Microbial agent and application thereof in prevention and treatment of rice blast and promotion of rice growth

Abstract

The invention discloses a microbial agent and application thereof in preventing and treating rice blast and promoting rice growth, active ingredients of the microbial agent are formed by compounding cold bacillus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO: 33492 and bacillus velezensis with the preservation number of CGMCC NO: 33493, and the microbial agent aims at solving the synergistic problem of preventing and treating rice blast and promoting rice growth. According to the complex microbial inoculant, through the synergistic interaction between the two strains, the metabolite of the complex microbial inoculant can completely inhibit the spore germination of magnaporthe oryzae; meanwhile, the fungicide can degrade cellulose, obviously promote the growth of plant height, fresh weight and dry weight of rice, and realize the integration of disease prevention and growth promotion. The invention further discloses a preparation method of the fungicide and an application method of the fungicide for preventing and treating rice blast and promoting growth in a root irrigation mode in the three-leaf stage of rice. The microbial agent disclosed by the invention has dual functions of efficiently preventing diseases and stably promoting growth, and an efficient and safe biological solution is provided for green and sustainable development of agriculture.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a compound microbial agent and its preparation method, particularly a microbial agent compounded from a specific Bacillus strain, and its application in controlling rice blast, inhibiting the germination of rice blast fungal spores, and promoting rice growth. Background Technology

[0002] Rice is the staple food for more than half of the world's population, and its safe production is directly related to national welfare, people's livelihood, and social stability. However, the production of rice by ascomycetes... Magnaporthe oryzae Rice blast, caused by the pathogen, has long been known as "rice cancer" due to its devastating epidemic potential and genetic variability. This pathogen infects rice leaves, nodes, panicles, and grains, causing leaf blast, node blast, and, most fatally, neck blast, often resulting in yield losses of 30%–50%, and even total crop failure in epidemic years. Traditionally, rice blast control has relied primarily on chemical fungicides (such as tricyclazole), a strategy that has historically contributed to food security. However, its inherent drawbacks are becoming increasingly apparent: the pathogen has evolved rapidly under strong selective pressure, developing resistance to many mainstream pesticides, leading to decreased efficacy and increased application costs; simultaneously, the overuse of chemical pesticides has caused ecological crises such as soil compaction, water pollution, and a sharp decline in biodiversity, threatening food safety and human health. Developing efficient, safe, and sustainable alternative control strategies has become an urgent need in the field of plant protection and a strategic task for ensuring national food and ecological security.

[0003] Against this backdrop, biological control—using beneficial microorganisms and their metabolites to suppress pathogens—is considered the most promising alternative. Early research focused primarily on single-strain and single-pathogen models; however, single strains are susceptible to environmental fluctuations in complex farmland environments, have limited functionality, and are insufficient to combat pathogens (such as...). M. oryzae High adaptability. Compound microbial agents, by combining multiple non-antagonistic beneficial microorganisms, can exert synergistic effects, replacing or reducing the use of chemical pesticides and fertilizers. These agents not only promote crop growth through mechanisms such as nitrogen fixation, phosphorus solubilization, and plant hormone secretion, but also enhance disease resistance through mechanisms such as inhibiting pathogens and inducing systemic resistance in plants. Currently, this technology has been widely applied in disease control, soil improvement, and crop quality enhancement, meeting the needs of sustainable development in green agriculture.

[0004] Microbial biological control holds great promise in agricultural production due to its advantages such as safety for humans and animals, environmental friendliness, and lack of pollution. By utilizing fungi, bacteria, actinomycetes, and their metabolites to prepare inoculants or biopesticides, diseases can be effectively controlled without easily developing resistance. In practical applications, antagonistic bacteria, bioremediation agents, and biofertilizers can be used to mitigate disease occurrence and ensure rice yield and quality. Utilizing biological control methods to control rice blast is an important direction for future agricultural development, with broad research and application prospects. Therefore, there is an urgent need to develop novel compound microbial agents, achieving efficient and sustainable disease control and crop yield increases through screening specific strains, optimizing formulations, and improving the stability and field adaptability of live bacteria. Summary of the Invention

[0005] The present invention aims to provide a compound microbial agent, particularly involving a specific combination of Bacillus amyloliquefaciens 1B-10 and Bacillus belyeis 8B-2, in order to overcome the technical defects of existing microbial agents in the prevention and control of rice blast and the promotion of rice growth, such as single strain function, insufficient synergistic effect, unstable control efficacy and limited growth-promoting effect.

[0006] The objective of this invention is achieved through the following technical means: A compound microbial agent, the active ingredients of which are *Bacillus belyesii* 1B-10 (CGMCC NO:33492) and *Cryptospira psychrophila* 8B-2 (CGMCC NO:33493). *Bacillus belyesii* 1B-10 is classified as... Bacillus velezensis The classification and naming of *Cryptospira 8B-2* is as follows: Peribacillus frigoritolerans .

[0007] As a more preferred technical solution of the present invention, the ratio of viable bacteria of Bacillus belyi 1B-10 to Bacillus cynomolgus 8B-2 is (0.8-1.2):1, preferably 1:1.

[0008] As a preferred embodiment of the present invention, the viable count of the *Bacillus belyssioides* 1B-10 is 1 × 10⁻⁶. 9 ~ 5×10 9 CFU / mL, the viable count of the *Cryptospira* 8B-2 was 1×10⁻⁶. 9 ~ 5×10 9 CFU / mL.

[0009] Another objective of this invention is to provide a method for preparing the above-mentioned composite microbial agent, comprising the following steps: (1) performing shake-flask fermentation on Bacillus belye 1B-10 with preservation number CGMCC NO:33492 and Cryospora 8B-2 with preservation number CGMCC NO:33493 respectively to obtain fermentation broth; (2) collecting the bacterial cells from step (1) by centrifugation and resuspending them with sterile water, and adjusting the bacterial cell concentrations of Bacillus belye 1B-10 and Cryospora 8B-2 to 1×10⁻⁶ respectively. 9 ~ 5×10 9 CFU / mL; (3) Mix the two bacterial solutions obtained in step (2) at a volume ratio of 1:1 to obtain the composite microbial agent.

[0010] As a more preferred technical solution of the present invention, the culture medium used for fermentation in step (1) is TSB culture medium, the fermentation temperature is 26-30℃, preferably 28℃, and the fermentation time is 40-56 hours, preferably 48 hours.

[0011] As a preferred technical solution of the present invention, the fermentation liquid culture medium used in the cultivation process is formulated as follows: 17.0g tryptone, 3.0g soybean papain hydrolysate, 5.0g sodium chloride, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, and pH 7.2.

[0012] As a preferred technical solution of the present invention, the two strains were activated by streaking on TSA plates and cultured in a constant temperature incubator at 28°C for 48 hours. Single colonies were picked and inoculated into TSB medium and cultured overnight with shaking at 28°C and 150 rpm / min to prepare seed liquid. The seed liquid was inoculated into fermentation medium at an inoculation rate of 2% and cultured with shaking at 28°C and 150 rpm / min for 48 hours to obtain the fermentation broth of the two strains.

[0013] Another objective of this invention is to provide the application of the above-mentioned compound microbial agent in inhibiting the germination of rice blast fungus spores and / or preventing rice blast disease.

[0014] Another objective of this invention is to provide the application of the above-mentioned compound microbial agent in promoting rice growth.

[0015] As a preferred technical solution of the present invention, the promotion of rice growth is manifested in increasing the plant height, fresh weight and dry weight of rice plants.

[0016] As a preferred technical solution of the present invention, the compound microbial agent is diluted 50-100 times before application.

[0017] As a preferred technical solution of the present invention, the root irrigation method is used for application.

[0018] As a better technical solution of the present invention, its application in inhibiting plant pathogenic fungi.

[0019] As a preferred technical solution of the present invention, the plant pathogenic fungus is selected from at least one of Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium oxysporum.

[0020] Another objective of this invention is to provide a method for preventing and controlling rice blast and promoting rice growth, wherein the compound microbial agent is diluted 50-100 times and applied as a root irrigation treatment at the 3-leaf stage of rice.

[0021] The beneficial effects are as follows: This invention, through experiments, has shown that the combination of Bacillus amyloliquefaciens 1B-10 and Bacillus belye 8B-2, with its sterile supernatant, can inhibit the germination of rice blast fungus spores by up to 100% within 12 hours. This indicates that the strain's metabolites contain highly effective antibacterial substances, and that the combination of the two strains produces a significant synergistic effect, far exceeding the control efficacy of single strains or conventional compound inoculants.

[0022] Both strains in the microbial agent of this invention have the ability to degrade cellulose, promoting the decomposition of soil organic matter and nutrient cycling. Pot experiments have confirmed that 21 days after inoculation with this microbial agent, the plant height, fresh weight, and dry weight of rice plants were significantly increased, achieving integrated application of "disease prevention and growth promotion" and effectively solving the problem of insufficient synergy of functional microbial agents.

[0023] This invention clarifies the optimal fermentation medium, fermentation conditions, and key parameters for field application of the microbial agent. Through process standardization and application standardization, it ensures that the microbial agent maintains high activity in storage and field environments, guaranteeing the stability and repeatability of disease prevention and growth promotion effects.

[0024] This invention screens and combines specific functional strains, and through innovative synergistic mechanisms and optimized application methods, systematically solves key technical bottlenecks in the prevention and healthy cultivation of rice blast. Compared with existing technologies, this invention achieves breakthroughs in control efficiency (100% spore germination inhibition rate), disease prevention and growth promotion, and technical operability, providing a more efficient and stable microbial technical solution for green agriculture. Attached Figure Description

[0025] Figure 1 This is a phylogenetic tree of strains Bacillus belyssae and Bacillus cyrtiformis based on the 16S rRNA gene.

[0026] Figure 2 This is a plate confrontation inhibition diagram of strains Bacillus belye and Bacillus cyrtolytica against rice blast fungus.

[0027] Figure 3This is an experiment on the cellulose degradation of Congo red transparent zones by strains Bacillus belyssae and Bacillus pyriformis.

[0028] Figure 4 This is a plate confrontation experiment between strains Bacillus belyssae and Bacillus cyrtiformis and other pathogens.

[0029] Figure 5 This is a diagram showing the inhibitory effect of the compound inoculant on the germination of rice blast fungus spores.

[0030] Figure 6 This is a diagram showing the inhibitory effect of the sterile supernatant of the compound inoculant on the germination of rice blast fungus spores.

[0031] Figure 7 It is the growth-promoting effect of compound microbial agents on potted rice. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and do not constitute a limitation thereof. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of the present invention.

[0033] Example 1: Strains Isolation, Identification and Phylogenetic Analysis This embodiment describes the isolation, screening, and identification process of two biocontrol strains, 1A and 2B. The specific steps include: (1) Isolation and screening of biocontrol strains Source of strain: Rhizosphere soil of Pinus tabuliformis from Northeast China.

[0034] Microbial Isolation Method: In this experiment, 10 trees were selected as sampling points in the Northeast Pinus tabuliformis forest of Shenhe District Botanical Garden, Shenyang City, Liaoning Province. The topsoil of each tree was removed, and fine pine roots were cut and preserved in sterile bags, temporarily stored at 4℃ for later use. The fine pine roots were placed in sterile 50mL centrifuge tubes, and an appropriate amount of sterile water was added to completely submerge the roots in a laminar flow hood. The tubes were sealed and incubated at 28℃ with shaking for 30 minutes. The rhizosphere soil was thoroughly washed off, and microorganisms were enriched. The fine roots were picked out, and the soil suspension was centrifuged at low temperature (4℃, 6000rpm, 15min). The supernatant was discarded, and the rhizosphere soil was collected. The collected rhizosphere soil was serially diluted, and 10... -4 10 -5 10 -6 The diluted solution was spread onto TSA medium, with each treatment repeated three times. The plates were inverted and incubated at 28°C. After 48 hours of incubation, the morphological characteristics of the colonies were observed. Single colonies with inconsistent morphology were promptly picked, numbered, and purified. Pure bacterial cultures were obtained by streaking 1-3 times. The purified strains were suspended in 20% glycerol and stored at -80°C.

[0035] (2) Identification of biocontrol strains Bacterial culture was used directly as a PCR template, and the 16S rRNA sequence was amplified using 27F / 1492R primers. A phylogenetic tree was constructed using MEGA10.0 software (neighbor-joining method). The phylogenetic tree based on 16S rRNA showed that the 16S rRNA gene copy of strain 1A, as shown in sequence 1 of the sequence listing, had the highest homology (99.71%) with the Bacillus belyssus standard strain CR-502; the 16S rRNA gene copy of strain 2B, as shown in sequence 2 of the sequence listing, had the highest homology (99.86%) with the Cryospora psychrophila standard strain DSM 8801. Figure 1 As shown. Therefore, the two strains were identified as Bacillus belyssus 1B-10 and Bacillus psychrophilus 8B-2, respectively.

[0036] (3) Growth inhibition of rice blast fungus by biocontrol strains Using a sterilized punch (5mm diameter), *Magnaporum oryzae* was prepared into mycelial cakes and inoculated onto the center of a 90mm diameter PDA plate using toothpicks. Three pure cultures of the same bacterium were then inoculated at equal intervals 3cm from the center of the plate using toothpicks. The control group consisted of pure cultures of *Magnaporum oryzae* on the PDA plate, with mycelial cakes inoculated at the corresponding positions. The plates were sealed with sealing film and incubated at 28℃ for 5 days. The presence of inhibition bands was observed, and the colony radii of the control and treatment groups were measured. Each colony treatment was repeated at least three times, and the average value was calculated. Results showed that strain 1A achieved an inhibition rate of 85.00% against *Magnaporum oryzae*, and strain 2B achieved an inhibition rate of 78.05%. The plate confrontation inhibition effects of strains *Bacillus belye* and *Cryptospira* against *Magnaporum oryzae* were as follows: Figure 2 As shown.

[0037] Inhibition rate (%) = (Coronation radius of control pathogen - Colony radius of treated pathogen) / Colony radius of control pathogen × 100 In the control group (CK), only rice blast fungus cakes were inoculated onto the plates. The results showed that the rice blast fungus grew uniformly radially on the PDA medium, almost covering the entire plate (90 mm in diameter) after 5 days. This indicates that rice blast fungus can grow and reproduce normally in the absence of biocontrol fungi, providing a clear control benchmark for subsequent treatment groups.

[0038] In treatment group 1A, a clear transparent inhibition zone (area without colony growth) appeared around each of the three mycelial cakes, indicating that the growth of *Magnapordica oryzae* was significantly inhibited. The inhibition only occurred in areas close to the biocontrol mycelial cakes, with no significant expansion in areas further away from the biocontrol mycelium. Combined with the data (inhibition rate 85.00%), this indicates that strain 1A can effectively secrete antimicrobial substances or inhibit *Magnapordica oryzae* growth through competitive action, demonstrating a significant inhibitory effect. Strain 2B was similar to 1A; an inhibition zone also appeared around the mycelial cakes, but the transparent area was slightly smaller than that of 1A, and a small amount of *Magnapordica oryzae* still grew in some areas. The data (inhibition rate 78.05%) shows that its inhibitory ability was slightly weaker than 1A, but it still exhibited a strong inhibitory effect.

[0039] The CK group showed no transparent inhibition zone, while groups 1A and 2B both showed clear inhibition zones, which directly verified the antagonistic effect of the biocontrol bacteria on rice blast fungus. The inhibition rate of 1A was higher than that of 2B.

[0040] (4) Experiment on the degradation of cellulose by biocontrol strains Prepare a solid culture medium using sodium carboxymethyl cellulose (CMC-Na) as the sole carbon source. Pour plates and inoculate two test strains onto each plate. Incubate upside down at 28°C for 1-3 days until colonies appear. Pour an appropriate amount of 1 mg / mL Congo red solution onto the surface of the plate, covering the entire plate, and incubate at room temperature for 15-30 minutes. Discard the Congo red staining solution and rinse the plate 2-3 times with 1M NaCl solution, incubating for 10-15 minutes each time, until the red background fades and a clear transparent zone is revealed. The results of the Congo red transparent zone degradation experiment of strains *Bacillus belyssae* and *Cryptospira* are shown below. Figure 3 As shown.

[0041] Congo red can combine with CMC-Na to form a red complex; if the strain secretes cellulase to degrade CMC-Na, a clear zone will form in the degradation area as the red complex disappears. The size of the clear zone can reflect the strain's ability to degrade cellulose (the larger the clear zone, the stronger the degradation ability is usually).

[0042] Figure 3 The cellulose degradation performance of strain 1A in the study is as follows: On the left plate, *Bacillus belychnophorus* successfully grew on a medium with CMC-Na as the sole carbon source, forming visible colonies (light-colored / white colonies on the plate). On the right plate (after staining), after staining with 1 mg / mL Congo red solution and elution with 1M NaCl solution, the background of the plate turned orange-red due to the binding of undegraded CMC-Na with Congo red; while the areas where *Bacillus belychnophorus* degraded CMC-Na formed clear zones (areas significantly lighter in color than the background) because the red complex was destroyed. The presence of clear zones directly proves that *Bacillus belychnophorus* can secrete cellulase to decompose CMC-Na, and the size of the clear zone reflects the strength of its cellulose degradation ability.

[0043] Figure 3 The cellulose degradation performance of *Cryptospira 2B* on the left plate is as follows: On the same medium, *Cryptospira 2B* showed relatively weak growth, and the visual presence of colonies on the plate was not as obvious as on the left plate of 1A. The right plate, after staining and washing, also showed a clear zone (marked by the white line in the image), indicating that *Cryptospira 2B* can also secrete cellulase to degrade CMC-Na. Both strains can degrade cellulose, which can improve the rhizosphere microenvironment, provide carbon sources for plants, or inhibit pathogen colonization by degrading cellulose in the soil.

[0044] (5) Inhibitory effect of biocontrol strains on other pathogenic fungi Using a sterilized punch (5 mm diameter), mycelial cakes of *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, and *Fusarium oxysporum* were prepared and inoculated onto the center of 90 mm diameter PDA plates using toothpicks. Three pure cultures of the same bacterium were then inoculated at equal intervals with toothpicks 3 cm from the center of the plate. The control group consisted of pure cultures of each pathogen on the PDA plate. The results of plate confrontation experiments between *Bacillus belyssioides* and *Cryptospira* strains and other pathogens are as follows: Figure 4 As shown.

[0045] Rhizoctonia solani ( Rhizoctonia solani ): In group CK, the bacterial colonies expanded rapidly radially from the center of the plate, almost covering the entire 90mm culture dish, indicating vigorous growth of the pathogen on PDA medium. In group 1A, the bacterial colonies were significantly smaller than those in CK, and a clear inhibition zone (a blank area without colony growth) appeared around the inoculation site 3cm from the center, indicating that group 1A could inhibit the growth of *Rhizoctonia solani* through secretion of antimicrobial substances or competitive action. In group 2B, the bacterial colony area was even smaller than that of group 1A, and the inhibition zone was wider, indicating that group 2B had a stronger antagonistic effect against *Rhizoctonia solani* than group 1A.

[0046] Sclerotium sclerotiorum ( Sclerotinia sclerotiorum ): In group CK, the colonies also expanded radially, covering most of the plate, and showed good growth. In group 1A, the pathogen colonies were divided into multiple discontinuous regions, and growth was significantly inhibited, with small inhibition zones appearing around the bacteria; however, the inhibitory effect was weaker than that of group 2B. In group 2B, *Sclerotinia sclerotiorum* grew only in the center with a small amount, and was strongly inhibited by bacteria around the periphery. The colony morphology was fragmented, and the inhibition zones were clearer and larger, indicating that group 2B had higher antagonistic activity against *Sclerotinia sclerotiorum*.

[0047] Fusarium oxysporum ( Fusarium oxysporum ): In group CK, colonies expanded outwards from the center (growth areas could be distinguished by the color characteristics of hyphae / spores), completely covering the plate. In group 1A, the expansion of pathogen colonies was inhibited, with inhibition zones appearing around the bacteria and slowed growth in the center. In group 2B, *Fusarium oxysporum* colonies had the smallest area and the most significant inhibition zones, indicating that group 2B also had a better inhibitory effect on *Fusarium oxysporum* than group 1A.

[0048] Both bacteria (1A and 2B) showed significant inhibitory effects on Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium oxysporum, demonstrating that Bacillus belye has the ability to antagonize multiple pathogens. The antagonistic ability of 2B was generally stronger than that of 1A. Among the treatment groups with multiple pathogens, 2B showed superior colony inhibition and inhibition zone integrity / range.

[0049] Example 2: Inhibition of rice blast fungal spore germination by compound inoculum.

[0050] This example demonstrates the inhibitory effect of two bacterial strains on *Magnapordica oryzae* and the inhibition of *Magnapordica oryzae* spore germination by a compound inoculant. The specific steps include: (1) Preparation of rice blast fungus spores The rice blast fungus was inoculated onto OMA medium (30 g of oats was boiled in 800 mL of distilled water for 25-30 min, filtered through three layers of gauze, and the filtrate was collected. 150 mL of tomato juice was added, stirred well, and 2% agar powder was added. The medium was then autoclaved at 121℃ for 20 min). The medium was cultured at 28℃ for 7-8 days. Once the mycelium had fully colonized the medium, it was broken with a sterile cotton swab to induce conidia. The medium was then covered with double layers of gauze and cultured at room temperature for 3-4 days. Sterile water was added to the culture dish, and the conidiophores, conidia, and mycelium were washed off with a sterile cotton swab. The conidia were then filtered into centrifuge tubes using a sterile three-layer paper funnel. The tubes were centrifuged at 4℃ and 4000 r / min for 15 min, the supernatant was discarded, and the precipitate was the conidia. The number of conidia was counted under a microscope using a hemocytometer. The rice blast fungus spores were diluted to a concentration of 5 × 10⁻⁶. 4 per mL.

[0051] (2) Preparation of compound microbial agents Two bacterial strains were activated by streaking on TSA plates and incubated at 28°C for 48 hours. Single colonies were picked and inoculated into TSB medium, and cultured overnight with shaking at 28°C and 150 rpm to prepare seed culture. The seed culture was then inoculated into fermentation medium at a 2% inoculum rate and cultured at 28°C and 150 rpm for 48 hours with shaking to obtain fermentation broth for both strains. The fermentation broth was collected and centrifuged at 4°C (6000 rpm, 15 min). The supernatant was discarded, and the bacterial cells were collected. The bacterial count was adjusted to 1 × 10⁻⁶ cells using sterile water. 9 CFU / mL, mix the two strains evenly in a 1:1 ratio.

[0052] (3) Inhibition of rice blast fungal spore germination by compound inoculant After resuspending the conidia in 1 mL of distilled water, the concentration was adjusted to 5 × 10⁻⁶. 4 For each bacterial cell / mL, pipette 30µL and drop it onto a hydrophobic membrane. Dilute the compound bacterial agent 100 times to a concentration of 1×10⁻⁶. 7 CFU / mL, 30µL was pipetted onto a hydrophobic membrane and mixed thoroughly with *Magnapordica oryzae* spores. The mixture was placed in a humidified chamber and incubated at 28℃. Germination was observed and photographed under a microscope at 12h and 24h. The compound inoculum showed a 100% inhibition rate of *Magnapordica oryzae* spore germination at 24h, with no spore germination observed at that time, significantly superior to the control group of 50µg / mL tricyclazole. The inhibitory effect of the compound inoculum on *Magnapordica oryzae* spore germination is as follows: Figure 5 As shown.

[0053] The compound inoculant group achieved highly efficient and long-lasting germination inhibition. At 12 hours, the spores of rice blast fungus were morphologically intact, without germ tubes, hyphae, or other hallmark structures of germination, indicating that spore germination was completely inhibited (inhibition rate reached 100%). At 24 hours, no signs of germination were observed (no germ tubes / hyphae extension), indicating that the inhibitory effect of the compound inoculant was long-lasting, continuously blocking the spore germination process within 24 hours.

[0054] The negative control group served as a reference for natural germination. At 12 hours, a large number of spores germinated, with clear germ tube structures visible, indicating that under sterile water treatment, rice blast fungus spores could normally initiate the germination process. At 24 hours, the germination rate further intensified, visually demonstrating the physiological pattern of natural spore germination without antibacterial substances.

[0055] The positive control group (50 μg / mL tricyclazole) showed that the traditional agent had limitations in inhibition. Although some spores failed to germinate at 12 h, the number of germinating spores was significantly higher than that of the compound fungal agent group, indicating that tricyclazole's inhibitory efficiency on spore germination was weaker than that of the compound fungal agent. At 24 h, the number of germinating spores further increased, showing that the antibacterial effect of tricyclazole decreased over time, in stark contrast to the long-lasting 100% inhibition of the compound fungal agent even after 24 h.

[0056] The compound microbial agent showed significantly better inhibitory effects on the germination of rice blast fungus spores than the traditional agent tricyclazole, and also possessed both high efficiency (100% inhibition rate in 12 hours) and long-lasting effect (inhibition effect maintained for 24 hours). This provides a more promising technical approach for the biological control of rice blast. Compared to chemical agents, the compound microbial agent not only achieves rapid germination inhibition but also reduces pesticide residues and environmental risks, demonstrating the application value of biological control methods.

[0057] (4) Inhibition of rice blast fungus spore germination by the sterile supernatant of the compound inoculant Preparation of sterile supernatant for the bacterial strain: The compound inoculum was centrifuged at 10,000 rpm for 20 min at 4℃ to remove bacterial cells. The supernatant was then filtered through a 0.22 µm filter membrane to obtain sterile supernatant. 30 µL of *Bacillus oryzae* spore suspension and the sterile supernatant of the compound inoculum were respectively transferred to a hydrophobic membrane, placed in a humidified chamber, and incubated at 28℃. Germination was observed and photographed under a microscope at 12 h and 24 h. The sterile supernatant of the compound inoculum also showed a strong inhibitory effect on the germination of *Bacillus oryzae* spores. The inhibitory effect of the sterile supernatant of the compound inoculum on the germination of *Bacillus oryzae* spores is shown in Figure 6, and the specific analysis is as follows: The compound bacterial agent group consisted of sterile supernatant containing the compound bacterial agent. The negative control group consisted of sterile water treatment as a blank control to eliminate the influence of solvents. The positive control group consisted of 50 μg / mL tricyclazole, a chemical bactericide, to verify the effectiveness of the experimental system.

[0058] The observation time point is: 12 hours: early stage of spore germination. 24 hours: late stage of spore germination. Observation indicators included spore germination rate, hyphal morphology (germ tube length, branching, cell integrity), and differences in inhibition effect (statistical differences in germination rate and hyphal morphology among different treatment groups).

[0059] The results of the 12-hour observation are as follows: The spore germination rate of the compound inoculant group was significantly reduced, with only a few spores forming short germ tubes (some of which were twisted or broken). Mycelial morphology was abnormal, with reduced branching, possibly accompanied by cell membrane damage. In the negative control group, spore germination was active, with long, thin germ tubes and normal branching, exhibiting typical germination characteristics of rice blast fungus. The germination rate was close to the natural state. The germination rate of the positive control group was significantly lower than that of the negative control group. Some spores germinated, but the germ tubes were short and thick, and mycelial extension was hindered. The compound inoculant showed a stronger inhibitory effect than chemical agents in the early germination stage (12 hours), possibly achieving rapid antibacterial activity by disrupting spore germination-related proteins or cell membranes.

[0060] The 24-hour observation results are as follows: The germination rate of the compound inoculant group further decreased, with most spores arresting in the early germination stage, and the hyphae being short and sparse. Cytoplasmic condensation or lysis of the hyphae may have been observed. The germination rate of the positive control group slightly increased compared to 12 hours later, but remained significantly lower than that of the compound inoculant group. Local hyphal extension occurred, but overall growth was inhibited, and hyphal breakage appeared in some areas. The sterile supernatant of the compound inoculant exhibited a strong and sustained inhibitory effect on the germination of rice blast fungus spores, especially in the early germination stage, outperforming the chemical agent tricyclazole. Its mechanism of action may involve multi-target interference, possessing the potential to be developed into a novel biopesticide.

[0061] Example 3: Application of compound microbial agent in potted rice This example uses the compound microbial agent prepared in Example 2 to verify its effectiveness in controlling rice blast and promoting growth in rice pots. Specific steps include: (1) Experimental setup and rice varieties used The potted experiments were conducted in 6×8cm pots. Rice seeds were surface-sterilized and then germinated. After sprouting, they were sown in sterilized potting soil, with 9 seeds sown in each pot. The pots were placed in a greenhouse for growth, with a light / dark ratio of 16 / 8 light / dark, a temperature of 25℃, and a humidity of 45%. The experimental group was treated with the compound microbial community prepared in Example 2, while the control group was inoculated with an equal volume of sterile water (CK). Each treatment was replicated three times. The rice variety used in the potted experiments was "Lijiang Xintuan Black Rice".

[0062] (2) Inoculation of rice seedlings with compound microbial agents Once the rice seedlings reach the 3-leaf stage, dilute the prepared compound microbial agent 100 times and apply it to the roots for root irrigation. Assuming 40g of soil is used per pot, apply 40mL of the compound microbial agent per pot. The final total bacterial count in the soil will be approximately 1×10⁻⁶. 7 CFU / mL.

[0063] (3) Measurement of growth indicators of rice plants Control group (CK): Rice not inoculated with the compound microbial agent; Treatment group: Rice inoculated with the compound microbial agent; Treatment period: 21 days after inoculation, growth traits (plant height, fresh weight, dry weight) were measured. 21 days after inoculation with the compound microbial agent, the rice was removed from the greenhouse, and its growth traits were investigated and statistically analyzed to evaluate the effect of the compound microbial agent on rice plant growth.

[0064] Rice inoculated with compound microbial agents showed improvements in plant height, fresh weight, and dry weight. Figure 7 This is the result of a growth-promoting experiment on rice potted plants using a compound microbial agent. The experiment explored the effects of the compound microbial agent on the growth traits of rice through a potted control experiment.

[0065] There was a highly significant difference in plant height between the control group (green column) and the treatment group (pink column). The average plant height of the treatment group was significantly higher than that of the control group. Combined with the scatter plot distribution (each point represents one replicate measurement), the plant height of most replicates in the treatment group was higher than that in the control group, indicating that the compound microbial agent significantly promoted the longitudinal growth of rice.

[0066] There was a significant difference in fresh weight between the two groups. The average fresh weight of the treatment group was higher than that of the control group. The scatter distribution also supports the conclusion that the overall fresh weight of the treatment group was higher, reflecting that the compound microbial agent can enhance the fresh accumulation of the overall biomass of rice.

[0067] There were also significant differences in dry weight between the two groups. The average dry weight of the treatment group was higher than that of the control group. Dry weight is a core indicator of the accumulation of photosynthetic products and the storage of substances in plants. This indicates that the compound microbial agent not only promotes the growth of "fresh matter" but also enhances the synthesis and storage of "dry matter (such as carbohydrates, proteins, etc.)".

[0068] Statistical analysis demonstrated that the differences between the treatment and control groups were not random fluctuations, but rather biological effects of the compound microbial agent treatment. The treatment group showed superior results in plant height (morphology), fresh weight (overall biomass), and dry weight (material storage), indicating that the compound microbial agent's growth-promoting effect on rice is systematic and multi-dimensional. After inoculation with the compound microbial agent, rice exhibited significant / extremely significant improvements in the three key growth traits of plant height, fresh weight, and dry weight, proving that this compound microbial agent has a clear growth-promoting effect on potted rice, providing a theoretical basis for subsequent field trials or microbial agent optimization.

[0069] The comparative experiment is as follows: Under the exact same experimental conditions as in Example 2, the inhibitory effects of each of the following groups on the germination of rice blast fungus spores were tested in parallel: For the Bacillus vesiculus single-strain group, only Bacillus vesiculus fermentation broth or its sterile supernatant was added.

[0070] For the single-strain group of Cryospora, only Cryospora fermentation broth or its sterile supernatant was added.

[0071] The germination of spores was observed and analyzed under a microscope, and the germination rate and germination inhibition rate of each group were calculated. Under the same potted plant conditions as in Example 2, the following experimental group was added: The Bacillus belysin monoculture treatment group was inoculated with only Bacillus belysin.

[0072] The Cryospora monoculture treatment group was inoculated with only Cryospora.

[0073] After 21 days of cultivation, key growth indicators such as plant height, fresh weight, and dry weight of each group were measured and recorded.

[0074] The results of spore germination inhibition rate and the increase rates of plant height and fresh weight are as follows: The compound microbial agent used in this invention has a strong inhibitory effect on the germination of *Magnapordica oryzae* spores, achieving a 100% inhibition rate within 48 hours. Both microorganisms possess the ability to degrade cellulose, thus having a fertilizing effect. The compound microbial agent also promotes rice growth, effectively increasing seedling height, fresh weight, and dry weight. In addition to inhibiting *Magnapordica oryzae*, the compound microbial agent also inhibits other plant pathogens, including but not limited to *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, and *Fusarium oxysporum*. The compound microbial agent exhibits high environmental compatibility and ecological safety, being harmless to humans and livestock. It effectively replaces chemical pesticides through biological control, avoiding the environmental pollution and residue risks associated with the latter; simultaneously, its growth-promoting function helps reduce fertilizer application, thereby saving farmers production costs.

Claims

1. A compound microbial agent, characterized in that, Its active ingredients include Bacillus belyssus 1B-10 with accession number CGMCC NO:33492 and Bacillus psychrophilus 8B-2 with accession number CGMCC NO:33493.

2. The compound microbial agent according to claim 1, characterized in that, The ratio of viable bacteria of Bacillus belyssus 1B-10 to Cryospora 8B-2 was 1:

1.

3. The compound microbial agent according to claim 1 or 2, characterized in that, The viable count of the *Bacillus belysinus* 1B-10 was 1 × 10⁻⁶. 9 -5×10 9 CFU / mL, the viable count of the *Cryptospira* 8B-2 was 1×10⁻⁶. 9 -5×10 9 CFU / mL.

4. A method for preparing the composite microbial agent according to any one of claims 1-3, characterized in that, The steps include: (1) performing shake-flask fermentation on Bacillus belye 1B-10 with preservation number CGMCC NO:33492 and Bacillus psychrophilus 8B-2 with preservation number CGMCC NO:33493 to obtain fermentation broth; (2) collecting the bacterial cells from step (1) by centrifugation and resuspending them with sterile water, and adjusting the bacterial cell concentrations of Bacillus belye 1B-10 and Bacillus psychrophilus 8B-2 to 1×10⁻⁶. 9 -5×10 9 CFU / mL; (3) Mix the two bacterial solutions obtained in step (2) at a volume ratio of 1:1 to obtain the composite microbial agent.

5. The preparation method according to claim 4, characterized in that, The fermentation culture medium used in step (1) is TSB medium, the fermentation temperature is 26-30℃, preferably 28℃, and the fermentation time is 40-56 hours, preferably 48 hours.

6. The use of the compound microbial agent according to any one of claims 1-3 in inhibiting the germination of rice blast fungus spores and / or controlling rice blast disease.

7. The application of the compound microbial agent according to any one of claims 1-3 in promoting rice growth.

8. The application according to claim 6 or 7, characterized in that, The compound microbial agent is diluted 50-100 times before application.

9. The application of the compound microbial agent according to any one of claims 1-3 in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi are selected from at least one of Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium oxysporum.

10. A method for preventing and controlling rice blast and promoting rice growth, characterized in that, The procedure includes the following steps: at the 3-leaf stage of rice, the compound microbial agent described in any one of claims 1-3 is diluted 50-100 times and then used for root irrigation.

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