Brevibacillus mat, microbial agent containing brevibacillus mat and application of brevibacillus mat

Abstract

The invention relates to the field of microorganisms and application, in particular to brevibacillus mat, a microbial agent containing the brevibacillus mat and application of the brevibacillus mat. The invention provides a brevibacillus sschleri strain KY987 which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the brevibacillus sschleri strain KY987 is CGMCC No. 37625. The strain not only has the activity of killing main pathogenic bacteria of soybean root rot, but also has broad-spectrum bactericidal activity on various plant pathogenic bacteria. In addition, the strain also has caenorhabditis elegans killing activity, is easy to grow, and is suitable for realizing large-scale industrial application.

Description

Technical Field

[0001] This application relates to the field of microbiology and its applications, specifically to a strain of *Bacillus schistosomiasis* (…). Brevibacillus schisleri ), Microbial agents containing it and their uses. Background Technology

[0002] Soybeans are a globally valuable dual-purpose crop, used for both food and oil, and are currently widely cultivated in more than 50 countries. Rich in high-quality protein, fat, various vitamins, and minerals, soybeans are a core source of vegetable oil and protein, and are widely used in edible oil processing and various food production (Horstmann J et al., 2020).

[0003] Soybean diseases are a key factor restricting high and stable soybean yields. Frequent outbreaks of various diseases caused by pathogens such as fungi, bacteria, nematodes, and viruses consistently result in significant yield reductions, severely impacting both quantity and quality (Jiang Chao, 2025). Among soybean diseases, soybean root rot, a prevalent soil-borne disease in soybean-producing areas, is considered one of the most threatening and devastating diseases in soybean production. Soybean root rot refers to a collective term for diseases affecting the roots and base of the stems of soybeans caused by multiple pathogens. The complexity of the pathogens presents a core challenge in the diagnosis and control of this disease (Ye Wenwu, 2020; Zhang FL, 2017). This disease primarily infects the roots and stem base of soybeans, affecting both seedlings and mature plants. Initially, small, irregularly shaped, light red lesions appear on the epidermis at the stem base. As the disease progresses, these lesions gradually turn reddish-brown and develop sunken necrosis. When the lesions encircle the stem base, the epidermal tissue dies, leading to poor root development and a significant reduction in the number of root nodules. Due to impaired root absorption, the above-ground parts of the plant exhibit stunted growth, pale green leaves, and ultimately, a sharp decrease in pod production and yield (Li Chengbai, 2023). This disease is extremely damaging; once it occurs, yields are typically reduced by 10%–30%, and in severely affected fields, losses can exceed 60%, or even result in total crop failure (Wang Shuang, 2023).

[0004] The core driving factor behind the high incidence and exacerbation of soybean root rot lies in the combined infection and synergistic pathogenic effects of multiple pathogens. Currently, there are four main pathogens widely recognized as causing soybean root rot, including Fusarium spp. (…). Fusarium spp. Phytophthora ( ) Phytophthora spp. ), Pythium genus ( Pythium spp. ) and Rhizoctonia solani ( Rhizoctonia solani(Li Leilei, 2024). Under natural field conditions, single pathogen infection is extremely rare. Fusarium, Phytophthora, Pythium, and Rhizoctonia solani often form complex infection systems, significantly increasing the probability and severity of disease occurrence through niche complementarity, synergistic infection processes, and interaction of pathogenic factors (Song Meiyan, 2021; Wang Shuchen, 2022; Zheng Peie, 2012; Yin Ling, 2013). The complex infection of these four pathogens is a crucial prerequisite for the difficulty in controlling soybean root rot, as they exhibit differentiated characteristics when infected individually. Under pure laboratory culture conditions, single infection of the four pathogens shows clear and specific pathogenic differentiation: Pythium (… Pythium The fungus *Phytophthora* prefers cool, humid soil conditions. Under suitable conditions, after infecting soybean roots, it forms brown, water-soaked lesions. As the disease progresses, the stems become thin and soft, the leaves turn yellow and wither, and eventually the plant collapses and dies (Yin Ling, 2013; PERINCHERRY L et al., 2019). Infection by *Phytophthora* fungi is highly dependent on high humidity. Their zoospores can move rapidly in the soil water film, targeting and infecting young root hairs and the epidermis at the base of the stem. After invading the host tissue, it causes rapid cell necrosis, and the diseased parts appear as dark brown, wet rot spots. This pathogen can survive in the soil for many years with diseased plant debris, and the probability of infection is significantly increased in fields with continuous cropping (Li Leilei et al., 2024; Wang Shuchen, 2022). Fusarium fungi are highly drought-resistant and can survive in the soil for more than 5 years as chlamydospores (NAEEM M et al., 2019; Gordon TR et al., 1997). They mainly invade plants through root wounds or lateral root tips, and after infecting the plant, they spread to the vascular tissue. The cell wall degrading enzymes they secrete destroy the host cell wall structure, and the Fusarium toxins they produce block the xylem vessels, hindering the transport of water and nutrients. The aboveground parts of the plant exhibit chronic wilting and yellowing of leaves, while longitudinal sectioning of the root system reveals obvious browning of the vascular bundles (PERINCHERRY L et al., 2019; RONCERO MI et al., 2000; ISLAM KT et al., 2017; Burgess LW et al., 2018; Ma Shumei, 2012). Rhizoctonia solani overwinters as mycelium or sclerotia in the soil or diseased plant debris. Under suitable conditions, it can directly invade the plant's stomata or wounds, harming the soybean throughout its entire growth cycle. Infection in seedlings leads to seed rot, browning and withering of the stem base, and the appearance of sunken, irregular, reddish-brown lesions. Infection in mature plants results in root rot, yellowing and stunting, and in severe cases, reduced seedling numbers and broken rows. The diseased parts are dry and lack mucus, a characteristic that contrasts sharply with the wet rot symptoms caused by Pythium and Phytophthora (YC Lin et al., 2022).

[0005] However, in natural disease outbreaks in the field, these pathogens rarely infect alone. They often infect in combination with *Pythium*-*Phytophthora* as pioneer infectors and *Fusarium*-*Rhizoctonia solani* as subsequent colonizers. The pioneer bacteria first disrupt the root epidermal barrier, secreting enzymes to degrade cell walls, while simultaneously altering the pH and nutrient composition of the root microenvironment, creating pathways for the subsequent bacteria to invade. The subsequent bacteria then invade the vascular bundles or deep tissues at the base of the stem through wounds, secreting toxins to further suppress the plant's defense response (Li Leilei et al., 2024; DONG C et al., 2019). Through niche complementarity, coordinated infection timing, and interaction of pathogenic factors, they form a stable and highly pathogenic complex infection system (Zhao Wei et al., 2018; HAO Y et al., 2022). Furthermore, there are reports indicating that *Fusarium* (…) To be cast Vitamin B6 produced by *Phytophthora indicum* can help *Phytophthora indicum* (soybean pustulosa) produce vitamin B6, which can help *Phytophthora indicum* (soybean pustulosa) (soybean pustulosa). Phytophthora sojae This circumvents soybean's disease resistance mechanisms, thereby enhancing its pathogenicity (WANG S et al., 2023). For growers, regardless of the combination of pathogens, the symptoms ultimately exhibited by the plants are highly similar—only typical root rot phenotypes such as reddish-brown sunken lesions at the stem base, brown and blackened roots, a sharp reduction in the number of root nodules, stunted plant growth, yellowing and chlorosis of leaves, sparse pod formation, and small, shriveled seeds can be observed. It is impossible to distinguish by the naked eye whether it is wet rot caused by Pythium, vascular bundle browning caused by Fusarium, or stem constriction caused by Rhizoctonia solani. This core contradiction between "pathogen diversity" and "symptom uniformity" directly leads to difficulties for farmers in carrying out targeted control. For example, if only metalaxyl-based agents are used to target Pythium, they cannot inhibit the damage caused by Fusarium and Rhizoctonia solani (Lou Tiancheng et al., 2021; Ma Shumei et al., 2015); if only carbendazim is used to control Fusarium, it is ineffective against Phytophthora; and blindly mixing multiple agents not only increases the cost of pesticides but may also reduce the efficacy due to pesticide antagonism, or even cause phytotoxicity (DODD SC et al., 2020; Li Changsong et al., 2018). Ultimately, control often fails due to incomplete or incorrect pesticide application, which is the core reason why soybean root rot is more difficult to control than diseases caused by a single pathogen.

[0006] The commonly used control methods at present have obvious limitations and are difficult to achieve efficient and sustainable control of soybean root rot: chemical control is an emergency measure with quick results, but it has the drawbacks of pesticide residue accumulation and destruction of beneficial soil microbial communities; and it is constrained by the complex infection characteristics of soybean root rot (Liu Zhanyun et al., 2025). The control spectrum of a single agent is narrow and cannot cover complex pathogens such as Pythium, Phytophthora, and Fusarium at the same time. Although the combination or continuous use of multiple agents can expand the control range, it is easy to aggravate pesticide residues, trigger antagonistic reactions between agents, and may also induce drug resistance mutations in pathogens (Dong Jiwei et al., 2024). Disease-resistant breeding is an economical and effective way to control soil-borne diseases, but its application in soybean root rot control is limited: Currently, no soybean germplasm resources have been discovered that are immune or highly resistant to this disease. Existing resistant varieties only exhibit partial resistance, and although the incidence rate in the field has decreased, complete control of the disease cannot be achieved. Furthermore, the resistance performance of resistant varieties is closely related to soil type, climate conditions, and cropping system in the planting area. The suitable varieties vary significantly across different production areas, resulting in poor universality and further increasing the difficulty of field application of disease-resistant breeding (Jiang et al., 2025; Yang Liu, 2022). Biological control has become a research hotspot due to its environmentally friendly, residue-free, and non-inducible resistance characteristics. However, most reported biocontrol strains target a single pathogen, and very few can simultaneously antagonize broad-spectrum biocontrol bacteria that infect complex fungal communities such as Pythium, Phytophthora, and Fusarium (Jiang et al., 2025; Chen et al., 2023).

[0007] Therefore, there is an urgent need to develop a new microbial strain that targets soybean root rot.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] This invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, this invention provides a strain of *Bacillus schistosomiasis* (…). Brevibacillus schisleri ), Microbial agents containing it and their uses.

[0010] In one aspect of the invention, a strain of *Bacillus schistosomiasis* (…) is provided. Brevibacillus scythes The strain KY987 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37625, and the deposit date is February 2, 2026. In one embodiment, the 16S rDNA sequence of the strain KY987 is shown in SEQ ID NO: 1.

[0011] This invention uses common soybean root rot pathogens (Fusarium solani and Rhizoctonia solani) as high-throughput screening indicator bacteria, and then conducts targeted screening for biocontrol microorganisms with fungicidal activity against other soybean root rot pathogens (Phytophthora soysarum and Pseudomonas aeruginosa). Then, considering production issues and subsequent application scenarios, the selected strains were further tested for growth rate, broad-spectrum anti-plant disease pathogen activity, and activity against Caenorhabditis elegans nematodes. Finally, a readily growing biocontrol microorganism strain, KY987, was obtained, capable of killing the main pathogen of soybean root rot, multiple crop disease pathogens, and Caenorhabditis elegans nematodes. Based on the 16sDNA sequence determination of strain KY987, and sequence alignment with the 16sRNA database (Chun's Lab) recognized by the International Committee for Bacteriology, combined with literature analysis, the taxonomic position of the target microorganism was determined. The alignment results indicate that this strain is identified as… Brevibacillus schisleri Its Chinese name is Bacillus schistosomiasis.

[0012] In another aspect, the present invention provides a microbial agent with both bactericidal and insecticidal functions, said microbial agent containing the aforementioned Bacillus schistosomiasis (…). Brevibacillus schisleri ) strain KY987, or containing the aforementioned Bacillus schistosomiasis ( Brevibacillus schisleri Fermentation products obtained from the fermentation of strain KY987.

[0013] In another aspect of the invention, the invention provides the use of the aforementioned Bacillus schistosomiasis strain KY987 or microbial agents containing it in the prevention and control of plant diseases.

[0014] In one embodiment, the plant disease includes one or more of the following: soybean root rot, cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, apple leaf spot, pear black spot, and tomato early blight.

[0015] In one embodiment, the soybean root rot is caused by Phytophthora soybeanum (… Phytophthora sojae Fusarium solani () Fusarium spp. ), large tofu mold ( Pythium spp. ) and Rhizoctonia solani ( Rhizoctonia nightshade It is caused by a single infection or multiple combined infections in one of the following.

[0016] In another aspect of the invention, the present invention provides the aforementioned *Bacillus schistosomiasis* (… Brevibacillus scythes The use of strain KY987 or microbial agents containing it in the control of plant nematodes.

[0017] In one embodiment, the plant nematode is selected from one or more of the following: Caenorhabditis elegans, soybean root-knot nematode, and soybean cyst nematode.

[0018] In another aspect, the present invention provides a method for simultaneously preventing and controlling plant diseases and plant pests, the method comprising administering the aforementioned *Bacillus schistosomiasis* (… Brevibacillus schisleri The strain KY987 or the aforementioned microbial inoculant was applied to the plant.

[0019] In one embodiment, the plant disease includes one or more of the following: soybean root rot, cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, apple leaf spot, pear black spot, and tomato early blight; the plant pest is soybean pathogenic nematode.

[0020] Preservation information: The *Bacillus schistosomiasis* provided in this application (… Brevibacillus schisleri The strain, named KY987, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is February 2, 2026, and the accession number is CGMCC No. 37625. It was confirmed as a viable strain by the collection center on February 2, 2026.

[0021] Beneficial effects Previously reported biocontrol strains often target a single pathogen. However, the *Bacillus schistosomiasis* strain KY987 isolated and screened in this invention exhibits fungicidal activity against major soybean pathogens, particularly *Phytophthora soybean*, *Fusarium soybean*, *Cryptospira spp.*, and *Rhizoctonia solani*. Furthermore, this strain shows antagonistic activity against various plant pathogenic fungi and insecticidal activity against soybean pathogenic nematodes (especially *C. elegans*). Therefore, this strain possesses dual efficacy in disease prevention and insect control, which is of great significance for the prevention and control of plant diseases, especially soybean diseases. The strain of this invention is a novel biocontrol strain resource with excellent comprehensive functions.

[0022] The method for simultaneously controlling plant diseases and plant pests provided by this invention offers a new approach to the control of fungal diseases and pests in plants. Furthermore, the strain KY987 described in this application can be used simultaneously to control plant pathogens and pathogenic nematodes with high efficiency. The method of this invention is simple and easy to promote and apply.

[0023] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The microorganisms that kill Fusarium solani or Rhizoctonia solani obtained by high-throughput screening are shown in the boxes in the figure (the radius of the inhibition zone produced by the microorganisms in the boxes is ≥1 mm). Figure 2 The microorganisms that kill pathogens associated with soybean root rot are shown. Four microorganisms in the figure can produce inhibition zones, and the radius of the inhibition zones is ≥1 mm. Figure 3 The image shows KY987 under a microscope (100x oil immersion). Figure 4 The results of the broad-spectrum fungicidal activity test of KY987 and the commercial strain Bacillus belyi are shown (Note: In order, the pathogens of soybean root rot are 4: Fusarium solani, Rhizoctonia solani, Phytophthora spp., and 12 major plant diseases: cotton wilt, watermelon wilt, banana wilt type 1, banana wilt type 2, banana wilt type 3, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, apple leaf spot, pear black spot, and tomato early blight). Figure 5 The growth, reproduction, and morphological response of Caenorhabditis elegans under treatment with three microorganisms in R2A medium are shown in the figure (the worms that are not easy to observe are specially marked, the red circles are dead worms, and the red squares are live worms).

[0025] Figure 6 The growth, reproduction, and morphological response of *C. elegans* treated with three microorganisms in NA medium are shown in the figure. (In the figure, worms that are not easily observed are specially marked. Dead worms are marked in red circles and red arrows, and live worms are marked in red boxes.) Figure 7 The growth, reproduction, and morphological response of *C. elegans* treated with three microorganisms in LB medium are shown in the figure. (Insecticidal worms that are difficult to observe are specially marked; dead worms are inside red circles, and live worms are inside red squares.) Figure 8 The growth, reproduction, and morphological response of *C. elegans* under treatment with three microorganisms in production medium 76 are shown in the figure. (In the figure, worms that are not easily observed have been specially marked; the red circles indicate dead worms, and the red squares indicate live worms.) Detailed Implementation The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] This invention proposes a strain of Bacillus schistosomiasis (Schilleria brevis) Brevibacillus schisleriThe following sections will describe in detail the microbial agents containing these agents and their uses.

[0027] In a first aspect, the present invention provides a strain of *Bacillus schistosomiasis* (… Brevibacillus scythes The strain KY987 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37625, and the deposit date was February 2, 2026. This strain is... Brevibacillus schisleri Its Chinese name is Bacillus schistosomiasis.

[0028] This invention reveals that this strain possesses both anti-plant pathogenic and insecticidal activities. Specifically, it exhibits fungicidal activity against four pathogens associated with soybean root rot (Phytophthora soybean, Fusarium, Pythium, and Rhizoctonia solani), and broad-spectrum fungicidal activity against 12 plant pathogens. Furthermore, its fungicidal activity surpasses that of Bacillus vesalivarius, a commercially recognized fungicide. In addition, this strain demonstrates excellent growth characteristics, with a growth rate exceeding that of the commercial control Bacillus subtilis 92068, indicating rapid propagation and facilitating widespread application. Moreover, this strain exhibits activity against *C. elegans* nematodes under various culture conditions, and its activity is stronger than that of *Bacillus vesalivarius* reported in the literature. Therefore, this strain is a soybean root rot-specific control strain that combines anti-*C. elegans* nematode activity, broad-spectrum crop pathogenic activity, and easy growth, demonstrating superior overall performance.

[0029] The 16S rDNA gene sequence of strain KY987 described in this invention is shown in SEQ ID NO:1. This application covers strains that have ≥95%, preferably ≥99.5%, sequence identity with SEQ ID NO:1.

[0030] Bacillus schistosomiasis ( Brevibacillus schisleri The type strain (ATCC 35690T = LMG17055T) was originally isolated from US soil and named by Johnson & Dunlap in 2019 (Johnson, Eric T., 2019). This strain was initially classified under the genus *Brucea*. Brevibacillus (This was later confirmed as a new species through phylogenetic genome analysis. It has been reported that...) Brevibacillus schisleri A key strain capable of efficiently degrading algal toxins has been developed for use in cyanobacterial composting (Wu Hainan, 2026). It can also degrade OTA and inhibit the growth of OTA-producing fungi. Its fermentation broth effectively inhibits the growth of OTA-producing fungi such as *Aspergillus niger*, *Aspergillus anthocyanin*, *Penicillium*, and *Aspergillus ochraceus*, forming a significant inhibition zone (Sun Changpo, 2026). This strain is eco-friendly and has enormous development potential. This application uses the screened *Bacillus schistosomiasis* (… Brevibacillus scythes Using KY987 as the research subject, subsequent multifaceted studies were conducted.

[0031] In a second aspect, the present invention provides a microbial agent with both bactericidal and insecticidal functions, said microbial agent containing the aforementioned Bacillus schistosomiasis (… Brevibacillus schisleri ) strain KY987, or containing the aforementioned Bacillus schistosomiasis ( Brevibacillus schisleri Fermentation products obtained from the fermentation of strain KY987.

[0032] In a specific implementation scheme, the microbial agent can be a solid agent or a liquid agent; when the microbial agent is a solid agent, the *Bacillus schistosomiasis* contained in the microbial agent ( Brevibacillus scythes The total viable count of strain KY987 is at least 1 × 10⁻⁶. -6 cfu·g -1 When the microbial agent is a liquid agent, the total viable count of *Brevibacillus schisleri* strain KY987 contained in the microbial agent is at least 1 × 10⁻⁶. -6 cfu·mL -1 .

[0033] In a specific implementation, the fermentation product obtained from the fermentation of strain KY987 can be a live bacterial preparation, freeze-dried bacterial powder, spore suspension, fermentation broth, fermentation supernatant, or inactivated bacterial cells. The fermentation product of strain KY987 can be obtained using conventional bacterial fermentation methods in the art, for example, by inoculating strain KY987 into a fermentation medium for fermentation culture to obtain the fermentation broth.

[0034] In a specific implementation, the microbial agent may also be a biocontrol agent containing the aforementioned strain KY987. In addition, the microbial agent may be used as a biofertilizer, etc. In a third aspect, the present invention provides the use of the *Bacillus schistosomiasis* strain KY987 or a microbial agent containing it in the control of plant diseases. This application has found that this strain is effective against *Fusarium solani* (soybean sclerosis fungus). Fusarium spp. Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Pythium ( Pythium spp. ), Phytophthora indicum ( Phytophthora sojae ), kills multiple crop pathogens (including fungal plant diseases: cotton wilt) Fusarium oxysporum f. sp. Vasinfectum Fusarium oxysporum wilt specific type), watermelon wilt disease ( Fusarium oxysporum f. sp. Niveum, Fusarium oxysporum (watermelon-specific type), three transformation types of banana wilt (… Fusarium oxysporum f. sp. Cubense,Fusarium oxysporum Cuban variant), wheat root rot ( Cochliobolus sativus, Cyclospora gracilis), rice sheath blight (Rhizoctonia solani) Rhizoctonia solani Kü h Wheat sheath blight ( Rhizoctonia cereale Rhizoctonia granatum), gray mold ( Botrytis cinerea Botrytis cinerea, early blight of tomato ( Alternaria solani, Alternaria alternata), pear black spot disease ( Alternaria alternata Alternaria, which only infects pear trees, and apple leaf spot disease (Alternaria alternata, which only infects pear trees). Alternaria alternata, Alternaria (which infects only apples) has activity against *Alternaria alternata*, and this strain also has anti-*C. elegans* (…). Caenorhabditis elegans The activity of this strain is reported for the first time in the world.

[0035] In this application, "controlling plant diseases" refers to the effect of strain KY987 in controlling plant diseases by inhibiting or killing pathogenic microorganisms that cause plant diseases. Therefore, this application also provides the application of strain KY987 or microbial agents containing it in inhibiting the growth of pathogens causing the aforementioned plant diseases. For example, strain KY987 can be prepared as a broad-spectrum fungicide or as a plant pathogen inhibitor.

[0036] In one embodiment, the plant disease includes one or more of the following: soybean root rot, cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, apple leaf spot, pear black spot, and tomato early blight.

[0037] In one embodiment, the soybean root rot is caused by Phytophthora soybeanum (… Phytophthora sojae Fusarium solani () Fusarium spp. ), large tofu mold ( Pythium spp. ) and Rhizoctonia solani ( Rhizoctonia nightshade It is caused by a single infection or multiple combined infections in one of the following.

[0038] In one specific embodiment, the present invention provides the use of the *Bacillus schistosomiasis* strain KY987 or a microbial agent containing it in the inhibition of plant pathogens. The plant pathogen is one that causes the aforementioned plant diseases. The plant is preferably soybean.

[0039] In a fourth aspect, the present invention provides the use of the aforementioned Bacillus schistosomiasis strain KY987 or microbial agents containing it in the control of plant nematodes.

[0040] In one embodiment, the plant nematode is selected from one or more of the following: Caenorhabditis elegans, soybean root-knot nematode, and soybean cyst nematode; preferably Caenorhabditis elegans.

[0041] In one embodiment, the strain KY987 of this application resulted in a mortality rate of over 80% for *C. elegans* under various culture conditions, and can be used in the preparation of nematicide formulations.

[0042] In a fifth aspect, the present invention provides a method for simultaneously preventing and controlling plant diseases and plant pests, the method comprising administering the aforementioned *Bacillus schistosomiasis* (… Brevibacillus schisleri The method involves applying strain KY987 or a microbial agent containing it to the plant during the cultivation of the plant (e.g., soybean).

[0043] In one embodiment, the plant diseases include one or more of the following: soybean root rot, cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, apple leaf spot, pear black spot, and tomato early blight; the plant pests are soybean pathogenic nematodes, especially *C. elegans*.

[0044] In one embodiment, the present invention provides the use of the said strain or a microbial agent containing it in the killing or control of target pests (pathogenic nematodes). In a specific embodiment, the lethality against *C. elegans* reaches more than 80%, enabling its application in the preparation of nematicidal formulations.

[0045] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0046] Example 1: Obtaining microorganisms that kill soybean root rot pathogens Soybean root rot is a common soil-borne disease in soybean producing areas across China, affecting soybeans throughout their entire growth cycle and causing yield losses exceeding 30% in severe cases. Literature reports a diverse range of pathogens, with key pathogens including *Phytophthora sojae*, *Fusarium sojae*, *Pythium sojae*, and *Rhizoctonia solani*. Outbreaks are often caused by a "combined infection" of these pathogens, rather than a single species. This combined infection not only renders single control measures ineffective but also makes it difficult to identify the specific pathogen based on appearance, as the disease ultimately presents with uniform symptoms of root rot, stunted growth, and yellowing and wilting leaves. To address this problem hindering the development of the soybean industry, this application comprehensively analyzes multiple typical diseased plant samples from soybean-producing fields in Dayangshu Town, Oroqen Autonomous Banner, Hulunbuir City, Inner Mongolia Autonomous Region. The results show that the aforementioned fungal pathogens reported in the literature can be isolated. Therefore, this invention utilizes the aforementioned pathogens (*Phytophthora sojae*, *Phytophthora sojae*, *Fusarium sojae*, *Phytophthora ... Phytophthora sojae Fusarium solani () Fusarium spp. ), Pythium ( Pythium spp. Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani As indicator bacteria, microorganisms that kill soybean root rot pathogens were screened and obtained. The specific operation is as follows: 1.1 High-throughput acquisition of microorganisms capable of killing Fusarium solani and Rhizoctonia solani Rhizoctonia solani and Fusarium oxysporum are the two main pathogens causing soybean root rot, a complex disease. They are closely related: Rhizoctonia solani primarily damages seedlings, infecting young stems and roots to cause damping-off and wilt, creating conditions for subsequent pathogen invasion; while Fusarium oxysporum damages throughout the entire growth period, especially in mature plants, causing not only root rot but also destroying the vascular bundles within the plant, resulting in typical wilting symptoms. In the field, these two pathogens often co-infect and work synergistically, forming a complex "root rot complex" along with other soil pathogens such as Pythium and Phytophthora, leading to root rot, wilting, and death in soybeans from seedling to mature stages, ultimately causing severe yield losses.

[0047] In view of the harm caused by Fusarium solani and Rhizoctonia solani to soybean production, this invention identifies Fusarium solani and Rhizoctonia solani as indicator strains, designs and establishes a targeted high-throughput screening method to obtain the target biocontrol microorganisms. The specific steps are as follows: 1.1.1 Sample Collection Soil sampling: A total of 82 soil samples were collected from all over the country, including various types of soil such as black soil, clay, and red soil, from forests, grasslands, wheat fields, and paddy fields. Each soil sample was labeled with its collection location (province, city, county), collection time, and collection source (forest, grassland, wheat field, paddy field, etc.).

[0048] 1.1.2 Obtaining Rhizoctonia solani and Fusarium solani 1.1.2.1 Obtaining Rhizoctonia solani A strain of Rhizoctonia solani (Latin name: Rhizoctonia solani) was obtained from China Agricultural University. Rhizoctonia solani Alternatively, other commercially available Rhizoctonia solani strains can be used for testing.

[0049] 1.1.2.2 Obtaining Fusarium solani (1) Samples: Soybean plant samples exhibiting typical root rot symptoms were collected from soybean fields in Dayangshu Town, Oroqen Autonomous Banner, Hulunbuir City, Inner Mongolia Autonomous Region. Parts with obvious lesion characteristics, such as diseased roots, were selected for subsequent pathogen isolation and identification. (2) Sample pretreatment: excise tissue from the junction of diseased and healthy tissue; (3) Surface disinfection (key: kill surface bacteria and protect internal pathogens): use a 95% alcohol cotton ball to evenly coat the tissue surface with 95% alcohol, and sterilize it by passing it over an alcohol lamp; (4) Cut the disinfected tissue into small pieces with sterile scissors, place it in a sterile mortar and grind it with sterile water; (5) Inoculation and culture: Dip an inoculation loop into the bacterial solution and streak it on a PDA medium containing chloramphenicol. Incubate at 30°C for 3-5 days and observe the colony growth. (6) Purification of strains: When white, pink or purple fluffy colonies (typical colony morphology of Fusarium) appear on the culture medium, use a sterile inoculation loop to pick up the hyphae on the edge of a single colony and transfer them to a new chloramphenicol-containing PDA medium. Repeat the transfer 1-2 times until a strain with uniform colony morphology and no contaminants is obtained. (7) Strain identification: 1. Morphological identification: Observe the colony color and hyphal morphology, pick hyphae to prepare slides, and observe the large conidia (falcate, multiseptate) and small conidia (elliptical, single-septate or unseptate) unique to Fusarium under a microscope. 2. Molecular identification: DNA was extracted from the strain, and the ITS sequence (internal transcribed spacer region) was amplified and compared with known Fusarium species of soybean (such as Fusarium oxysporum). F. oxysporum Fusarium solani F.solani Sequence alignment was performed to identify the species.

[0050] 1.1.3 High-throughput method for obtaining microorganisms with the function of killing Rhizoctonia solani and Fusarium solani. (1) Take 5 soil samples (0.2 g of each soil sample), mix them and place them in 50 mL of sterile water. After shaking well, take a small amount of the liquid and dilute it 10 times, 100 times and 1000 times respectively. Take 100 μL of each and spread it evenly on R2A solid medium. Incubate at 30℃ for 2-4 days. (The solid R2A medium is prepared as follows: 0.5 g peptone, 0.5 g yeast, 0.5 g tryptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate, 1000 mL H2O, 15 g agar. Autoclave at 121.0℃ for 30 min.) (2) Pick the colonies from the culture dish and transfer them to a 96-well plate containing R2A solid medium and incubate at 30°C for 2 days.

[0051] (3) Scrape the mycelium of Fusarium solani and place it in sterile water. Shake well to make a bacterial suspension. Then take a small amount of bacterial suspension and spread it evenly on R2A solid medium to make selective medium A for later use.

[0052] (4) Scrape the sclerotia of Rhizoctonia solani and place them in a sterile mortar. Grind the sclerotia into powder and add sterile water to make a bacterial suspension. Then take a small amount of bacterial suspension and spread it evenly on R2A solid medium to make selective medium B for later use.

[0053] (5) Using a sterilized microplate replicator (96 wells), collect microorganisms from the 96-well microplates and sequentially transfer them to selective medium A and selective medium B. Incubate at 30°C for 2-3 days and observe the growth of microorganisms and the formation of inhibition zones. If inhibition zones are formed around the colonies, it indicates that the microorganisms have antifungal activity. Specific phenomena are as follows: Figure 1 As shown. Microorganisms that produce inhibition zones with a radius ≥1 mm were selected and purified by streaking on R2A solid medium to obtain microbial strains with the function of killing Phytophthora soybeanis.

[0054] 1.1.4 Duplicate Validation The obtained microbial strains with the function of killing soybean Phytophthora are inoculated again on selective medium A and selective medium B to eliminate false positive microorganisms that cannot produce inhibition zones on specific mediums, and finally obtain microorganisms with the function of killing soybean Fusarium or Rhizoctonia solani.

[0055] 1.1.5 Results A fungus was isolated from soybean plants with rotten roots collected from a soybean field in Dayangshu Town, Oroqen Autonomous Banner, Hulunbuir City, Inner Mongolia Autonomous Region. The morphology of this fungus was observed (results are shown in Table 1), and its DNA was analyzed. The ITS sequence or coxII gene was amplified and compared with known sequences, confirming its identity as a fungus. Fusarium sp.(Fusarium) This strain can infect soybeans and cause soybean root rot. Note that there are no restrictions on the Fusarium strain used for testing; other commercially available Fusarium strains can also be used.

[0056] Eighty-two soil samples (nearly 27,800 microorganisms) were screened using high-throughput microbial methods, resulting in 666 microorganisms with the ability to kill either Fusarium solani or Rhizoctonia solani. The classification of these functional microorganisms is shown in Table 2. 312 strains were found to be effective against Fusarium solani, and 354 strains were effective against Rhizoctonia solani. Among these, 89 strains were effective against both Fusarium solani and Rhizoctonia solani. To screen for target microorganisms with better control effects against soybean root rot caused by multiple pathogens, this invention prioritizes microbial strains with the ability to kill both Fusarium solani and Rhizoctonia solani for further research.

[0057] Table 1. Morphological description of fungi obtained from soybean root rot plants

[0058] Table 2. Statistical table of microorganisms verified to have the ability to kill Fusarium solani or Rhizoctonia solani.

[0059] 1.2 Obtaining microorganisms with the function of killing soybean root rot pathogens Soybean root rot is a soil-borne disease caused by the combined infection of multiple pathogens. Several major known pathogens exist, such as Fusarium, Rhizoctonia solani, Phytophthora soybeanii, and Pythium. This invention provides new scientific evidence for the complex etiology of soybean root rot. To obtain biocontrol microorganisms that can inhibit the major pathogens of soybean root rot, this experiment used the aforementioned pathogens (Phytophthora soybeanii, Fusarium soybeanii, Pythium, and Rhizoctonia solani) as indicator fungi, conducting confrontation and inhibition tests. The specific procedures are as follows: 1.2.1 Obtaining pathogens from soybean plants with root rot 1.2.1.1 Obtaining the mold for large tofu (1) Samples: Soybean plants with root rot were collected from soybean fields in Dayangshu Town, Oroqen Autonomous Banner, Hulunbuir City, Inner Mongolia Autonomous Region, and parts of soybean plants with typical symptoms were selected. (2) Cut the sterilized tissue into 2-3 pieces with sterile scissors and place them evenly on PDA medium containing chloramphenicol; (3) Invert the petri dish and place it in a constant temperature incubator at 20-23℃ (the optimal temperature for Pythium; growth is inhibited above 28℃). Incubate in the dark for 1-2 days and observe colony growth daily.

[0060] (4) Purification of strains 1. Typical colony characteristics of Pythium: Initially, it is white, transparent, cottony or fluffy, and may turn grayish-white later. The colony spreads quickly (faster than Fusarium) and has no obvious pigment (distinguishing it from the pink / purple colonies of Fusarium). 2. Using a sterile inoculation loop, pick up the hyphae from the edge of a single colony and transfer them to a fresh chloramphenicol-containing PDA medium. Incubate at 20-23°C for 3-5 days. 3. Repeat the transfer 1-2 times until a pure strain with uniform colony morphology and no contaminating bacteria (no colonies of other colors, no mucus-like bacterial colonies) is obtained.

[0061] (5) Strain confirmation: 1. Morphological observation: Typical colony characteristics of Pythium: Initially, it is white, transparent, cottony or fluffy, and may turn grayish-white later. The colony spreads quickly (faster than Fusarium) and has no obvious pigment (distinguishing it from the pink / purple colonies of Fusarium). It can be preliminarily identified.

[0062] 2. Molecular identification: Extract DNA from the strain, amplify the ITS sequence or coxII gene, and compare it with the sequences of known *C. tomentosa* species to accurately determine the species. This invention does not limit the type of *C. tomentosa* used; commercially available *C. tomentosa* species can also be used for testing.

[0063] 1.2.1.2 Obtaining Phytophthora soybeanense A strain of the pathogen causing soybean blight was obtained from China Agricultural University. It is named *Phytophthora sojae* (Latin: *Phytophthora sojae*). Phytophthora sojae This invention does not restrict the use of Phytophthora soybeani, and commercially available Phytophthora soybeani can also be used for testing.

[0064] 1.2.2 Obtaining biocontrol microorganisms that kill the pathogenic fungus of soybean root rot *Phytophthora indicum* and *Phytophthora indicum* were used as indicator strains. The 89 microorganisms verified to possess fungicidal activity against *Fusarium solani* and *Rhizoctonia solani* in section 1.1.5 were used as test strains. Indicator strain mycelial discs were inoculated in the center of R2A solid medium, and test strains were inoculated simultaneously at four points 3 cm away from the mycelial discs in four directions. The medium was incubated at 30°C for 8 days. After incubation, microbial growth and inhibition zone formation were observed. The formation of inhibition zones around the colonies indicated that the microorganism possessed fungicidal activity. Specific phenomena were observed as follows: Figure 2 As shown. Microorganisms that produce inhibition zones with a radius ≥1 mm were selected and purified by streaking on R2A solid medium to obtain microbial strains with the function of killing soybean root rot pathogens.

[0065] 1.2.3 Results 1.2.3.1 Identification results of pathogens obtained from soybean plants with root rot A fungus was isolated from soybean plants with rotten roots collected from a soybean field in Dayangshu Town, Oroqen Autonomous Banner, Hulunbuir City, Inner Mongolia Autonomous Region (commercially available Pythium mold can also be used for testing). The morphology of this fungus was observed (results are shown in Table 3), and its DNA was extracted. The ITS sequence or coxII gene was amplified and compared with known sequences to confirm its identity. for Phytopythium helicoides ; Phytopythium helicoides ( *Phytophthora*, originally classified as a genus of *Pythium* ( Pythium However, later taxonomic revisions classified it as a separate genus, Phytophthora. Phytopythium It is a plant pathogenic oomycete that mainly infects crop roots, causing root rot and damping-off. It can infect soybeans, mainly causing root rot. It often co-infects with other soil-borne pathogens (such as Fusarium), which aggravates the damage.

[0066] Table 3. Morphological description of fungi obtained from soybean root rot plants

[0067] (The figure shows that 4 microorganisms can produce inhibition zones, and the radius of each inhibition zone is ≥1mm) 1.2.4. Obtain the results of biocontrol microbial experiments that can comprehensively inhibit various pathogens causing soybean root rot. Confrontation and inhibition tests were conducted on 89 microorganisms with verified fungicidal activity against Fusarium solani and Rhizoctonia solani (targeting Fusarium solani, Rhizoctonia solani, Pythium, Phytophthora spp., and newly isolated pathogenic bacteria). The results are shown in Table 4. Among the 89 microorganisms with verified fungicidal activity against Fusarium solani and Rhizoctonia solani, 62 strains were effective against Pythium spp., and 21 strains were effective against Pythium spp.; moreover, 8 strains showed fungicidal activity against all four pathogens and could inhibit the main pathogens of soybean root rot. These 8 microorganisms meet the criteria for being the target microorganisms of this invention (microorganisms for controlling soybean root rot). Therefore, these 8 microorganisms with verified activity against soybean root rot pathogens were selected for further verification.

[0068] Table 4. Statistical table of microorganisms verified to have the ability to kill pathogens causing soybean root rot.

[0069] Example 2. Obtaining easily growing biocontrol microorganisms that can prevent and control soybean root rot Easy growth is a core prerequisite for the industrial application of biocontrol microorganisms. These strains can complete high-density fermentation using inexpensive basal culture media, significantly reducing the raw material and equipment costs for large-scale production. Their short reproduction cycle significantly improves fermentation efficiency, shortens product preparation cycles, and meets the production capacity requirements of commercial production. Simultaneously, easily cultivated strains often exhibit strong environmental adaptability, resulting in more stable shelf-life inoculants and lower losses during storage, transportation, and field application. This allows for rapid transformation from laboratory screening to field application, providing economical and efficient technical support for the green control of soybean root rot. In biocontrol microorganism research, rapid reproduction is one of the core characteristics of easy growth, manifested in the short generation time and rapid biomass accumulation of strains under suitable conditions. This reduces the time cost of large-scale fermentation and enables faster colonization in the crop rhizosphere to exert biocontrol effects. Therefore, this invention tested the rapid reproduction of the eight biocontrol microorganisms obtained in "1.2.4" that can control soybean root rot, selecting the commercially recognized easy-growing strain—Bacillus subtilis—as a control. The specific operation and results are as follows: 2.1 Rapid reproductive capacity assessment (1) Bacillus subtilis was set as the control strain, and the above 8 biocontrol microorganisms that can prevent soybean root rot were used as test strains; (2) Scrape the activated test strain and control strain into sterile water to prepare a bacterial suspension, inoculate it into R2A liquid medium and allow the inoculated liquid OD to rise. 600nm= Incubate at 0.05 g, 30°C, and 200 rpm for 48 hours; (Liquid R2A medium is prepared as follows: 0.5 g peptone, 0.5 g yeast, 0.5 g tryptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate, 1000 mL H2O, autoclave at 121.0°C for 30 min.) (3) Take samples after the culture is completed and measure the OD. 600nm Enter the information in Table 5; 2.2 Results of rapid reproductive performance testing The rapid reproduction capacity of eight biocontrol microorganisms effective against soybean root rot is shown in Table 5. Compared to the control strain Bacillus subtilis 92068, the OD values ​​of five microorganisms (565, 213-2, J208, 769, and J195-874) after 48 h of culture were significantly higher. 600nm The values ​​were all lower than those of the reference strain Bacillus subtilis 92068, indicating a lower growth rate; the OD values ​​of the three microorganisms (J320, J314, and KY987) during the same period were also lower. 600nm The values ​​were all higher than those of the reference strain Bacillus subtilis 92068, indicating good rapid reproduction characteristics.

[0070] Table 5. OD of Bacillus subtilis and 8 biocontrol microorganisms effective in controlling soybean root rot after 48 hours of culture. 600nm Value comparison

[0071] 2.3 Determination of growth rate by viable cell count method For the three strains exhibiting excellent rapid reproductive capacity, this study used viable cell counting to more accurately determine their growth rate in order to clarify their proliferation characteristics. The procedure is as follows: (1) Bacillus subtilis was set as the control strain, and the above three strains that showed excellent rapid reproduction ability were used as test strains; (2) Scrape the activated test strain and control strain into sterile water to make a bacterial suspension, inoculate it into the production fermentation liquid culture medium, and make the inoculated liquid OD 600nm =0.05, incubated at 30℃ and 200rpm for 48 hours; (3) After the culture period ends, take samples and perform serial dilutions, spread them on plates to complete the viable count, and fill the results in Table 6.

[0072] 2.4 Results of growth rate determination by viable cell count method Table 6 shows the viable cell counts of Bacillus subtilis 92068 and three strains exhibiting excellent rapid reproduction capabilities. Among them, KY987 achieved a viable cell count of 1.03 billion / mL after 48 hours of culture, significantly higher than that of Bacillus subtilis 92068, which is currently capable of large-scale production, demonstrating excellent growth characteristics and potential for large-scale production. Therefore, KY987 was selected as the subject of further research in this study.

[0073] Table 6. Comparison of viable cell counts of various strains under fermentation liquid culture medium conditions in production media

[0074] Example 3. Obtaining an easily culturable microorganism with broad-spectrum anti-pathogenicity against both crop pathogens and the main pathogen of soybean root rot. To explore the application scope of strain KY987 in the field of crop disease biological control and to screen easily culturable microbial resources that possess both broad-spectrum killing of crop pathogens and high-efficiency inhibition of the main pathogen of soybean root rot, a confrontation experiment was designed to test the inhibitory effect of strain KY987 on broad-spectrum crop pathogens. The specific operation steps are as follows: 3.1 Test for broad-spectrum anti-crop pathogens Twelve pathogens of diseases were selected as indicator strains: three specialized types of cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, tomato early blight, pear black spot, and apple leaf spot. The easily culturable microorganism KY987, which has been verified to have fungicidal activity against the pathogen of soybean root rot in section 2.4, was selected as the test strain. The test strain and each indicator strain were simultaneously inoculated onto R2A solid medium and cultured at 30℃ for 7 days. (The solid R2A medium was prepared as follows: 0.5 g peptone, 0.5 g yeast extract, 0.5 g tryptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate, 1000 mL H2O, 15 g agar, autoclaved at 121.0℃ for 30 min.) After cultivation, the antagonistic effect of strain KY987 on pathogenic fungi was observed. The width of the bactericidal zone (the width of the transparent inhibition zone between the colonies of the test strain and the indicator strain, with 0 mm recorded when there is no inhibition zone) and the radius of the test strain colony were measured and recorded using the cross-cross method. The fungicidal intensity (E) was then calculated based on the above indicators to evaluate the antagonistic ability of the test strain against each indicator pathogenic fungus (the larger the fungicidal intensity value, the stronger the bactericidal ability of the microorganism against the corresponding pathogenic fungus; the smaller the fungicidal intensity value, the weaker the bactericidal ability of the microorganism against the corresponding pathogenic fungus). The results are shown in Table 7.

[0075]

[0076] 3.2 Results A confrontation experiment was designed to test KY987, an easily cultivated microorganism with proven fungicidal activity against soybean root rot pathogens, against 12 major crop pathogens. The results showed that KY987 exhibited fungicidal activity greater than 0 against all 12 pathogens, demonstrating stable and excellent activity. This aligns with the target microorganism of this invention, making it an easily cultivated microorganism with broad-spectrum activity against both crop pathogens and all pathogens causing soybean root rot. KY987 possesses significant biocontrol potential and market application prospects; therefore, it has been designated as the patented strain for this invention and will be further studied.

[0077] Table 7. Results of the fungicidal activity of KY987 against 12 plant pathogens.

[0078] Example 4. Identification of strain KY987 4.1 Preparation of DNA Template Pick a purified single colony and place it at the bottom of an EP tube. Add 200 μL of 5% (w / v) BT-chelex 100 (prepared with distilled water and sterilized at 121℃ for 30 min). Boil in a water bath for 15 min, then quickly freeze at -20℃ or -80℃. Thaw at room temperature, centrifuge at 6000 rpm for 3 min, and use 2 μL of the supernatant as a template. Amplify the 16S gene according to the 16S amplification system. The sequence of primer 27F, SEQ ID NO:2, is 5'-AGAGTTTGATCCTGGCTCAG-3'; the sequence of primer 1492R, SEQ ID NO:3, is 5'-GGTTACCTTGTTACGACTT-3'.

[0079] 16S PCR amplification system: 25 μL Green taqMix 12.5 μL DDH2O 9.5 μL AE 0.5 μL 1492 R0.5 μL 27F 0.5 μL DNA template 2.0 uL Total: 25 μL PCR amplification program: Step 1: 95℃, 5 min; Step 2: 94℃, 1 min; 55℃, 1 min; 72℃, 1.5 min, 35 cycles; Step 3: 72℃, 10 min; 4℃, hold.

[0080] 4.2. 16S rDNA Sequencing Results of KY987 The 16S rDNA sequence determination result of strain KY987 is shown in SEQ ID NO:1 below:

[0081] Based on the obtained 16S rDNA sequence of KY987, homologous sequences were searched in GenBank and compared with each other. Simultaneously, sequence alignment was performed with the 16S RNA database recognized by the International Committee for Bacteriology, and combined with literature analysis to determine the taxonomic position of the target microorganism (Yoon, SH, Ha, SM, Kwon, S., Lim, J., Kim, Y., Seo, H. and Chun, J. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol.67:1613-1617). The results showed that the 1438-base sequence of this strain is similar to that of *Bacillus schistosomiasis* (…). Brevibacillus schisleri It has a high degree of homology, with a similarity of up to 99.36%, and has been deposited in the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37625.

[0082] Example 5. Observation of strain morphology 5.1 Operation The selected strains were inoculated onto R2A plates and incubated at 30°C for 2 days. The size, shape, color, gloss, viscosity, raised shape, transparency, edge characteristics, and presence or absence of spores of the colonies were observed.

[0083] 5.2 Results of Observation of Strains' Morphology Observation of strain KY987 ( Brevibacillus schisleri After culturing on R2A medium for 2 days, the colonies were yellowish-gray, opaque, flat, smooth, round, with serrated edges. Microscopic examination (100x oil immersion) revealed a cell length of approximately 1.53–2.00 μm. Figure 3 ).

[0084] Example 6. Comparison of KY987 and Bacillus belysinus Bacillus belesiensis ( Bacillus velezensisKY987 is currently a leading strain in the field of biocontrol, possessing broad-spectrum antibacterial activity and easy large-scale production, and is widely used as a reference strain for biocontrol function verification. To further clarify the biocontrol potential and application value of strain KY987, and in accordance with the research objectives of this study, a plate confrontation experiment was conducted, using *Bacillus belyssinus* as a control. The inhibition zone size and other indicators of KY987 against 16 crop pathogens were systematically compared, and the fungicidal intensity was calculated to quantitatively evaluate its fungicidal ability and biocontrol advantages. The *Bacillus belyssinus* strain used here is a conventional commercial strain. Specific procedures are as follows: 6.1 Comparison of the antimicrobial activity of KY987 and Bacillus belysin against plant pathogens Sixteen plant disease pathogens were selected as indicator strains, including four pathogens of soybean root rot (Phytophthora soybata, Fusarium wilt, Pythium spp., and Rhizoctonia solani), three specialized strains of cotton wilt, watermelon wilt, and banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, tomato early blight, pear black spot, and apple leaf spot. Bacillus berberis and KY987 were used as test strains. A confrontation experiment was conducted on R2A solid medium to test the fungicidal activity: the test strains and each indicator strain were simultaneously inoculated on R2A solid medium and cultured at 30°C (Pythium spp. cultured for 3 days, and the others for 7 days). After cultivation, the strength of the antagonistic effect of the strains against pathogenic fungi was observed. The width of the bactericidal zone (the width of the transparent inhibition zone between the colonies of the test strain and the indicator strain, with 0 mm recorded when there is no inhibition zone) and the radius of the test strain colony were measured and recorded using the cross-cross method. The fungicidal intensity (E) was then calculated based on the above indicators to evaluate the strength of the antagonistic ability of the test strains against each indicator pathogenic fungus (the larger the fungicidal intensity value, the stronger the bactericidal ability of the microorganism against the corresponding pathogenic fungus; the smaller the fungicidal intensity value, the weaker the bactericidal ability of the microorganism against the corresponding pathogenic fungus).

[0085]

[0086] 6.2 Results The broad-spectrum fungicidal activity of Bacillus belye, which is recognized in the market for its fungicidal activity, and the strain KY987 of this invention were tested to determine their activity against crop pathogens. The results are shown in Table 8. Figure 4 As shown in Table 8. Figure 4The results showed that biocontrol microorganism KY987 exhibited fungicidal activity against all 16 tested crop pathogens, with fungicidal intensity values ​​all greater than 0. Bacillus bellis, on the other hand, showed fungicidal activity against 14 of these pathogens, with corresponding fungicidal intensity values ​​greater than 0. The comparison revealed that KY987 has a significantly broader fungicidal spectrum than Bacillus bellis. Furthermore, KY987 demonstrated higher fungicidal intensity against all 16 crop pathogens compared to Bacillus bellis, indicating superior fungicidal activity. In conclusion, KY987 is a biocontrol microorganism with a broad fungicidal spectrum, excellent fungicidal activity, and is easily culturable, possessing significant potential for market development.

[0087] Table 8. Comparison of fungicidal activity of KY987 and Bacillus belye against 16 plant pathogenic fungi.

[0088] Example 7. Obtaining an easy-to-grow biocontrol microorganism that also kills Caenorhabditis elegans Nematodes are important pathogens in agricultural production, among which soybean root-knot nematodes and soybean cyst nematodes are particularly harmful to the soybean industry. They not only cause root malformation and impaired absorption function, but also trigger secondary pathogen infections, resulting in yield reductions of 20%-50%, and in severe cases, complete crop failure. Currently, screening biocontrol microorganisms with both broad-spectrum inhibitory ability against plant pathogens and highly effective nematode-killing activity is a key approach to solving the problem of green control of soybean nematode diseases and ensuring safe production in the soybean industry. *C. elegans* (a type of nematode) Caenorhabditis elegans Due to its clear genetic background, short lifespan (2-3 days), sensitivity to nematicidal active substances, and high homology with plant pathogenic nematodes in physiological metabolic pathways (such as nerve conduction and epidermal structure formation), it is a classic model organism for initial screening of nematicidal activity in biocontrol bacteria. Its test results can effectively provide a reference for subsequent activity verification against soybean pathogenic nematodes. Given that previous studies have confirmed that KY987 has broad-spectrum inhibitory effects against various pathogenic bacteria and fungi, this experiment used *C. elegans* as the test subject, setting up the strain KY987 as the treatment group, *Bacillus subtilis* as the negative control group, and *Bacillus belyss*, which has been reported to have nematicidal activity, as the positive control group. A specific activity test against *C. elegans* was conducted to clarify its nematicidal potential and lay the foundation for subsequent research on the control of soybean pathogenic nematodes. The specific operation is as follows: 7.1 Nematicidal Activity Test (1) Preparation before the experiment: Caenorhabditis elegans: N2 wild-type nematodes were inoculated into NA medium filled with Escherichia coli OP50 (as food for Caenorhabditis elegans) and cultured at 23℃ for 3-5 days to obtain synchronized L4 stage larvae (stable activity and sensitive to nematicides) for later use. (2) KY987 was set as the treatment group, Bacillus subtilis (92068) was set as the latent control group, and Bacillus vesiculosus was set as the positive control group. Each group was repeated 3 times. (3) KY987, Bacillus subtilis and Bacillus belye were inoculated onto solid culture media R2A, NA, LB and production medium 76 respectively and streaked, and incubated at 30°C for 48 hours. The preparation methods for solid R2A medium are as follows: 0.5 g peptone, 0.5 g yeast, 0.5 g tryptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate, 1000 mL H2O, 15 g agar, autoclaved at 121.0℃ for 30 min; the preparation methods for solid LB medium are as follows: 10.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000 mL H2O, 15 g agar, autoclaved at 121.0℃ for 30 min; the preparation methods for solid NA medium are as follows: 10.0 g peptone, 5.0 g sodium chloride, 3.0 g beef meal, 1000 mL H2O, 15 g agar, autoclaved at 121.0℃ for 30 min; the preparation method for production medium No. 76 is as follows: potato flour 10 10 g soybean meal extract, 0.5 g sodium chloride, 0.5 g calcium chloride dihydrate, 2 g magnesium sulfate, 0.3 g sodium pyruvate, 0.1 g ferrous sulfate, 1000 mL H2O, 15 g agar, autoclave at 121.0℃ for 30 min.

[0089] (4) Take the same volume of Caenorhabditis elegans worm cakes and place them on the plate cultured in “7.1 (3)” above; (5) Place the plates in a 23℃ constant temperature incubator and co-culture in the dark. Observe and record the results at 0h and 18h ​​of incubation. (6) Calculate and record the mortality rate; (7) Data processing and result determination: 1. The mean and standard deviation of mortality rates for each group were calculated using Excel, and a one-way ANOVA was performed to compare the significant differences between the treatment group and the control group and the positive control group (P < 0.05 was considered significant, and P < 0.01 was considered extremely significant). 2. Result determination: If the nematode mortality rate in the KY987 treatment group was significantly higher than that in the blank control group, and there was no significant difference (or a shorter mortality rate) compared with the positive control group, it indicates that KY987 has clear nematode-killing activity.

[0090] 7.2 Results Caenorhabditis elegans was used as the test subject. The strain KY987 described in this application was used as the treatment group, Bacillus subtilis as the negative control group, and Bacillus belye, which has been reported to have nematicidal activity, was used as the positive control group. A specific test was conducted to determine the nematicidal activity against Caenorhabditis elegans. The results are as follows: Figures 5 to 8 And as shown in Table 9.

[0091] (1) From the table Figures 5 to 8 As corroborated by the phenotypic evidence provided in Table 9, the following results were obtained: When testing the three strains (Bacillus subtilis, KY987, and Bacillus belyssus) for their effectiveness against *C. elegans*, at the start point (0h), the size, number, and viability of *C. elegans* introduced into all treatment groups were almost identical. After 18h of co-culture, differences emerged among the treatment groups, with particularly pronounced differences between strains. Treatment with *Bacillus subtilis* did not affect the viability or population size of *C. elegans*; the worms were numerous, exhibited continuous S-shaped body movements, crawled quickly, and turned flexibly. Under certain culture medium conditions (R2A, NA, LB), [further details needed]. The development of larvae into adults: After treatment with KY987, the vast majority of *C. elegans* died, their bodies became rigid, and under certain culture conditions (LB, production medium 76), the worms decomposed, with the remaining few worms showing significantly reduced vitality, slow movement, or even cessation. After treatment with *Bacillus belyssioides*, dead worms were observed only under certain culture conditions (LB, production medium 76). Under production medium 76, most *C. elegans* were dead, and a significant number of dead worms were also observed under LB medium. No dead worms were observed under R2A and NA medium conditions, and the surviving worm populations were large and agile. In summary, *Bacillus subtilis* is not harmful to *C. elegans*, *Bacillus belyssioides* exhibits certain *C. elegans*-killing activity under specific conditions, and KY987 exhibits *C. elegans*-killing activity under multiple conditions, with its activity being stronger than that of *Bacillus belyssioides* reported in the literature.

[0092] (2) According to the mortality statistics provided in Table 10, Bacillus subtilis showed a mortality rate of 0, indicating that it had no activity against C. elegans. Bacillus belyss could produce a mortality rate of over 40% under specific culture conditions (LB, production medium 76), while the mortality rate was 0 under R2A and NA culture conditions. Bacillus belyss has a certain activity against C. elegans, but it is subject to environmental regulation. KY987 caused a mortality rate of over 80% against C. elegans under all four culture conditions. KY987 has the activity against C. elegans and its activity is stronger than that of Bacillus belyss.

[0093] (3) The mortality rates of KY987 (treatment group), Bacillus subtilis (negative control), and Bacillus vesicularis (positive control) were statistically analyzed, and the differences between groups were assessed using one-way ANOVA (see Table 11). The results showed that there were extremely significant statistical differences in the mortality rates of *C. elegans* among the different strain treatment groups (P<0.01). Further pairwise comparisons between groups were performed using the least significant difference (LSD) method. The results showed that in R2A, NA, and LB media, the mortality rates of *C. elegans* in the KY987 and Bacillus subtilis treatment groups were extremely significant (P<0.01); the mortality rates of *C. elegans* in the KY987 and Bacillus vesicularis treatment groups were extremely significant (P<0.01); in production medium 76, the mortality rates of *C. elegans* in the KY987 and Bacillus subtilis treatment groups were extremely significant (P<0.01); there was no significant difference in the mortality rates of *C. elegans* in the KY987 and Bacillus vesicularis treatment groups (P=0.43).

[0094] In summary, KY987 exhibits significantly better killing effects against *C. elegans* than *Bacillus subtilis*, which has no effect on *C. elegans*. In R2A, NA, and LB media, KY987 shows significantly better killing effects against *C. elegans* than *Bacillus belye*, which has been reported to have killing effects against *C. elegans*. In production medium 76, the killing effect of KY987 against *C. elegans* is comparable to that of *Bacillus belye*, which has been reported to have killing effects against *C. elegans*, demonstrating outstanding application advantages.

[0095] Table 9. Effects of three microorganisms on the growth, reproduction, and behavior of *C. elegans* under different culture medium conditions.

[0096] Table 10. Mortality rate (%) of three strains of Caenorhabditis elegans after treatment under different culture medium conditions

[0097] Table 11. Statistical analysis of the killing effect (mortality rate) of three strains against Caenorhabditis elegans under four culture medium conditions.

[0098] Conclusion: This application used common soybean root rot pathogens (Fusarium and Rhizoctonia solani) as high-throughput screening indicator bacteria to selectively screen for biocontrol microorganisms with fungicidal activity against other soybean root rot pathogens (Phytophthora soysarum and Pyrophyte). Considering production needs and future application scenarios, the target strain underwent further testing including growth rate determination, broad-spectrum killing of crop pathogens, and activity against Caenorhabditis elegans. Ultimately, a readily growing biocontrol microorganism, KY987, was screened, capable of killing the main pathogen of soybean root rot, multiple crop pathogens, and Caenorhabditis elegans. 16S rDNA confirmation confirmed that strain KY987 of this application is... Brevibacillus schisleri The strain, known in Chinese as *Bacillus schistosomiasis*, exhibits good safety and demonstrates significant fungicidal effects against pathogens causing soybean root rot and various crop diseases. It also possesses excellent insecticidal activity against *C. elegans* nematode and exhibits easy growth characteristics, making it highly promising for market applications and of significant practical importance for promoting the healthy and sustainable development of the soybean industry.

[0099] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0100] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A strain of Bacillus schistosomiasis ( Brevibacillus schisleri strain KY987, characterized in that, The strain KY987 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37625, and the deposit date is February 2, 2026.

2. The *Bacillus schistosomiasis* brevis according to claim 1 (… Brevibacillus schisleri strain KY987, characterized in that, The 16S rDNA sequence of strain KY987 is shown in SEQ ID NO:

1.

3. A microbial agent with both bactericidal and insecticidal functions, characterized in that, The microbial agent contains *Bacillus schistosomiasis* as described in claim 1 or 2. Brevibacillus schisleri ) strain KY987, or containing *Bacillus schistosomiasis* as described in claim 1 or 2 ( Brevibacillus schisleri Fermentation products obtained by fermentation of strain KY987.

4. The *Bacillus schistosomiasis* brevis according to claim 1 or 2 (… Brevibacillus schisleri The use of strain KY987 or the microbial agent according to claim 3 in the prevention and control of plant diseases.

5. The use according to claim 4, characterized in that, The plant diseases include one or more of the following: Soybean root rot, cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, apple leaf spot, pear black spot, and tomato early blight.

6. The use according to claim 5, characterized in that, The soybean root rot is caused by Phytophthora soybean (… Phytophthora sojae Fusarium solani () Fusarium spp. ), large tofu mold ( Pythium spp. ) and Rhizoctonia solani ( Rhizoctonia solani It is caused by a single infection or multiple combined infections in one of the following.

7. The *Bacillus schistosomiasis* brevis according to claim 1 or claim 2 (… Brevibacillus schisleri The use of strain KY987 or the microbial agent according to claim 3 in the control of plant nematodes.

8. The use according to claim 7, wherein the plant nematode is selected from one or more of the following: Caenorhabditis elegans, soybean root-knot nematode, and soybean cyst nematode.

9. A method for simultaneously preventing and controlling plant diseases and plant pests, characterized in that, The method includes administering the *Bacillus schistosomiasis* as described in claim 1 or 2 (…). Brevibacillus schisleri The strain KY987 or the microbial agent of claim 3 is applied to the plant.

10. The method according to claim 9, characterized in that, The plant diseases include one or more of the following: soybean root rot, cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, apple leaf spot, pear black spot, and tomato early blight; the plant pests are soybean pathogenic nematodes.

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