifically defined as follows: The process begins in the early stage of jointing, when the first internode at the base of the rice plant begins to elongate significantly, and ends in the late heading stage, when the spikelets at the top of the uppermost panicle begin to emerge from the sheath of the flag leaf.

6. The method for controlling rice blast with a compound microbial fermentation broth according to claim 1, characterized in that, In step 3, the foliar spraying operation is carried out 2 to 3 times during the entire jointing to heading stage time window; The specific timing of the second to third spraying applications is as follows: The first spraying was carried out at the early stage of rice jointing, that is, when the first internode at the base of the rice has elongated by 1 to 2 cm; each spraying used the newly prepared compound microbial fermentation broth preparation with a consistent concentration of live bacteria.

7. The method for controlling rice blast with a compound microbial fermentation broth according to claim 1, characterized in that, In step S3, the total concentration of live bacteria in the compound microbial fermentation broth preparation should reach [a certain level] during spraying. CFU / mL to CFU / mL; This concentration is achieved by adjusting the prepared bacterial cell concentration to... The compound microbial fermentation broth preparation with a concentration of CFU / mL was obtained by diluting it 10 times with clean, room-temperature water.

8. The method for controlling rice blast with a compound microbial fermentation broth according to claim 1, characterized in that, In step S3, the spraying amount is specifically 600 to 900 liters of the diluted compound microbial fermentation broth preparation per hectare of paddy field for each spraying. The spraying equipment requires that the diameter of the spray droplets be controlled within the range of 100 micrometers to 200 micrometers; Spraying operations should be carried out in sunny and windless weather conditions, with the best time being before 9:00 a.m. and after 4:00 p.m., avoiding the hottest and most sunny period of daytime. If rain washes off the leaves within 24 hours of spraying, re-spraying should be carried out promptly after the leaves have dried.

9. A method for controlling rice blast using a compound microbial fermentation broth according to claim 1, characterized in that, The composite microbial fermentation broth preparation prepared in step S1 does not contain any synthetic bactericides, antibiotics, chemically synthesized surfactants, or other chemical auxiliaries. The mechanism by which the biological protective layer inhibits the attachment and germination of rice blast fungus spores relies entirely on the physical shielding effect produced by the colonization of the microbial cells on the leaf surface and their metabolic activities, the competitive consumption of nutrients on the leaf surface, and the secretion of antibacterial substances that inhibit the activity of rice blast fungus spores. The antibacterial substances include lipopeptide compounds, polyketide compounds, and antibacterial proteins.

10. A method for controlling rice blast using a compound microbial fermentation broth according to claim 1, characterized in that, This method is applicable to rice production systems that follow green control principles or organic rice planting standards; by spraying the compound microbial fermentation liquid preparation during the rice jointing to heading stage, biological control of rice blast is achieved without the use of chemically synthesized pesticides.

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