Bacillus sp. NWAFU 21-25 and application thereof

Abstract

The application discloses a garlic leaf blight biocontrol and growth promoting bacterium Bacillus sp.NWAFU 21-25 and application thereof, and belongs to the technical field of microbial biocontrol agents. Bacillus sp.NWAFU 21-25 was preserved in China Center for Type Culture Collection on January 23, 2026, and the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2026211; has the functions of nitrogen fixation, potassium dissolution, organic phosphorus dissolution, proteinase production and cellulase production; through garlic seedling anti-leaf blight experiments and garlic, cucumber and tomato seedling growth promotion experiments, it is shown that the biocontrol strain can significantly promote the growth and development of garlic, cucumber and tomato seedlings, significantly alleviate the damage symptoms of garlic leaf blight, and has good broad-spectrum resistance to plant diseases, thereby providing a beneficial reference for breeding of strains suitable for resisting garlic leaf blight and promoting growth.

Description

Technical Field

[0001] This invention relates to the field of microbial biocontrol agents, and in particular to a biocontrol and growth-promoting bacterium for garlic leaf blight. Bacillus sp. NWAFU 21-25 and its applications. Background Technology

[0002] garlic( Allium sativum Garlic (Allium chinense) is a biennial herbaceous plant belonging to the genus Allium in the family Amaryllidaceae. It is widely cultivated worldwide and, due to its antibacterial, antioxidant, and anticancer properties, is commonly used as a vegetable, condiment, or medicinal plant, making it popular with consumers. Currently, garlic production mainly utilizes bulb-based asexual reproduction. Long-term continuous cropping has further 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, and in the middle and later stages, a black mold layer appears on the diseased leaves, eventually leading to the death of the entire leaf. 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, infected fields generally experience a 20%-30% yield reduction, while severely affected fields suffer yield reductions of over 50% or even total crop failure, causing serious economic losses to garlic farmers.

[0003] Currently, the control of garlic leaf blight in production relies on comprehensive measures, primarily 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 ecologically 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.

[0004] 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

[0005] The purpose of this invention is to provide a biocontrol and growth-promoting bacterium for garlic leaf blight. Bacillussp. NWAFU 21-25 and its application can significantly promote the growth and development of garlic, cucumber and tomato seedlings, and have good broad-spectrum resistance to plant diseases, especially garlic leaf blight.

[0006] To achieve the above objectives, this invention provides a biocontrol and growth-promoting bacterium for garlic leaf blight. Bacillus sp.NWAFU21-25, Bacillus sp. NWAFU 21-25 was deposited on January 23, 2026 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026211.

[0007] This invention also provides the above-mentioned biocontrol and growth-promoting bacteria for garlic leaf blight. Bacillus Application of sp. NWAFU 21-25 in the prevention and control of plant diseases.

[0008] Preferably, 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.

[0009] This invention also provides the above-mentioned biocontrol and growth-promoting bacteria for garlic leaf blight. Bacillus Application of sp. NWAFU 21-25 in the preparation of microbial inoculants for the prevention and control of plant diseases; in microbial inoculants Bacillus The effective viable count of sp. NWAFU 21-25 is 10. 8 cfu / mL; 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.

[0010] This invention also provides the above-mentioned biocontrol and growth-promoting bacteria for garlic leaf blight. Bacillus Application of sp. NWAFU 21-25 in promoting the growth of plant seedlings.

[0011] Preferred, Bacillus sp. NWAFU 21-25 promotes the growth of garlic plants and the accumulation of dry matter.

[0012] Preferred, Bacillus sp. NWAFU 21-25 promotes the growth of cucumber seedlings.

[0013] Preferred, Bacillus sp. NWAFU 21-25 promotes the growth of tomato seedlings.

[0014] This invention also provides the above-mentioned biocontrol and growth-promoting bacteria for garlic leaf blight. Bacillus The application of sp. NWAFU 21-25 in the preparation of microbial inoculants to promote the growth of plant seedlings; the effective viable count of Bacillus NWAFU 21-25 in the microbial inoculant is 10. 8 cfu / mL.

[0015] Therefore, this invention provides a biocontrol and growth-promoting bacterium for garlic leaf blight. Bacillus sp. NWAFU 21-25 and its application have the following beneficial effects: Bacillus sp. NWAFU 21-25 possesses nitrogen-fixing, potassium-solubilizing, organophosphate-solubilizing, protease-producing, and cellulase-producing functions. Experiments on garlic seedling resistance to leaf blight and growth-promoting experiments on garlic, cucumber, and tomato seedlings show that this strain can significantly promote the growth and development of garlic, cucumber, and tomato seedlings, and has a good alleviating effect on garlic leaf blight. It also has good broad-spectrum resistance, providing a useful reference for the breeding of strains suitable for resistance to garlic leaf blight and growth promotion.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention 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.

[0018] Figure 1 The image shows the inhibition of Bacillus NWAFU 21-25 against the pathogen causing garlic leaf blight; where a is the control group and b is the treatment group. Figure 2 The image shows the growth of Bacillus NWAFU 21-25 on LB solid medium; where a is a morphological image of the strain and b is a single colony image. Figure 3 Phylogenetic tree of Bacillus NWAFU 21-25; Figure 4 The results show the detection results of extracellular enzyme inhibition activity and growth-promoting characteristics of Bacillus NWAFU 21-25; where a is the result of nitrogen fixation ability, b is the result of organophosphorus solubilization ability, c is the result of protease production ability, d is the result of potassium solubilization ability, and e is the result of cellulase production ability. Figure 5 Results of growth promotion of garlic seedlings by inoculation with Bacillus NWAFU 21-25; where a represents the aboveground growth promotion result and b represents the underground growth promotion result. Figure 6 The results of cucumber seedling growth promotion after inoculation with Bacillus NWAFU 21-25 are shown; where a is a side view of cucumber seedlings in the control group and the treatment group, and b is a top view of cucumber seedlings in the control group and the treatment group. Figure 7 Results of growth promotion in tomato seedlings after inoculation with Bacillus NWAFU 21-25; Figure 8 The effect of Bacillus NWAFU 21-25 inoculation on the resistance of garlic seedlings to leaf blight; Figure 9 This is a diagram showing the broad-spectrum antibacterial activity of Bacillus NWAFU 21-25 against different pathogens; where a represents garlic leaf blight, b represents gray mold, c represents cucumber wilt, d represents wheat stem base disease, e represents potato dry rot, f represents apple anthracnose, g represents grape gray mold, h represents wheat take-all disease, i represents tomato wilt, j represents garlic root rot, and k represents cucumber anthracnose. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0022] The culture medium used in the examples is as follows: 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, 20g glucose, and distilled water to a final volume of 1L. Set the pH to its natural value and sterilize at 121℃ for 21 minutes before use.

[0023] PDB liquid culture medium: 200g potato, 20g sucrose, 1000mL distilled water, pH 7.0, sterilized at 121℃ for 21min before use.

[0024] LB solid medium: 5.0g yeast extract, 10g peptone, 10g sodium chloride, 18g agar, pH 7.0±0.1, distilled water to a final volume of 1L, autoclave at 121℃ for 15min.

[0025] LB liquid medium: 5.0g yeast extract, 10g peptone, 10g sodium chloride, pH 7.0±0.1, distilled water to a final volume of 1L, autoclave at 121℃ for 15min.

[0026] 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, distilled water to a final volume of 1L, autoclave at 121℃ for 15min.

[0027] 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.

[0028] 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 distilled water to a final volume of 1L and autoclave at 121℃ for 15 minutes.

[0029] 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 to a final volume of 1L, autoclave at 121℃ for 15min.

[0030] Cellulase detection medium: 10.0g peptone, 10.0g yeast extract, 10.0g sodium carboxycellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, 18g agar, distilled water to a final volume of 1L, autoclaved at 121℃ for 15min.

[0031] Silicate bacterial 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. Weigh 23.6g of this product, dilute with distilled water to 1L, and autoclave at 121℃ for 15min.

[0032] 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 to a final volume of 1L, autoclaved at 116℃ for 30min.

[0033] 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, autoclaved at 121℃ for 21min.

[0034] Preparation of colloidal chitin: Dissolve 20g of chitin in 350mL of concentrated hydrochloric acid, place at 4℃ for 24h, filter with glass wool, add 2L of ice-cold anhydrous ethanol to the filtrate and incubate at -20℃ overnight, centrifuge at 10000r / min for 20min, wash the precipitate continuously with running tap water until the pH is neutral, and store in a sealed container at -20℃.

[0035] Example 1 Using garlic from Qixian County preserved in the Vegetable Physiology and Biotechnology Laboratory as the test material, the collected garlic samples were peeled, 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 diluted with sterile water to a concentration of 10. -1 10 -2 10 -3 10 -4 10 -5 Spread each dilution of the sample onto LB agar plates and incubate at 28°C for 3-5 days. Use sterile water from the final rinsing of the sample during surface disinfection as a control, spreading it onto LB and NA plates to check the thoroughness of surface disinfection. After single colonies have grown, use a sterile inoculation loop to pick single colonies that differ in color, morphology, and size, streak them, and re-incubate them. Store the purified bacteria in 50% glycerol at -20°C for later use.

[0036] The pathogen causing garlic leaf blight was selected as the target bacterium for antagonistic bacteria screening. Using a 5mm diameter punch, holes were made along the edge of the pathogen colony to obtain 5mm diameter mycelial discs, which were then placed in the center of PDA medium. Colonies were then inoculated using an inoculation loop at approximately 2.5cm from the top, bottom, left, and right of the mycelial disc, with four inoculations per PDA medium and three replicates. A control (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] The antagonistic effect of the bacterial strains selected in the initial screening was further verified (the operation method in the early stage was the same as that in the initial screening): four holes were made at a distance of about 2.5 cm from the top, bottom, left and right of the bacterial cake using a 5 mm diameter punch, and 30 μL of bacterial fermentation broth was inoculated. The blank control (CK) was inoculated only with 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 of control pathogen - Coronavirus colony diameter of treatment pathogen) / Coronavirus colony diameter of control pathogen × 100%.

[0039] The inhibition diagram of Bacillus NWAFU 21-25 against the pathogen causing garlic leaf blight is shown below. Figure 1 As shown in the figure, a is the control group and b is the rescreening group.

[0040] The inhibition diameter and inhibition rate of Bacillus NWAFU 21-25 against the pathogen causing garlic leaf blight are shown in Table 1.

[0041] Table 1. Inhibition diameter and inhibition rate against garlic leaf blight pathogen.

[0042] As shown in Table 1, Bacillus NWAFU 21-25 inhibited mycelial growth of garlic leaf blight by 83.11% in the initial screening and 81.98% in the rescreening. The inhibition rates in the initial and rescreening were both above 80%, demonstrating strong antibacterial ability.

[0043] Example 2 NWAFU strain 21-25 was streaked on LB solid medium, sealed with sealing film, and incubated in a biochemical incubator at 28℃ for 2 days. After 2 days, the culture dish was removed and the basic morphological characteristics such as colony morphology, size, color, texture viscosity, gloss, transparency, and edge smoothness were observed and recorded by taking pictures.

[0044] The results are as follows Figure 2 As shown, a is a morphological image of the bacterial strain, and b is a single colony image. Morphological observation shows that the bacterial strain appears white and irregular on LB medium, with serrated edges, a rough and wrinkled surface, and is opaque. The bacterial cells are slightly moist, viscous, and easy to pick up.

[0045] Example 3 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.1) and 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO.2). 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.

[0046] The ITS sequence of Bacillus NWAFU 21-25 is shown in SEQ ID NO.3.

[0047] SEQ ID NO.3:

[0048] Phylogenetic tree of Bacillus NWAFU 21-25 as follows Figure 3 As shown. Based on the homology comparison analysis results, the gene sequence of Bacillus NWAFU 21-25 is similar to... Bacillus halotolerans strain SY1836 is in the same branch. Based on the phylogenetic tree results, it is inferred that NWAFU 21-25 belongs to halophilic Bacillus. Bacillus halotolerans ).

[0049] Bacillus ( Bacillus sp.) NWAFU 21-25 was deposited on January 23, 2026 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026211.

[0050] Example 4 Single colonies of Bacillus NWAFU 21-25 were picked using a sterile inoculation loop and inoculated into LB broth. The culture was then incubated on a shaker at 28°C and 200 rpm for 16 hours to obtain the NWAFU 21-25 fermentation broth, which was stored for later use. 10 μL of the NWAFU 21-25 fermentation broth was spot-inoculated approximately 2.5 cm above, below, to the left, and to the right of the center of each plate. Each medium was inoculated four times, with three replicates. The plates were incubated at 28°C for 2–7 days. The presence of a clear zone was observed, and the colony diameter (d) and the clear zone diameter (D) were measured. The presence or absence of a clear zone indicated enzyme activity; enzyme activity was positively correlated with the HC value (D / d).

[0051] Protease production capacity: Bacillus NWAFU 21-25 was inoculated onto a protein detection medium plate, and the presence or absence of a clear zone was observed after 3 days.

[0052] Cellulase production capacity: Bacillus NWAFU 21-25 was inoculated onto cellulase detection medium plates. After 2-3 days, 1 mg / mL Congo red solution was added for staining for 30 min. The staining solution was discarded, and the plates were washed with 1 mol / L sodium chloride for 30 min. The presence or absence of a clear zone was observed.

[0053] Chitinase production capacity: Bacillus NWAFU 21-25 was inoculated onto chitinase detection medium plates and cultured for 7 days. Observe whether a clear zone appears.

[0054] The growth-promoting properties of Bacillus NWAFU 21-25 were detected, including its nitrogen fixation capacity, ability to dissolve inorganic phosphorus and organic phosphorus, potassium solubilization, siderophore production, and IAA production capacity.

[0055] Nitrogen fixation capacity: Bacillus NWAFU 21-25 was inoculated into Assoube medium and cultured for 7 days to observe whether there was bacterial growth.

[0056] Phosphate-solubilizing ability: Bacillus NWAFU 21-25 was inoculated onto solid organic and inorganic phosphate-solubilizing medium plates and cultured for 7 days to observe whether a transparent phosphate-solubilizing zone appeared.

[0057] Siderogenetic carrier: Bacillus NWAFU 21-25 was inoculated onto chromium azure CAS plates, and the presence of a yellow-green halo was observed after 7 days.

[0058] IAA detection: Take 1 mL of the cultured NWAFU 21-25 bacterial suspension and inoculate it into a liquid medium containing L-tryptophan. Incubate at 28℃ and 200 r / min for 24 h. Take 100 μL of the bacterial suspension 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.

[0059] Potassium solubilization capacity: Bacillus NWAFU 21-25 was inoculated into silicate medium and cultured for 7 days to observe whether it produced an oily liquid.

[0060] The results of the detection of extracellular enzyme inhibition activity and growth-promoting properties of Bacillus NWAFU 21-25 are shown in Table 2 and Figure 4 As shown.

[0061] Table 2. Results of detection of extracellular enzyme inhibitory activity and growth-promoting properties of Bacillus NWAFU 21-25

[0062] Note: "+" sign indicates that the ability exists, and "-" sign indicates that the ability does not exist.

[0063] From Table 2, Figure 4 It can be seen that the extracellular enzyme inhibition activity of Bacillus NWAFU 21-25 is mainly manifested in the function of producing protease and cellulase, but not in the function of producing chitinase, and the protease activity value is 0.79, and the cellulase activity value is 0.78; the growth-promoting characteristics are manifested in the functions of nitrogen fixation, potassium solubilization and organic phosphorus dissolution, but not in the function of inorganic phosphorus dissolution.

[0064] Example 5 To verify the growth-promoting effect of Bacillus NWAFU 21-25 on garlic, cucumber, and tomato seedlings, the tested varieties were "Soviet Red Skin Garlic (Improved Garlic)" garlic, "Bonai" cucumber, and "Ailsa Craig" tomato, which were preserved in the Vegetable Cultivation Physiology, Ecology and Biotechnology Laboratory of the College of Horticulture, Northwest A&F University.

[0065] The supernatant from the centrifuged Bacillus NWAFU 21-25 fermentation broth was diluted with ddH2O to adjust the OD. 600 The value is 0.8 (10) 8 (cfu / mL) and set aside for later use.

[0066] 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%.

[0067] (1) Experiment on promoting the growth of garlic seedlings: Healthy garlic cloves of uniform size were selected and sown in seedling pots containing high-pressure moist heat sterilization substrate. The diameter of the seedling pot × bottom diameter × height of the pot was 7cm × 5cm × 7.3cm. One garlic seedling with uniform growth was planted in each pot. When the garlic seedlings grew to 3 leaves, the roots were evenly irrigated with Bacillus subtilis NWAFU 21-25 fermentation liquid around the base of the seedling stem. 10mL of Bacillus subtilis was irrigated per seedling. CK was the same amount of water only. 21 seedlings were inoculated in each treatment, and the treatment was repeated 3 times. The bacterial solution was inoculated again after 7 days. 7 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.

[0068] The formula for calculating the seedling strength index is as follows: Seedling strength index = (stem diameter / plant height + root dry weight / aboveground dry weight) × total plant dry weight.

[0069] Seven days after the second inoculation, the growth of garlic seedlings was observed, and the results were as follows: Figure 5 As shown in Table 3.

[0070] Table 3. Growth-promoting effects of Bacillus NWAFU 21-25 on garlic seedlings.

[0071] Note: Different lowercase letters in the same column indicate significant differences. P <0.05).

[0072] Depend on Figure 5As shown in Table 3, compared with the control group, the fresh weight and dry weight of garlic seedlings treated with NWAFU 21-25 fermentation broth increased significantly, by 31.65% and 30.16%, respectively. Meanwhile, inoculation with NWAFU 21-25 also promoted the growth of garlic stem diameter, leaf area, and leaf number.

[0073] Therefore, Bacillus NWAFU 21-25 has the characteristics of a rhizosphere growth promoter (PGPR), which can promote the growth and development of garlic plants and the accumulation of dry matter.

[0074] (2) Growth-promoting experiment of 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 diameter of the seedling pot × bottom diameter × height of the pot was 7cm × 5cm × 7.3cm. When the cucumber and tomato seedlings had three leaves and one heart, seedlings with uniform growth were selected and 10mL of Bacillus subtilis NWAFU 21-25 fermentation liquid was poured into the base of the stem within 2cm. Each treatment was inoculated with 21 seedlings, and the results were repeated 3 times. The control group was inoculated with an equal amount of water, and after 7 days, the bacterial solution was inoculated again. 7 days after the second inoculation, 10 representative plants were randomly selected from each treatment, and the plant height, stem diameter, leaf area, number of leaves, dry weight, and fresh weight of cucumber and tomato seedlings were measured, and the seedling vigor index was calculated.

[0075] Strong seedling index = (stem diameter / plant height + root dry weight / aboveground dry weight) × total plant dry weight.

[0076] ① The results of the experiment on the effect of Bacillus NWAFU 21-25 fermentation broth on cucumber seedling biomass are as follows: Figure 6 As shown in Table 4.

[0077] Table 4. Growth-promoting effects of Bacillus NWAFU 21-25 on cucumber seedlings.

[0078] Note: Different lowercase letters in the same column indicate significant differences. P <0.05).

[0079] From Table 4 and Figure 6 It was found that the growth traits of cucumber seedlings treated with Bacillus NWAFU 21-25 fermentation broth were significantly better than those of the control, and it significantly promoted the growth of cucumber seedlings. Among them, the fresh weight and dry weight of cucumber seedlings increased significantly, by 16.91% and 29.61% respectively compared with the control. Bacillus NWAFU 21-25 can promote the growth of cucumber seedlings.

[0080] ② The growth-promoting effect of Bacillus NWAFU 21-25 on tomato seedlings is as follows: Figure 7 As shown in Table 5.

[0081] Table 5. Growth-promoting effects of Bacillus NWAFU 21-25 on tomato seedlings.

[0082] Note: Different lowercase letters in the same column indicate significant differences. P <0.05).

[0083] Depend on Figure 7 As shown in Table 5, compared with the control, Bacillus NWAFU 21-25 promoted the growth indicators of tomato seedlings to varying degrees. Treatment with NWAFU 21-25 fermentation broth increased the plant height, stem diameter, and dry weight of tomato seedlings by 16.97%, 12.03%, and 34.57%, respectively, compared to the control. This indicates that NWAFU 21-25 is a rhizosphere growth promoter (PGPR) that can promote the growth of tomato seedlings.

[0084] Example 6 The control effect and broad-spectrum resistance of garlic leaf blight by inoculation with Bacillus NWAFU 21-25.

[0085] Garlic leaf blight pathogen ( 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 dilute the concentration to 10 with sterile water. 5 CFU / mL is prepared for later use.

[0086] Surface-sterilized garlic bulbs were planted in sterile nutrient soil. When the garlic bulbs reached the 3-leaf stage, an inoculation experiment using NWAFU 21-25 was conducted; the endophytic bacteria to be tested in the garlic were prepared as OD (Organic Dioxide) samples. 600 After preparing a bacterial suspension with a pH of 0.8, place it in a spray bottle and spray the mixed endophytic bacterial solution evenly onto the leaves. Spray once every three days, and then allow the leaves to rest for another three days after each spraying. Establish 20 parallel strains per plant for the endophytic bacteria. Then, inoculate with a spore suspension of the garlic leaf blight pathogen.

[0087] Add a concentration of 10% to a sterilized 100mL spray bottle. 5 A spore suspension of *Pseudomonas aeruginosa* (containing 1 drop of Tween 20 to aid adsorption) was applied to the plantlets (approximately 5 mL per plant). Each treatment was replicated 20 times, with sterile water serving as a control. After inoculation, the plants were placed in the dark and kept moist to allow spore germination, and then allowed to develop naturally at approximately 21°C.

[0088] After inoculation with the pathogen of garlic leaf blight, the disease status of garlic leaves and the disease status of plants were observed at 7, 9, 11 and 13 days of growth, and the incidence rate and disease index of garlic were calculated.

[0089] The grading standards for leaf blight are as follows: Grade 0, no lesions on the leaves; Grade 1, lesions covering less than 5% of the total leaf area; Grade 3, lesions covering 6%-25% of the total leaf area; Grade 5, lesions covering 26%-50% of the total leaf area; Grade 7, lesions covering 51%-75% of the total leaf area; Grade 9, lesions covering more than 76% of the total leaf area.

[0090] Then, calculate the disease index using the following method: .

[0091] The control effect of Bacillus NWAFU 21-25 on garlic leaf blight is as follows: Figure 8 As shown in Table 6.

[0092] Table 6. Control efficacy of Bacillus NWAFU 21-25 against garlic leaf blight.

[0093] Depend on Figure 8 As shown in Table 6, there were differences in the disease incidence of garlic seedlings under different treatments. Compared with the treatment of inoculation with the garlic leaf blight pathogen alone, the NWAFU 21-25 treatment group significantly alleviated the disease incidence in garlic seedlings. When inoculated with the garlic leaf blight pathogen alone, the average disease index of garlic leaf blight was as high as 83.07%, while after application of NWAFU 21-25 and subsequent pathogen infection treatment, the disease index of garlic leaf blight was 23.81%, indicating that Bacillus NWAFU 21-25 has a therapeutic effect on garlic leaf blight, and the therapeutic effect is relatively good.

[0094] Example 7 The broad-spectrum resistance to NWAFU 21-25 was verified using 11 fungal pathogens: 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. The testing method is as shown in Example 1.

[0095] The test results are shown in Table 7 and Figure 9 As shown.

[0096] Table 7 Broad-spectrum resistance to Bacillus NWAFU 21-25

[0097] From Table 7 and Figure 9It was found that Bacillus NWAFU 21-25 exhibited significant inhibitory effects against a variety of pathogens. Specifically, it showed an inhibition rate of 74.91% against grape gray mold, 80.38% against wheat take-all, and 70.56% against garlic root rot, all exceeding 70%. Furthermore, it also showed inhibition rates of over 50% against other tested pathogens, indicating that Bacillus NWAFU 21-25 possesses broad-spectrum resistance and excellent antibacterial effects.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A plant with garlic leaf blight biocontrol and growth-promoting bacteria Bacillus sp.NWAFU 21-25, characterized in that: Bacillus sp. NWAFU 21-25 was deposited on January 23, 2026 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026211; Bacillus The ITS sequences of sp. NWAFU 21-25 are shown in SEQ ID NO.

3.

2. The plant-based biocontrol and growth-promoting bacteria for garlic leaf blight as described in claim 1 Bacillus Sp. NWAFU 21-25 is used in the prevention and control of plant diseases.

3. The application according to claim 2, characterized in that: 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.

4. The plant-based biocontrol and growth-promoting bacteria for garlic leaf blight as described in claim 1 Bacillus The application of sp. NWAFU 21-25 in the preparation of microbial agents for the prevention and control of plant diseases is characterized by: Microbial agents Bacillus The effective viable count of sp. NWAFU21-25 is 10. 8 cfu / mL; 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.

5. The plant-based biocontrol and growth-promoting bacteria for garlic leaf blight as described in claim 1 Bacillus Application of sp. NWAFU 21-25 in promoting the growth of plant seedlings.

6. The application according to claim 5, characterized in that: Bacillus sp. NWAFU 21-25 promotes the growth of garlic plants and the accumulation of dry matter.

7. The application according to claim 5, characterized in that: Bacillus sp. NWAFU 21-25 promotes the growth of cucumber seedlings.

8. The application according to claim 5, characterized in that: Bacillus sp. NWAFU 21-25 promotes the growth of tomato seedlings.

9. A plant-based biocontrol and growth-promoting bacterium for garlic leaf blight as described in claim 1 Bacillus The application of sp. NWAFU 21-25 in the preparation of microbial agents that promote the growth of plant seedlings is characterized by: Microbial agents Bacillus The effective viable count of sp. NWAFU 21-25 is 10. 8 cfu / mL.

Images