Bacillus safensis and application thereof in preventing and treating plant diseases

Abstract

The application belongs to the field of agricultural microbial technology, and particularly relates to a plant endophytic Bacillus safensis and application thereof in plant disease prevention and treatment. The plant endophytic Bacillus safensis has a strain number of BSA15, a classification name of Bacillus safensis Bacillus safensis , a preservation number of CGMCC No. 36637, and is preserved in the China General Microbiological Culture Collection Center on November 17, 2025. The Bacillus safensis has a broad-spectrum inhibitory activity on pathogenic fungi and oomycetes, and can produce a significant inhibitory effect on plant pathogenic microorganisms through multiple mechanisms. In addition, the Bacillus safensis BSA15 can be stably colonized in the plant body as an endophyte, further enhancing the disease prevention and control effect. Using the strain for disease prevention and treatment can significantly reduce the use amount of chemical pesticides, reduce pesticide residues and environmental pollution, and meet the requirements of green agriculture and sustainable development.

Description

Technical Field

[0001] This application belongs to the field of agricultural microbiology technology, and specifically relates to a plant endophytic Bacillus sabolicii and its application in the prevention and control of plant diseases. Background Technology

[0002] Plant diseases are a significant factor hindering the sustainable development of global agricultural production, especially soil-borne diseases caused by fungi and oomycetes, which are characterized by their insidious onset, rapid spread, and difficulty in control. Common plant diseases such as wilt, root rot, blight, bacterial blight, and gray mold not only lead to substantial crop yield reductions but also severely impact the quality and economic benefits of agricultural products. Currently, agricultural production still relies heavily on chemical pesticides for disease control, but the long-term excessive use of chemical agents has triggered a series of serious problems, including increased pathogen resistance, soil microecological imbalance, excessive pesticide residues, environmental pollution, and ecosystem damage. With increasing global emphasis on food safety, ecological protection, and sustainable agricultural development, the development of efficient, safe, and environmentally friendly biological control technologies has become an important direction for the field of plant protection.

[0003] Microbial fungicides, as an important component of biological control, have attracted widespread attention in research and application both domestically and internationally due to their advantages such as wide availability, diverse mechanisms of action, and good environmental compatibility. Bacillus spp. ( Bacillus Bacteria, due to their ability to produce heat- and dry-resistant spores, possess strong environmental adaptability and storage stability, giving them a significant advantage in the development of microbial pesticides. Among them, *Bacillus safranin* (… Bacillus safensis As a Gram-positive bacterium widely distributed in soil, plant rhizosphere, and extreme environments, *Bacillus sabdariffa* has been reported to possess various biological activities, including antagonizing plant pathogens, promoting plant growth, and enhancing plant stress resistance. Its antibacterial mechanisms mainly include secreting antimicrobial peptides (such as surfactants and iturobacillusin), cell wall degrading enzymes (chitinases and glucanases), competing for ecological niches and nutrients, and inducing systemic resistance in plants. *Bacillus sabdariffa* can not only survive in soil and rhizosphere but also colonize as an endophyte within plant tissues such as roots, stems, and leaves. By establishing stable colonization within the plant, it enhances the persistence and stability of pathogen inhibition. This endophytic characteristic gives it broader application potential in plant disease control.

[0004] However, significant differences exist among different strains of *Bacillus safranin* in terms of genetic background, metabolite composition, antimicrobial spectrum, and field efficacy. While some *Bacillus safranin* strains have been reported to inhibit certain plant pathogens, their effectiveness in practical applications is generally limited by a narrow inhibitory spectrum, unstable efficacy, and limited environmental adaptability. Furthermore, the colonization ability, persistence, and stress resistance of existing inoculants in complex field environments require further improvement.

[0005] Therefore, screening for new strains of Bacillus sabinatus with broad-spectrum, high-efficiency, and stable antibacterial activity, suitable for different crops and agricultural ecosystems, is key to promoting the industrialization and field application of this type of microbial fungicide. Summary of the Invention

[0006] This application, through systematic strain screening and identification, obtained a *Bacillus sabinatus* strain BSA15 with broad-spectrum antibacterial activity. This strain exhibits significant inhibitory effects against various important plant pathogenic fungi and oomycetes, and its fermentation broth also demonstrates stable antibacterial effects. Furthermore, it can effectively colonize within plants, providing endogenous protection for crops and possessing excellent development potential. This application aims to provide this strain and its application in plant disease control, offering new microbial resources and technical support for green crop control.

[0007] Firstly, this application provides a plant endophytic Bacillus saforticus strain, the strain numbered BSA15 and classified as Bacillus saforticus. Bacillus safensis The specimen, with accession number CGMCC No.36637, was deposited at the China General Microbiological Culture Collection Center on November 17, 2025.

[0008] Secondly, this application also provides a microbial bactericide containing the *Bacillus salsa* and / or a culture of the *Bacillus salsa* described in the first aspect.

[0009] Furthermore, the culture is a substance obtained by culturing the *Bacillus sarfusca* in a microbial culture medium.

[0010] In this application, "culture" refers to a general term for liquid or solid products (i.e., fermentation products) that have grown microbial communities after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms or contain a certain amount of culture medium, metabolites, and / or other components produced during the cultivation process. "Culture" also includes passaged cultures obtained by subculturing microorganisms, which can be a culture of a single generation or a mixture of several generations.

[0011] In this application, the metabolites can be obtained from the fermentation broth of *Bacillus salsaformis*. The fermentation broth of *Bacillus salsaformis* can be sterile or contain bacteria. The sterile fermentation broth is obtained by fermenting *Bacillus salsaformis* in a liquid culture medium and then filtering the fermentation broth using a bacterial filter.

[0012] In this application, the microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material or a biological material; the mineral material may be at least one selected from peat moss, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material may be at least one selected from various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal manure; the liquid carrier may be water.

[0013] In this application, the microbial agent can be in various dosage forms, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules. Depending on the need, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc., may also be added to the agent.

[0014] Thirdly, this application also provides the application of the plant endophytic Bacillus safoetida described in the first aspect or the microbial bactericide described in the second aspect, wherein the application is any one of the following:

[0015] A1) Promotes plant growth;

[0016] A2) Prepare products that promote plant production;

[0017] A3) Inhibits plant pathogens;

[0018] A4) Prepare products that inhibit plant pathogens.

[0019] Furthermore, the plant pathogens include pathogenic fungi and pathogenic oomycetes.

[0020] Furthermore, the pathogenic fungus is selected from Fusarium graminearum (Fusarium graminearum). Fusarium gramineae Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Fusarium graminearum ( Fusarium pseudograsses ), gray mold ( Botrytis cinerea Verticillium dahliae Verticillium dahliae ), Flathead anthrax bacteria ( Colletotrichum truncatum ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum One or more of the following.

[0021] Furthermore, the pathogenic oomycete is selected from *Pythium tertrum* (…). The last Pythium ), Phytophthora capsici ( Phytophthora capsicum ), Phytophthora ( Phytophthora cactorum ), Phytophthora parasiticum ( Phytophthora parasitic ), soybean phytotoxicum ( Phytophthora sojae One or more of the following.

[0022] Fourthly, this application also provides a method for preventing and controlling plant diseases, the method comprising treating plants or plant culture media with Bacillus sabensis described in the first aspect or a microbial fungicide described in the second aspect.

[0023] Furthermore, the plant disease is caused by at least one pathogen described in the third aspect.

[0024] Furthermore, the plant can be a monocotyledonous or dicotyledonous plant. The monocotyledonous plant can be wheat, rice, corn, sugarcane, etc., and the dicotyledonous plant can be soybean, potato, alfalfa, tobacco, tomato, cucumber, apple, etc. Preferably, the plant can be soybean, alfalfa, or pine.

[0025] Preservation Information

[0026] Classification and nomenclature: Bacillus safranin Bacillus safensis

[0027] Accession number: CGMCC No. 36637

[0028] Deposit date: November 17, 2025

[0029] Preservation Institution: China General Microbiological Culture Collection Center (CGMCC)

[0030] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] Compared with the prior art, this application has the following technical effects.

[0032] The *Bacillus safoetida* BSA15 strain provided by this invention has significant technical advantages and application value in the biocontrol of plant diseases. This strain exhibits broad-spectrum and highly effective inhibitory effects against a variety of important plant pathogenic fungi and oomycetes, effectively blocking the infection process of pathogens by producing antibacterial metabolites. Through antibacterial experiments, pot experiment efficacy tests, and field application verification, this strain has shown stable control effects under different environmental conditions, demonstrating good field adaptability and application reliability. Simultaneously, *Bacillus safoetida* BSA15 can stably colonize within plants as an endophyte, establishing a protective barrier within tissues such as roots, stems, and leaves, further enhancing disease control. Using this strain for disease control can significantly reduce the amount of chemical pesticides used, reducing pesticide residues and environmental pollution, meeting the requirements of green agriculture and sustainable development. Furthermore, this strain possesses good fermentation characteristics and formulation potential, providing an important strain resource and technical foundation for the development of novel microbial fungicides. Attached Figure Description

[0033] Figure 1A phylogenetic tree for Bacillus salsa BSA15 was constructed based on whole-genome sequencing results.

[0034] Figure 2 Morphological characteristics of Bacillus safranin BSA15; including A. colony morphology and B. Gram staining.

[0035] Figure 3 The image shows the inhibitory effect of Bacillus sabovellae BSA15 on pathogenic fungi; from left to right in the image are Fusarium graminearum (…). Fusarium gramineae Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Fusarium graminearum ( Fusarium pseudogramineum ), gray mold ( Botrytis gray Verticillium dahliae Verticillium dahliae ), Flathead anthrax bacteria ( Colletotrichum truncated ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium solani () Fusarium solani ).

[0036] Figure 4 The image shows the inhibitory effect of Bacillus salsa BSA15 on pathogenic oomycetes; from left to right in the image are Pythium cerevisiae (…). The last Pythium ), Phytophthora capsici ( Phytophthora capsicum ), Phytophthora ( Phytophthora cast ), Phytophthora parasiticum ( Phytophthora parasitica ), soybean phytotoxicum ( Phytophthora sojae ).

[0037] Figure 5 The inhibitory effect of Bacillus salsa BSA15 fermentation broth on pathogens (fungi and oomycetes).

[0038] Figure 6 This indicates the colonization status of Bacillus salsa BSA15 in plants.

[0039] Figure 7 The efficacy of Bacillus salsa BSA15 in controlling disease in potted plants after artificial inoculation of soybean seeds with the pathogen was evaluated.

[0040] Figure 8 The efficacy of Bacillus sabolicii BSA15 in potted plants artificially inoculated with the pathogen in alfalfa and Pinus massoniana.

[0041] Figure 9 This study investigated the field efficacy of Bacillus salsa BSA15 in soybean fields. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0043] Example 1: Strain BSA15 is Bacillus safranin.

[0044] Strain BSA15 was isolated from soybean roots and phylogenetic analysis was performed using the Type (Strain) Genome Server (TYGS) platform. Based on whole-genome sequence alignment, the genomic distance between the tested strain and known type strains and closely related strains in the database was calculated. Combined with genomic similarity indicators such as digital DNA-DNA hybridization (dDDH), the phylogenetic relationships between strains were quantitatively analyzed. Based on this, a phylogenetic tree was constructed using a phylogenetic algorithm.

[0045] The results are as follows Figure 1 The results showed that strain BSA15 clustered with the Bacillus safensis type strain in the same phylogenetic clade, indicating that it has a close phylogenetic relationship with Bacillus safensis at the genomic level, and it was identified as Bacillus safensis.

[0046] Example 2 Morphological identification

[0047] Bacillus safranin BSA15 was inoculated onto LB solid medium and cultured at 37°C for 1 day. Its morphological characteristics were then observed. Results are as follows: Figure 2 The colonies are nearly round, grayish-white or light-colored, with a smooth and moist surface; Gram staining results are positive.

[0048] Example 3: Bacillus salsa BSA15 exhibits broad-spectrum resistance

[0049] The inhibitory activity of strain BSA15 against pathogenic fungi, oomycetes, and bacteria was determined using the plate confrontation method. The selected pathogenic fungus was *Fusarium graminearum* (Gynostemma pentaphyllum). Fusarium gramineae Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Fusarium graminearum ( Fusarium pseudograminearum) Botrytis cinerea ( Botrytis cinerea Verticillium dahliae Verticillium dahliae ), Flathead anthrax bacteria ( Colletotrichum truncated ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium solani () Fusarium solani ); Pathogenic oomycetes: Pythium cerevisiae ( The last Pythium ), Phytophthora capsici ( Phytophthora capsicum ), Phytophthora ( Phytophthora cactorum ), Phytophthora parasiticum ( Phytophthora parasitica ), soybean phytotoxicum ( Phytophthora sojae ).

[0050] The method for detecting pathogenic fungi and oomycetes is as follows: Add 20 mL of PDA / V8 medium to each 90 mm diameter petri dish. Use a 6 mm diameter punch to create filter paper discs, stacking two layers together and attaching them around the perimeter of the plate. Add 20 μL LOD to each disc. 600 The control group was prepared with a 1.0 g / L Bacillus safranin BSA15 bacterial suspension. An equal volume of dd water was added to the control group, and a 6 mm diameter pathogen block was inoculated in the center of the plate.

[0051] The results are as follows Figure 3 and Figure 4 The results showed that Bacillus safranin BSA15 had a good inhibitory effect on both pathogenic fungi and oomycetes.

[0052] Example 4: Antibacterial activity of Bacillus salsa metabolites BSA15

[0053] BSA15 strain was inoculated into NB liquid medium at 30°C and shaken at 180 rpm for 72 hours. The resulting bacterial culture was centrifuged at 12000g for 15 min, and the supernatant was filtered through a 0.22μm bacterial filter to obtain sterile fermentation broth.

[0054] The fermentation broth was mixed with NB medium at ratios of 45% and 65%, and the mixture was poured onto plates. Soybean Phytophthora was then inoculated into the center of each plate. Phytophthora sojae ), Phytophthora parasiticum ( Phytophthora parasitica ), Phytophthora ( Phytophthora cast Fusarium oxysporum ( Fusarium oxysporum ).

[0055] The results are as follows Figure 5 The results showed that the sterile fermentation broth had a good inhibitory effect on both pathogenic fungi and oomycetes.

[0056] Example 5: Colonization of Bacillus salsa BSA15 in plants

[0057] First, induce Bacillus salsa BSA15 to produce rifampicin-resistant mutant strains.

[0058] To further investigate the colonization of Bacillus saefolius BSA15 in different parts of soybean, 10 sterilized seeds were sown into sterilized vermiculite and irrigated with 50 mL of BSA15 bacterial solution labeled with resistance. Eight days after root irrigation, samples were taken: 0.6 g each of roots, stems, and leaves were cut and surface-sterilized by soaking in 70% alcohol for 30 seconds, 2% sodium hypochlorite for 1 minute, and rinsing three times with sterile water. The samples were then ground and mixed in a mortar with 6 mL of sterile water. The supernatant was allowed to settle for 10 minutes and then diluted 10-fold to four concentrations (T1–T4). 0.1 mL of each concentration was spread onto LB agar plates containing 50 μg / mL rifampicin, with three replicates per concentration. The plates were incubated at 28°C for 2 days, and the colony count was recorded. The colony count per gram of fresh soybean tissue was calculated to evaluate colonization ability.

[0059] The results are as follows Figure 6 The results showed that Bacillus safoetida BSA15 can transfer to the stems and leaves after entering the soybean roots, demonstrating a certain transfer ability and avoiding external environmental stress.

[0060] Example 6: Bacillus sabinatus BSA15 can control diseases caused by various pathogens on a variety of plants.

[0061] The pathogenic fungus Fusarium graminearum ( Fusarium graminearum ) and soybean phytotoxicum ( Phytophthora sojae After the colonies have fully grown on 90mm plates, cut them into small pieces and mix them evenly into vermiculite. Inoculate two dishes of pathogens into each pot and sow 10 soybean seeds. Apply OD fertilizer on the day of inoculation and the fourth day after inoculation. 600 Use a 1.0 BSA15 bacterial solution and pour 50 mL of the solution into each pot.

[0062] Similar to the above method, alfalfa was inoculated with Fusarium solani (… Fusarium solani ) and Fusarium oxysporum ( Fusarium oxysporum ), and inoculation of Fusarium oxysporum into Masson pine ( Fusarium oxysporum ).

[0063] The results are as follows Figure 7 and Figure 8 The results show that the application of Bacillus salsa BSA15 can effectively prevent and control diseases.

[0064] Example 7 Field efficacy test of Bacillus salsa BSA15

[0065] A 3m*3m plot was set up in the field, with two treatments. Treatment one was the OD (Original Demand) phase. 600Treatment 1.0% bacterial solution, Treatment 2% Bacillus subtilis diluted 800 times, and Control% water were applied together with the seeds at the time of sowing. After the soybeans matured, 10 plants were randomly selected from each plot, and the results were repeated three times. The number of pods per plant, the weight of grains per plant, the number of grains per plant, the weight of 100 grains, the mold rate, the shriveling rate, the plot yield, and the yield per acre were recorded.

[0066] The statistical results are shown in Table 1.

[0067] Table 1 Statistical Results of Field Indicators

[0068]

[0069] Meanwhile, the disease index and prevention and control effects were statistically analyzed based on the incidence and infection levels of purpura (Table 2).

[0070] Table 2 Infection Severity

[0071]

[0072] The results are as follows Figure 9 The results showed that soybean yield increased and disease control was better when Bacillus salsa BSA15 was applied.

[0073] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A plant endophytic Bacillus saforticus, characterized in that, The plant endophytic Bacillus sarfusense strain is numbered BSA15 and classified as Bacillus sarfusense (BSA15). Bacillus safensis The accession number (CGMCC No. 36637) was deposited at the China General Microbiological Culture Collection Center on November 17, 2025.

2. A microbial bactericide, characterized in that, The microbial bactericide contains Bacillus sarfusae as described in claim 1.

3. The application of the plant endophytic Bacillus saforticus according to claim 1 or the microbial fungicide according to claim 2, characterized in that, The application is any of the following: A1) Promotes soybean growth; A2) Prepare products that promote soybean production; A3) Inhibits plant pathogens; A4) Prepare products that inhibit plant pathogens; The plant pathogen is a pathogenic fungus or a pathogenic oomycete; the pathogenic fungus is selected from Fusarium graminearum (…). Fusarium graminearum Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani ( ) Rhizoctonia solani ), Fusarium graminearum ( Fusarium pseudograminearum ), gray mold ( Botrytis cinerea Verticillium dahliae Verticillium dahliae ), Flathead anthrax bacteria ( Colletotrichum truncatum ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium solani () Fusarium solani One or more of the following: the pathogenic oomycetes are selected from *Pythium tertrum* (…). Pythium ultimum ), Phytophthora capsici ( Phytophthora capsici ), Phytophthora ( Phytophthora cactorum ), Phytophthora parasiticum ( Phytophthora parasitica ), soybean phytotoxicum ( Phytophthora sojae One or more of the following.

4. A method for preventing and controlling plant diseases, characterized in that, The method includes treating plants with Bacillus saffron as described in claim 1 or a microbial fungicide as described in claim 2; the plant disease is caused by at least one pathogen as described in claim 3.

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