Bacillus sp. NWAFU56-41 and application thereof in preventing and treating plant diseases and promoting plant growth

Abstract

This invention discloses a Bacillus NWAFU56-41 and its application in the prevention and control of plant diseases and the promotion of plant growth, belonging to the field of microbial application technology, and particularly relating to a Bacillus ( Bacillus Bacillus sp. NWAFU56-41 was deposited at the China Center for Type Culture Collection (CCTCC) on January 23, 2026, with accession number CCTCC NO: M 2026213. This Bacillus sp. NWAFU56-41 is used in the control of plant diseases. The Bacillus sp. NWAFU56-41 provided by this invention can alleviate garlic leaf blight and significantly promote the growth of garlic, cucumber, and tomato seedlings, and exhibits good broad-spectrum resistance to plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and in particular relates to a Bacillus NWAFU56-41 and its application in the prevention and control of plant diseases and the promotion of plant growth. Background Technology

[0002] garlic( Allium sativum Garlic (Allium chinense) is a biennial herbaceous plant belonging to the genus Allium in the family Amaryllidaceae. Widely cultivated worldwide, it is popular as a vegetable, condiment, or medicinal plant due to its antibacterial, antioxidant, and anticancer properties. Currently, garlic production primarily utilizes bulb-based asexual reproduction. Long-term continuous cropping has exacerbated pathogen accumulation and varietal degeneration, leading to a rapid increase in foliar diseases. Among these, fungi of the genus *Allium* (*L.*) are particularly prevalent. Stemphylium Leaf blight caused by this disease is particularly severe, posing a significant threat to production safety. In the early stages, leaves show small white spots or withered leaf tips; as infection worsens, the withered area gradually expands. In the middle and later stages, a black mold layer appears on the diseased leaves, and in severe cases, the entire leaf dies. The pathogen typically first infects garlic at the 2-3 leaf stage after emergence, and then reinfects from February to mid-April of the following year, severely hindering bolting and yield formation. Under continuous cropping conditions, diseased fields generally experience yield reductions of 20%-30%, while severely affected fields suffer yield reductions of over 50%, or even total crop failure, causing severe economic losses to garlic farmers.

[0003] Currently, the control of garlic leaf blight in production mainly relies on integrated measures, primarily using chemical agents, such as selecting disease-resistant varieties, soil disinfection, and crop rotation. However, long-term and excessive use of chemical agents easily leads to a series of problems, including environmental pollution, pesticide residues, disruption of ecological balance, and increased drug resistance in pathogens. Developing environmentally friendly, cost-effective, and efficient alternative control methods has become an urgent need for garlic production. In recent years, beneficial microorganisms have attracted much attention due to their green, safe, and eco-compatible characteristics, as well as their multiple functions. They not only have significant antagonistic and inhibitory effects on pathogens but also directly promote plant growth through nitrogen fixation, phosphorus and potassium solubilization, secretion of plant hormones, and induction of systemic resistance. They can effectively alleviate disease stress and improve crop yield and quality, perfectly aligning with the concepts of green agricultural development and integrated pest management. However, research on identifying beneficial endophytic bacteria from garlic itself and applying them to disease control is still very limited. Therefore, isolating and screening endophytic bacteria from healthy garlic bulbs that have both highly effective antagonistic activity against leaf blight pathogens and significant growth-promoting effects is of great theoretical and practical significance for reducing dependence on chemical pesticides, ensuring garlic yield and quality, and protecting the ecological environment. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a Bacillus NWAFU56-41 and its application in the prevention and control of plant diseases and the promotion of plant growth. The Bacillus NWAFU56-41 provided by this invention can be used to control garlic leaf blight and can significantly promote the growth of garlic, cucumber, and tomato seedlings, exhibiting good broad-spectrum resistance to plant diseases.

[0005] To achieve the above objectives, the present invention provides a Bacillus ( Bacillus sp.) NWAFU56-41, the Bacillus sp. NWAFU56-41, was deposited on January 23, 2026 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026213.

[0006] The present invention also provides the application of the Bacillus NWAFU56-41 in the prevention and control of plant diseases.

[0007] Preferably, the plant diseases include garlic leaf blight, gray mold, cucumber wilt, wheat stem base disease, potato dry rot, apple anthracnose, grape gray mold, wheat take-all disease, tomato wilt, garlic root rot, and cucumber anthracnose.

[0008] The present invention also provides the application of the Bacillus NWAFU56-41 in plant growth promotion.

[0009] Preferably, the Bacillus NWAFU56-41 promotes the growth of garlic plants and the accumulation of dry matter.

[0010] Preferably, the Bacillus NWAFU56-41 promotes the growth of cucumber seedlings.

[0011] Preferably, the Bacillus NWAFU56-41 promotes the growth of tomato seedlings.

[0012] This invention also provides a microbial agent for the prevention and control of plant diseases, wherein the microbial agent comprises Bacillus NWAFU56-41, and the effective viable count of Bacillus NWAFU56-41 in the microbial agent is 10. 8 cfu / mL.

[0013] Preferably, the plant diseases controlled by the microbial inoculant for preventing and controlling plant diseases include garlic leaf blight, gray mold, cucumber wilt, wheat stem base disease, potato dry rot, apple anthracnose, grape gray mold, wheat take-all disease, tomato wilt, garlic root rot, and cucumber anthracnose.

[0014] The present invention also provides a microbial inoculant for promoting plant growth, wherein the microbial inoculant includes Bacillus NWAFU56-41, and the effective viable count of Bacillus NWAFU56-41 in the microbial inoculant is 10. 8 cfu / mL.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a Bacillus NWAFU56-41 and its application in the prevention and control of plant diseases and the promotion of plant growth. The Bacillus NWAFU56-41 provided by this invention has the functions of nitrogen fixation, potassium solubilization, organophosphate dissolution, iron carrier production, protease production, and cellulase production. It can prevent and control garlic leaf blight and significantly promote the growth of garlic, cucumber, and tomato seedlings. In addition, it has good broad-spectrum resistance to plant diseases such as gray mold, cucumber wilt, wheat stem base disease, potato dry rot, apple anthracnose, grape gray mold, wheat take-all disease, tomato wilt, garlic root rot, and cucumber anthracnose. The control effect on garlic leaf blight is the most outstanding. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The images show the growth of strain NWAFU56-41 on LB solid medium, where a is a morphological image of the strain and b is a single colony image. Figure 2 Phylogenetic tree of strain NWAFU56-41; Figure 3 The image shows the inhibition of strain NWAFU56-41 against the pathogen causing garlic leaf blight. In the image, a represents the control group and b represents the treatment group. Figure 4 The results of the growth-promoting characteristics detection of strain NWAFU56-41 are shown in the following figures: a) nitrogen fixation result, b) organic phosphorus solubilization result, c) protease production result, d) potassium solubilization result, e) cellulase production result, and f) siderophore production result. Figure 5 The results of inoculating garlic seedlings with strain NWAFU56-41 are shown. In the figure, a represents the aboveground growth promotion result, b represents the underground growth promotion result, CK represents the control group, and NWAFU56-41 represents the treatment group. Figure 6The results of cucumber seedling growth promotion after inoculation with strain NWAFU56-41 are shown in the figure. In the figure, a represents the whole plant growth promotion result, b represents the leaf growth promotion result, CK represents the control group, and NWAFU56-41 represents the treatment group. Figure 7 The results of tomato seedling growth promotion after inoculation with strain NWAFU56-41 are shown in the figure. CK represents the control group and NWAFU56-41 represents the treatment group. Figure 8 The effect of inoculation with strain NWAFU56-41 on resistance to leaf blight in garlic seedlings is shown in the figure. CK represents the control group and NWAFU56-41 represents the treatment group. Figure 9 This image shows the broad-spectrum antibacterial activity of strain NWAFU56-41 against different pathogens. Specifically, a) shows the efficacy against garlic leaf blight, b) against gray mold, c) against cucumber wilt, d) against wheat stem base disease, e) against potato dry rot, f) against apple anthracnose, g) against grape gray mold, h) against wheat take-all disease, i) against tomato wilt, j) against garlic root rot, and k) against cucumber anthracnose. In each small image, the left petri dish represents the pathogen growth status of the control group, and the right petri dish represents the pathogen growth status of the treatment group inoculated with strain NWAFU56-41. Detailed Implementation

[0018] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials associated with those documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail. Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0020] The culture medium used in this invention has the following formulation: PDA medium: Wash and peel potatoes, weigh 20g and cut into small pieces. Boil in distilled water for 20 minutes, filter through three layers of gauze and collect the filtrate. Add 20g agar and 20g glucose, and bring the volume to 1000mL. Set the pH to its natural value and sterilize at 121℃ for 21 minutes before use.

[0021] PDB liquid culture medium: 200g potato, 20g sucrose, 1000mL distilled water, pH 7.0, sterilized at 121℃ for 21min before use.

[0022] LB solid medium: 5.0g yeast extract, 10g peptone, 10g sodium chloride, 18g agar, pH 7.0±0.1, 1L distilled water, autoclaved at 121℃ for 15min.

[0023] LB liquid medium: 5.0g yeast extract, 10g peptone, 10g sodium chloride, pH 7.0±0.1, 1L distilled water, autoclaved at 121℃ for 15min.

[0024] Assumption medium: 0.2g potassium dihydrogen phosphate, 10.0g mannitol, 0.2g sodium chloride, 0.1g calcium sulfate, 5.0g calcium carbonate, 15.0g agar, 0.2g magnesium sulfate, pH 7.0±0.1, 1L distilled water, autoclaved at 121℃ for 15min.

[0025] Protein detection culture medium: 10g skim milk powder, 20g agar, heated and dissolved in 1L distilled water, autoclaved at 105℃ for 20min to avoid the formation of flocculent protein.

[0026] Organophosphate bacteria culture medium: glucose 10.0g, lecithin 0.2g, ammonium sulfate 0.5g, yeast extract 0.5g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, calcium carbonate 1.0g, sodium chloride 0.3g, agar 15.0g, pH 7.0-7.5. Add 1L of distilled water and autoclave at 121℃ for 15min.

[0027] Inorganic phosphorus bacteria culture medium: glucose 10.0g, ammonium sulfate 0.5g, yeast extract 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, calcium phosphate 5.0g, agar 15.0g, pH 7.0-7.5, distilled water 1000mL, autoclave at 121℃ for 15min.

[0028] Cellulase detection medium: 10.0g peptone, 10.0g yeast extract, 10.0g sodium carboxycellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, 18g agar, 1L distilled water, autoclaved at 121℃ for 15min.

[0029] Silicate bacteria culture medium: sucrose 5.0g, magnesium sulfate 0.5g, calcium sulfate 0.1g, disodium hydrogen phosphate 2.0g, ferric chloride 0.005g, glass powder 1.0g, agar 15.0g, pH 7.0±0.2, distilled water 1000mL, autoclave at 121℃ for 15min.

[0030] CAS detection medium: Chromium azurite S (CAS) 0.06g, hexadecyltrimethylammonium bromide (HDTMA) 0.07g, ferric chloride hexahydrate 0.002g, sodium dihydrogen phosphate dihydrate 0.3g, disodium hydrogen phosphate dodecahydrate 0.3g, ammonium chloride 0.1g, potassium dihydrogen phosphate 0.04g, sodium chloride 0.06g, agar 9g, pH 6.8±0.1, distilled water 1000mL, autoclaved at 116℃ for 30min.

[0031] Chitinase detection culture medium: 1% colloidal chitin, 1g ammonium dihydrogen phosphate, 0.2g potassium chloride, 0.2g magnesium sulfate, 20g agar, and sterile water to a final volume of 1000mL, pH 7. Autoclave at 121℃ for 21min. Preparation of colloidal chitin: Dissolve 20g chitin in 350mL concentrated hydrochloric acid, incubate at 4℃ for 24h, filter with glass wool, add 2L of ice-cold anhydrous ethanol to the filtrate, incubate overnight at -20℃, centrifuge at 10000r / min for 20min, continuously wash the precipitate with running tap water until pH is neutral, and store in a sealed container at -20℃.

[0032] The Soviet red-skinned garlic (improved garlic), Cangshan garlic, Qixian garlic, purple-skinned garlic, and white-skinned garlic used in this invention are germplasm materials preserved by the Vegetable Physiology and Biotechnology Laboratory of Northwest A&F University.

[0033] Example 1 I. Isolation, identification, and preservation of strain NWAFU56-41: 1. Sample collection: The garlic variety tested was Soviet red-skinned garlic (improved garlic) preserved by the Vegetable Physiology and Biotechnology Laboratory of Northwest A&F University.

[0034] 2. Isolation and preservation of rhizosphere bacteria: Endophytic antagonistic bacteria were isolated using a tissue homogenization method. Different types of garlic from various regions, including Soviet red-skinned garlic (improved garlic), Cangshan garlic, Qixian garlic, as well as purple-skinned and white-skinned garlic, were used as test materials. After peeling, the collected garlic samples were rinsed with sterile water, air-dried indoors, and then transferred to a laminar flow hood. The surface-sterilized samples were placed in a sterile mortar, and an appropriate amount of sterile water was added for thorough grinding. After standing, the supernatant was collected and repeatedly diluted with sterile water to a concentration of 10. -1 10 -2 10 -3 10 -4 10-5 Dilutes of each gradient were spread onto LB agar plates and incubated at 28°C for 4 days. Sterile water from the final rinsing of the sample during surface disinfection was used as a control, spread onto LB and NA agar plates to check the thoroughness of surface disinfection. After single colonies grew, single colonies of different colors, morphological characteristics, and sizes were picked with a sterile inoculation loop and streaked for reculturing. The purified bacterial cultures were then stored at -20°C in 50% glycerol for later use.

[0035] II. Determination of the antibacterial and growth-promoting properties of strain NWAFU56-41: 1. Determination of the antibacterial activity of strain NWAFU56-41: Preparation of fermentation broth for strain NWAFU56-41: A single colony of strain NWAFU56-41 was picked with a sterile inoculation loop and inoculated into LB liquid medium. The culture was then incubated on a shaker at 28°C and 200 r / min for 16 h. The resulting culture was stored for later use.

[0036] Initial screening stage: The pathogen causing garlic leaf blight was selected as the target bacterium for antagonistic bacteria screening. A 5mm diameter punch was used to create 5mm diameter mycelial cakes along the edge of the pathogen colonies, which were then placed in the center of PDA medium. Colonies were then inoculated using an inoculation loop from purified NWAFU56-41 single colonies at approximately 2.5cm above, below, left, and right of the mycelial cake. Each PDA medium was inoculated four times, with three replicates. A control group (CK) was used, inoculated only with the target bacterium. After sealing, the medium was incubated at 28℃ for 7 days, and the inhibition rate was measured using the cross-hatching method.

[0037] Secondary screening stage: The following method was used to further verify the antagonistic effect of the bacterial strains selected in the preliminary screening (the operation method in the early stage was the same as that in the initial screening): Four holes were made about 2.5 cm away from the top, bottom, left and right of the bacterial cake using a 5 mm diameter punch, and 30 μL of NWAFU56-41 fermentation broth was inoculated. The control group (CK) was inoculated with only the target bacteria. The bacteria were cultured at 28 ℃ in a biochemical incubator for 7 days. Each treatment was repeated 3 times. The inhibition radius was recorded and the inhibition rate was calculated.

[0038] Inhibition rate (%) = (Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group) / Coronavirus colony diameter in control group × 100%.

[0039] 2. Determination of the antibacterial extracellular enzyme activity of strain NWAFU56-41: The activities of protease, chitinase, and cellulase in strain NWAFU56-41 were determined: Using the fermentation broth of NWAFU56-41 prepared by the aforementioned method, 10 μL of the broth was spotted approximately 2.5 cm above, below, to the left, and to the right of the center of each plate. Each medium was spotted four times, with three replicates. The plates were incubated at 28℃ for 2–7 days. The presence or absence of a clear zone was observed, and the colony diameter (d) and the diameter of the clear zone (D) were measured. The presence or absence of enzyme activity was determined by the presence or absence of a clear zone on the medium; enzyme activity was positively correlated with the HC value (D / d).

[0040] Protease production capacity: The strain NWAFU56-41 was inoculated onto a protein detection medium plate, and the presence or absence of a clear zone was observed after 3 days.

[0041] Cellulase production capacity: Inoculate strain NWAFU56-41 onto cellulase detection medium plates. After 2-3 days, add 1 mg / mL Congo red solution for staining for 30 min. Discard the staining solution and wash with 1 mol / L sodium chloride for 30 min. Observe whether a clear zone appears.

[0042] Chitinase production capacity: The strain NWAFU56-41 was inoculated onto a chitinase detection medium plate and cultured for 7 days. The presence or absence of a clear zone was then observed.

[0043] 3. Determination of growth-promoting functions of strain NWAFU56-41: The growth-promoting characteristics of strain NWAFU56-41 were tested, including nitrogen fixation capacity, inorganic phosphorus and organic phosphorus solubility, potassium solubilization, siderophore production, and IAA production capacity.

[0044] Nitrogen fixation capacity: The strain NWAFU56-41 was inoculated into Assumption medium and cultured for 7 days to observe whether there was cell growth.

[0045] Phosphorus solubility: The strain NWAFU56-41 was inoculated onto solid organic and inorganic phosphorus solubilizing medium plates and cultured for 7 days. The presence or absence of transparent phosphorus solubilizing zones was observed.

[0046] Siderogenetic vector: Inoculate strain NWAFU56-41 onto chromium azure CAS plates and observe for the appearance of yellow-green halos after 7 days.

[0047] IAA detection: Take 1 mL of the cultured NWAFU56-41 fermentation broth and inoculate it into a liquid medium containing L-tryptophan. Incubate at 28℃ and 200 r / min for 24 h. Take 100 μL of NWAFU56-41 fermentation broth and drop it onto a white ceramic plate. Add the same volume of Salkowski colorimetric reagent and mix well. React in the dark at room temperature for 30 min. If the color turns red, it indicates that the strain has the ability to produce IAA; otherwise, it does not.

[0048] Potassium solubilization capacity: The strain NWAFU56-41 was inoculated into silicate medium and cultured for 7 days to observe whether it produced an oily liquid.

[0049] III. Results: 1. Results of inhibition rate determination of strain NWAFU56-41: Preliminary and secondary screening of strains were conducted using the pathogen of garlic leaf blight, such as... Figure 3 As shown, Figure 3 a and Figure 3 Table b shows the antagonistic screening of the treatment group (NWAFU56-41) against garlic leaf blight and the pathogen plate photograph of the control group (CK). As shown in Table 1, strain NWAFU56-41 exhibited an initial mycelial growth inhibition rate of 83.58% against garlic leaf blight, and a secondary inhibition rate of 84.09%. Both the initial and secondary inhibition rates were above 80%, demonstrating strong antibacterial ability.

[0050] Table 1. Inhibition diameter and inhibition rate of strain NWAFU56-41 against garlic leaf blight pathogen.

[0051] 2. Results of assays on the extracellular enzyme inhibition activity and growth-related functions of strain NWAFU56-41: From Table 2 below and Figure 4 a~ Figure 4 As can be seen from the results, the extracellular enzyme inhibition activity of strain NWAFU56-41 is mainly manifested in the production of protease and cellulase, but not in the production of chitinase, with a protease activity value of 0.57 and a cellulase activity value of 0.54; the growth-promoting characteristics are manifested in the functions of nitrogen fixation, potassium solubilization, organic phosphorus solubilization, and iron carrier production, but not in the function of inorganic phosphorus solubilization.

[0052] Table 2. Results of the detection of extracellular enzyme inhibitory activity and growth-promoting properties of strain NWAFU56-41

[0053] Note: "+" indicates that the ability exists, while "-" indicates that the ability does not exist.

[0054] 3. Morphological and molecular identification: Take the purified strain, streak it on LB solid medium, seal it with sealing film, and incubate it in a biochemical incubator at 28℃ for 2 days. Then take out the culture dish and observe the basic morphological characteristics such as colony morphology, size, color, texture viscosity, gloss, transparency, and edge smoothness. Take pictures and record them.

[0055] Single colonies of the purified strain were picked and dissolved in 10 μL of sterile water, and bacterial DNA was extracted using the boiling lysis method. Specifically, the lysis was performed at 100℃ for 10 min to obtain bacterial DNA. PCR amplification of the selected strain was performed using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO. 3). The PCR reaction mixture consisted of 50 μL of the following: template (genomic DNA) 4 μL, primers (27F and 1492R) 2 μL each, ddH2O 17 μL, and MIX (2×Taq plus Master Mix) 25 μL. PCR amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min; 35 cycles, with a final extension at 72℃ for 10 min. PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The obtained bacterial sequences were compared using BLAST in the NCBI database, and a phylogenetic tree was constructed using MAGA-X software.

[0056] Morphological observation revealed that strain NWAFU56-41 appeared white, round, with smooth, non-serrated edges, a glossy, wrinkled, and opaque surface on LB medium. The cells were slightly moist, viscous, and easily picked up. (Specific details are as follows...) Figure 1 As shown, Figure 1 a and Figure 1 Figure b shows the colony morphology of NWAFU56-41 on LB medium.

[0057]

[0058] Sequencing results were used to locate sequences from different genera within the same species in the NCBI database for multiple alignment. A phylogenetic tree was then constructed using MEGA-X software for comparison. The results are as follows: Figure 2 As shown, based on the homology comparison analysis results, the gene sequence of strain NWAFU56-41 is similar to... Bacillus mojavensis strain C265 is in the same branch, and based on the phylogenetic tree results, it is inferred that NWAFU56-41 belongs to Bacillus mojavei (…). Bacillus mojavensis ), named NWAFU56-41.

[0059] Strain NWAFU56-41 is a Bacillus ( Bacillus (sp.), deposited on January 23, 2026 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: M 2026213.

[0060] Example 2 The growth-promoting effect of strain NWAFU56-41 on garlic, cucumber, and tomato seedlings was tested. The tested varieties were “Soviet Red-skinned Garlic (Improved Garlic)”, “Bonai” cucumber, and “Ailsa Craig” tomato, which were preserved in the Vegetable Cultivation Physiology, Ecology and Biotechnology Laboratory of the College of Horticulture.

[0061] Using the fermentation broth of strain NWAFU56-41 prepared in Example 1, the supernatant after centrifugation was diluted with ddH2O to adjust the OD. 600 The value is 0.8 (10) 8 (cfu / mL) and set aside for later use.

[0062] The pot experiment was conducted in April 2025 in the artificial climate chamber of Northwest A&F University. The culture conditions for cucumber and tomato seedlings were: light intensity of 360 µmol·photons·m -2 ·s -1 The temperature was 25±1℃ / 18±1℃ (day / night), the photoperiod was 16h / 8h (day / night), and the relative humidity was 60%~80%. The cultivation conditions for garlic seedlings were: light intensity of 200µmol·photons·m -2 ·s -1 The temperature is 22±1℃ / 17±1℃ (day / night), the photoperiod is 16h / 8h (day / night), and the relative humidity is 50%~70%.

[0063] Growth promotion experiment of garlic seedlings: Healthy garlic cloves of uniform size were selected and sown in seedling pots filled with autoclaved substrate. The pot diameter × bottom diameter × height was 7cm × 5cm × 7.3cm, and one garlic seedling of uniform growth was planted in each pot. When the garlic seedlings grew to 3 leaves, the treatment group (NWAFU56-41) was treated by evenly drenching the roots of the seedlings within 2cm of the base of the stem with fermentation broth of strain NWAFU56-41, with 10mL of broth per seedling. The control group (CK) was inoculated with only an equal amount of water. Each treatment was inoculated with 21 seedlings, and the results were repeated 3 times. Seven days later, the seedlings were inoculated with the bacterial solution again. Seven days after the second inoculation, 6 representative plants were randomly selected from each treatment, and the seedling height, leaf length, leaf width, stem diameter, number of leaves, dry weight, and fresh weight were measured. The seedling vigor index was calculated using the following formula: Seedling vigor index = (stem diameter / plant height + root dry weight / aboveground dry weight) × total plant dry weight.

[0064] Growth-promoting experiments on cucumber and tomato seedlings: After disinfection, soaking, and germination, cucumber and tomato seeds were sown in seedling pots containing high-pressure moist heat sterilization substrate. The pot diameter × bottom diameter × height was 7cm × 5cm × 7.3cm. When the cucumber and tomato seedlings reached the three-leaf stage, seedlings with uniform growth were selected. The treatment group (NWAFU56-41) was irrigated with 10mL of NWAFU56-41 fermentation broth within 2cm of the stem base. Each treatment was inoculated with 21 seedlings, and the inoculation was repeated 3 times. The control group (CK) was inoculated with only an equal amount of water, and then inoculated with the bacterial solution again after 7 days. Seven days after the second inoculation, 10 representative plants were randomly selected from each treatment. The plant height, stem diameter, leaf area, number of leaves, plant dry weight, and plant fresh weight of cucumber and tomato seedlings were measured, and the seedling vigor index was calculated using the following formula: Seedling vigor index = (stem diameter / plant height + root dry weight / aboveground dry weight) × total plant dry weight.

[0065] Table 3. Growth-promoting effect of strain NWAFU56-41 on garlic seedlings

[0066] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0067] Seven days after the second inoculation, observe the growth of the garlic seedlings (e.g., Figure 5 a and Figure 5 As shown in Figure b and Table 3), compared with the control group, treatment of garlic seedlings with NWAFU56-41 fermentation broth significantly increased both fresh weight and dry weight, by 31.85% and 29.03%, respectively. Simultaneously, inoculation with NWAFU56-41 also promoted stem diameter, leaf area, and leaf number in garlic. Therefore, strain NWAFU56-41 exhibits characteristics of a rhizosphere growth-promoting bacterium (PGPR), capable of promoting garlic plant growth and dry matter accumulation.

[0068] Table 4. Growth-promoting effect of strain NWAFU56-41 on cucumber seedlings

[0069] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0070] The results of the experiment on the effect of fermentation broth of strain NWAFU56-41 on the biomass of cucumber seedlings are shown in Table 4. The relevant growth traits of cucumber seedlings treated with fermentation broth of strain NWAFU56-41 were significantly better than the control, and it significantly promoted the growth of cucumber seedlings. The fresh weight and dry weight of the cucumber seedlings increased significantly, increasing by 26.58% and 39.66% respectively compared to the control. Strain NWAFU56-41 can promote the growth of cucumber seedlings (e.g., Figure 6 a and Figure 6 (b)

[0071] Table 5. Growth-promoting effect of strain NWAFU56-41 on tomato seedlings

[0072] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0073] Compared with the control group, strain NWAFU56-41 promoted the growth indicators of tomato seedlings to varying degrees. Treatment with NWAFU56-41 fermentation broth increased the plant height, stem diameter, and dry weight of tomato seedlings by 30.21%, 10.55%, and 36.36%, respectively, compared to the control. This indicates that NWAFU56-41 is a rhizosphere growth promoter (PGPR) that can promote the growth of tomato seedlings. (See Table 5 and...) Figure 7 ).

[0074] Example 3 Control efficacy and broad-spectrum resistance assay of inoculated strain NWAFU56-41 against garlic leaf blight: 1. Preparation of spore suspension of garlic leaf blight pathogen: The pathogen of garlic leaf blight (preserved in the laboratory) Stemphylium vesicarium Using a punch, take a 5mm diameter mycelial cake and inoculate it in the center of a PDA medium. After incubating at 28℃ for 7 days, scrape off the mycelium from the plate, add sterile water and shake. Filter the mycelium with gauze to obtain the supernatant, i.e., the sporangium suspension. Take 10μL of the filtered sporangium suspension and drop it onto a hemocytometer. Count the spore concentration under a microscope, and then dilute the concentration to 10 with sterile water. 5 CFU / mL is prepared for later use.

[0075] 2. Preparation of fermentation broth for strain NWAFU56-41: Preparation of fermentation broth for strain NWAFU56-41: Single colonies of strain NWAFU56-41 were picked with a sterile inoculation loop and inoculated into LB liquid medium. The culture was then incubated on a shaker at 28°C and 200 rpm for 16 h to obtain the culture broth. After centrifugation, the supernatant was diluted with ddH2O to adjust the OD. 600 The value is 0.8 (10) 8 (cfu / mL) and set aside for later use.

[0076] 3. Determination of the control effect of fermentation broth of strain NWAFU56-41 on garlic leaf blight: Garlic seedling cultivation and management methods: Pot cultivation was carried out in an artificial climate chamber under the following conditions: light intensity of 200 µmol·photons·m -2 ·s -1 The temperature is 22±1℃ / 17±1℃ (day / night), the photoperiod is 16h / 8h (day / night), and the relative humidity is 50%~70%.

[0077] Garlic bulbs that have undergone surface sterilization were planted in sterile nutrient soil. When the garlic bulbs reached the 3-leaf stage, an inoculation experiment with NWAFU56-41 was conducted. The endophytic bacteria to be tested were prepared into a bacterial suspension with an OD value of 0.8, placed in a spray bottle, and the mixed endophytic bacteria solution was evenly sprayed onto the leaves every three days, for a total of three sprays, followed by a 3-day incubation period. Twenty replicates were set up for each endophytic bacteria plant. Subsequently, a suspension of pathogenic spores from the cultured garlic leaf blight pathogen was prepared for inoculation. The live inoculation method is as follows: Using a sterilized knife, a small wound approximately 0.5 cm long was made perpendicular to the vein in the middle of the garlic leaf, and then one drop of a 10% concentration was applied. 5 A spore suspension of *Garlic leaf blight* fungus was prepared at a concentration of 1 spore / mL. Each treatment was replicated six times, with a control group consisting of one drop of sterile water applied to the cut area. Subsequent treatments followed the spore suspension spraying method. Leaf disease and plant lesion status were observed at 7, 9, 11, and 13 days after inoculation with the garlic leaf blight pathogen, and the incidence and disease index were calculated. The grading criteria for leaf blight are as follows, and the disease index was calculated.

[0078] The grading standards for leaf blight are as follows: Grade 0, leaves without disease spots; Grade 1: Lesions cover less than 5% of the total leaf area; Grade 3, with lesions covering 6% to 25% of the total leaf area; Level 5, with lesions covering 26% to 50% of the total leaf area; Level 7, with lesions covering 51% to 75% of the total leaf area; Level 9, with lesions covering more than 76% of the total leaf area.

[0079] Then, calculate the disease index using the following method: .

[0080] 4. Determination of broad-spectrum resistance of strain NWAFU56-41: The broad-spectrum resistance of NWAFU56-41 was verified using 11 fungal pathogens preserved in the laboratory. The 11 fungal pathogens were garlic leaf blight, gray mold, cucumber wilt, wheat stem base disease, potato dry rot, apple anthracnose, grape gray mold, wheat take-all disease, tomato wilt, garlic root rot, and cucumber anthracnose.

[0081] Preparation of fermentation broth for strain NWAFU56-41: A single colony of strain NWAFU56-41 was picked with a sterile inoculation loop and inoculated into LB liquid medium. The culture was then shaken on a shaker at 28°C and 200 r / min for 16 h. The resulting culture broth was the fermentation broth and was stored for later use.

[0082] Initial screening stage: The pathogen was selected as the target bacterium for antagonistic screening. A 5mm diameter punch was used to create 5mm diameter mycelial discs along the edge of the pathogen colony, which were then placed in the center of PDA medium. The treatment group (NWAFU56-41) was then inoculated using an inoculation loop from purified NWAFU56-41 single colonies, approximately 2.5cm above, below, to the left, and to the right of the mycelial disc. Each PDA medium was inoculated four times, with three replicates. A control group (CK) was used, inoculated only with the target bacterium. After sealing, the cultures were incubated at 28℃ for 7 days, and the inhibition rate was measured using the cross-hatching method.

[0083] Secondary screening stage: The following method was used to further verify the antagonistic effect of the bacterial strains selected in the initial screening (the operation method in the early stage was the same as that in the initial screening): Four holes were made in the treatment group (NWAFU56-41) with a 5 mm diameter punch at a distance of about 2.5 cm from the top, bottom, left and right of the bacterial cake, and 30 μL of NWAFU56-41 fermentation broth was inoculated. The control group (CK) was inoculated with only the target bacteria. The bacteria were cultured in a biochemical incubator at 28℃ for 7 days. Each treatment was repeated 3 times. The inhibition radius was recorded and the inhibition rate was calculated.

[0084] Inhibition rate (%) = (Coronavirus colony diameter of control pathogen - Coronavirus colony diameter of treatment pathogen) / Coronavirus colony diameter of control pathogen × 100%.

[0085] 5. Results: Table 6. Control efficacy of strain NWAFU56-41 against garlic leaf blight

[0086] As shown in Table 6, the disease incidence in garlic seedlings varied among different treatments. Compared to treatments involving inoculation with the garlic leaf blight pathogen alone, the NWAFU56-41 treatment group significantly alleviated the disease incidence in garlic seedlings. Figure 8 As shown, when treated with garlic leaf blight pathogen alone, the average disease index of garlic leaf blight was as high as 83.07%, while after treatment with pathogen infection following application of NWAFU56-41, the disease index of garlic leaf blight was 20.95%, indicating that strain NWAFU56-41 has a therapeutic effect on garlic leaf blight, and the therapeutic effect is good.

[0087] Table 7 Broad-spectrum resistance of strain NWAFU56-41

[0088] Strain NWAFU56-41 exhibits significant inhibitory effects against a variety of pathogens (as shown in Table 7 and...). Figure 9 a~ Figure 9 (As shown in k). Among them, the inhibition rate against grape gray mold reached 75.72%, the inhibition rate against wheat take-all disease reached 80.11%, and the inhibition rate against garlic root rot reached 69.79%. In addition, the inhibition rate against other tested pathogens also reached more than 50%, indicating that strain NWAFU56-41 has broad-spectrum resistance and excellent antibacterial effect.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A Bacillus (Bacillus sp.) NWAFU56-41, characterized in that, Bacillus The Bacillus NWAFU56-41 was deposited at the China Center for Type Culture Collection (CCTCC) on January 23, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026213. ​ 2. The application of Bacillus NWAFU56-41 as described in claim 1 in the prevention and control of plant diseases.

3. Use according to claim 2, characterized in that, The plant diseases mentioned include garlic leaf blight, gray mold, cucumber wilt, wheat stem base disease, potato dry rot, apple anthracnose, grape gray mold, wheat take-all disease, tomato wilt, garlic root rot, and cucumber anthracnose.

4. The application of Bacillus NWAFU56-41 as described in claim 1 in plant growth promotion.

5. Use according to claim 4, characterized in that, The Bacillus NWAFU56-41 promotes the growth of garlic plants and the accumulation of dry matter.

6. The application according to claim 4, characterized in that, The Bacillus NWAFU56-41 promotes the growth of cucumber seedlings.

7. The application according to claim 4, characterized in that, The Bacillus NWAFU56-41 promotes the growth of tomato seedlings.

8. A microbial inoculant for controlling plant diseases, characterized in that, The microbial agent comprises the Bacillus of claim 1, and the effective viable cell number of the Bacillus NWAFU56-41 in the microbial agent is 10 8 cfu / mL.

9. The microbial inoculant for controlling plant diseases according to claim 8, characterized in that, The plant diseases controlled by the microbial agents include garlic leaf blight, gray mold, cucumber wilt, wheat stem base disease, potato dry rot, apple anthracnose, grape gray mold, wheat take-all disease, tomato wilt, garlic root rot, and cucumber anthracnose.

10. A microbial inoculant for promoting plant growth, characterized in that, The microbial agent comprises the Bacillus of claim 1, and the effective viable cell number of the Bacillus NWAFU56-41 in the microbial agent is 10 8 cfu / mL.

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