Bacillus siamensis capable of producing volatile broad-spectrum antibacterial substances and application thereof

Abstract

This invention relates to a strain of *Bacillus sicca* capable of producing volatile broad-spectrum antibacterial substances and its applications, belonging to the field of biological control technology. To address the shortcomings of chemical control of tomato gray mold, this invention provides a strain of *Bacillus sicca* and its applications. This strain, isolated from honeysuckle, produces IAA (inhibitory acetic acid), antimicrobial peptides, and volatile organic compounds. The volatile organic compounds produced by this strain exhibit broad-spectrum antibacterial activity, achieving a 100% inhibition rate against *Botrytis cinerea*, *Fusarium oxysporum*, *Fusarium graminearum*, and *Cucumis mellitus* var. *cubiclesii*. Furthermore, this strain demonstrates excellent control efficacy against tomato gray mold caused by *Botrytis cinerea* and can be used for the control of gray mold in tomato plants and for fruit preservation. Compared to traditional control measures, the *Bacillus sicca* strain provided by this invention offers advantages such as high efficiency, broad-spectrum disease resistance, easy spread and transmission, wide coverage, and low likelihood of developing drug resistance, making it a promising candidate for controlling diseases in greenhouse fruits and vegetables.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to a strain of Bacillus sicca that can produce volatile broad-spectrum antibacterial substances and its applications. Background Technology

[0002] Gray mold, a common tomato disease, primarily affects the flowers, fruits, leaves, and stems of tomatoes. Infected flowers cause flower drop, preventing normal fruit set and impacting the fruit-setting rate. Infected fruits have significantly reduced commercial value; young fruits will fall off directly, while mature fruits will rot and become inedible. Leaf infection reduces photosynthetic area, hindering plant growth and nutrient accumulation. Severe stem infection damages the plant's vascular tissues, leading to the entire plant's wilting and death. Under suitable environmental conditions, gray mold can cause severe yield losses. Without control, yield losses can reach 30%-50% in years with widespread outbreaks. Furthermore, this loss not only manifests as reduced yield in the current season but can also have lasting adverse effects on subsequent planting seasons due to the accumulation of the pathogen in the soil and other environmental environments.

[0003] To prevent tomatoes from being threatened by post-harvest diseases such as gray mold, chemical pesticides are commonly used for control. However, the use of chemical pesticides has many adverse effects, such as environmental damage, threats to human health, induction of drug resistance in pathogens, and slow degradation, posing significant risks to human health and well-being. Biological control, on the other hand, offers advantages. Using Bacillus subtilis for biological control has advantages such as low cost, environmental friendliness, harmlessness to humans, easy degradation, no residue, and no induction of resistance. Therefore, biological control is attracting increasing attention.

[0004] In nature, some microorganisms produce volatile antimicrobial organic compounds that can effectively inhibit the growth of pathogens even at extremely low concentrations. Furthermore, these compounds possess advantages such as strong permeability, high diffusion efficiency, no residue, easy degradation, and no induction of resistance. Therefore, utilizing these volatile organic compound-producing microorganisms for biological control is an effective alternative to chemical control. Promoting this technology can simultaneously inhibit multiple pathogens, reduce the environmental impact of chemical drugs, and safeguard human health. Summary of the Invention

[0005] To address the shortcomings of chemical control methods for tomato gray mold, this invention provides a strain of Bacillus siamensis NEAU-ZGX24 that produces volatile broad-spectrum antibacterial substances and its applications. The specific technical solution is as follows:

[0006] The first objective of this invention is to provide a strain of Bacillus siamensis NEAU-ZGX24, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 20242176, deposited on October 14, 2024, at Wuhan University, Wuhan, China.

[0007] Further specifying, the Bacillus NEAU-ZGX24 of Siam can produce IAA, antimicrobial peptides and volatile antimicrobial substances; the volatile antimicrobial substances include 3-methylbutyric acid, ethyl heptaate, and valeric acid.

[0008] The second objective of this invention is to provide the application of the above-mentioned Bacillus NEAU-ZGX24 in inhibiting the mycelial growth of Botrytis cinerea.

[0009] A third objective of this invention is to provide the application of the antimicrobial peptide produced by the above-mentioned Bacillus NEAU-ZGX24 in inhibiting the mycelial growth of Botrytis cinerea.

[0010] A fourth objective of this invention is to provide the application of the volatile organic compounds produced by the above-mentioned Bacillus NEAU-ZGX24 in inhibiting the growth of plant pathogenic fungi.

[0011] In one embodiment of the present invention, the plant pathogenic fungi include, but are not limited to, *Botrytis cinerea*, *Fusarium moniliforme*, *Fusarium graminearum*, *Fusarium solani*, *Stagonosporopsis cucurbitacearum*, *Bipolaris sorokiniana*, *Fusarium oxysporum*, *Fusarium subglutinans*, *Fusarium verticillioides*, *Verticillium dahliae*, *Colletotrichum orbiculare*, and *Magnaporthe oryzae*.

[0012] In one embodiment of the present invention, the volatile organic compounds include 14 compounds, among which the substances that exert the disease-resistant effect are 3-methylbutyric acid, ethyl heptaate, and valeric acid.

[0013] The fifth objective of this invention is to provide the application of the above-mentioned Bacillus NEAU-ZGX24 in the prevention and control of postharvest gray mold in tomatoes, wherein the application method of Bacillus NEAU-ZGX24 is fumigation.

[0014] The sixth objective of this invention is to provide the application of the above-mentioned Bacillus NEAU-ZGX24 in the prevention and control of gray mold infection during tomato growth, wherein the application method of Bacillus NEAU-ZGX24 is fumigation.

[0015] The seventh objective of this invention is to provide an application of a fungicide containing the above-mentioned Bacillus NEAU-ZGX24 in the prevention and control of postharvest gray mold of tomatoes or gray mold infection during tomato growth.

[0016] The beneficial effects of this invention are:

[0017] The Bacillus thymosus NEAU-ZGX24 provided by this invention was isolated from honeysuckle and is an endophytic bacterium of medicinal plants that is harmless to the environment. This invention demonstrates through plate confrontation experiments that *Bacillus sicca* NEAU-ZGX24 exhibits good inhibitory effects against *Botrytis cinerea*, with an inhibition rate reaching 54.10%. It was also found that this strain can produce indoleacetic acid, antimicrobial peptides, and volatile organic compounds (VOCs). Furthermore, the antimicrobial peptides produced by this strain also have inhibitory effects on *Botrytis cinerea*, and the VOCs produced have strong inhibitory effects on 12 plant pathogenic fungi, with an inhibition rate of 100% against *Botrytis cinerea*, *Fusarium oxysporum*, *Fusarium graminearum*, and *Cucumis melo* wilt. Furthermore, this invention identified and tested the inhibitory activity of the VOCs produced by strain NEAU-ZGX24, finding that among 14 VOCs, 3-methylbutyric acid, ethyl heptanoate, and valeric acid showed the most significant inhibitory effects against *Botrytis cinerea*, with these three substances achieving a 100% inhibition rate at a concentration of 100 μL / L.

[0018] Furthermore, this invention investigated the biocontrol effects of strain NEAU-ZGX24 on tomato fruits, detached tomato leaves, and tomato plants. It was found that fumigation with strain NEAU-ZGX24 effectively controlled postharvest gray mold in tomatoes, significantly improving the edible and marketable fruit rates, with a control efficacy of 82.73%. Fumigation with strain NEAU-ZGX24 also effectively controlled gray mold in tomato leaves, achieving a control efficacy of 94.81%, and effectively controlled gray mold in tomato plants as well, with a control efficacy exceeding 70%.

[0019] Furthermore, the volatile substances produced by Bacillus thymosus NEAU-ZGX24 have a good preservative effect on tomato fruits. Compared with traditional control measures, using Bacillus thymosus NEAU-ZGX24 to control plant diseases has advantages such as high efficiency, broad-spectrum disease resistance, easy spread and wide coverage, and low likelihood of developing resistance. It has broad application prospects in the control of diseases in greenhouse fruits and vegetables. Attached Figure Description

[0020] Figure 1 A phylogenetic tree constructed based on the 16S rRNA sequence of strain NEAU-ZGX24;

[0021] Figure 2 The figure shows the results of determining the inhibitory activity of strain NEAU-ZGX24 against Botrytis cinerea using the plate confrontation method.

[0022] Figure 3 The figure shows the results of determining the inhibitory activity of lipopeptides produced by strain NEAU-ZGX24 against Botrytis cinerea using the plate confrontation method.

[0023] Figure 4 The image shows the inhibitory effect of VOCs produced by strain NEAU-ZGX24 on 12 plant pathogenic fungi, as determined by the plate-to-plate assay.

[0024] Figure 5 A statistical chart showing the inhibition rate of VOCs produced by strain NEAU-ZGX24 against 12 plant pathogenic fungi;

[0025] Figure 6 The image shows the detection results of indoleacetic acid production by strain NEAU-ZGX24.

[0026] Figure 7 The effect of fumigation using VOCs produced by strain NEAU-ZGX24 to control postharvest gray mold in tomatoes is illustrated.

[0027] Figure 8 The effect of using the fumigation of VOCs produced by strain NEAU-ZGX24 to control tomato leaves infected with tomato gray mold is shown in the figure.

[0028] Figure 9 The figure shows the statistical results of the inhibition rates of 14 VOCs produced by strain NEAU-ZGX24 against Botrytis cinerea. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. In the art, any embodiments obtained by those skilled in the art without creative effort are protected by this invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0031] The Bacillus siamensis NEAU-ZGX24 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242176, deposit date October 14, 2024, and address of the depositary institution: Wuhan University, Wuhan, China.

[0032] The culture media and their formulations involved in the following implementation examples are as follows:

[0033] PDA medium: Wash 200g of potatoes, cut them into small pieces, boil for about 30 minutes, filter through four layers of gauze, add tap water to make up to 1000mL of the filtrate, add 20g of glucose and 20g of agar.

[0034] LB medium: 1g tryptone, 0.5g yeast extract, 1g sodium chloride, 100mL distilled water. In addition, if preparing solid medium, 2g of agar should be added.

[0035] Example 1: Isolation and identification of Bacillus neuraminidum NEAU-ZGX24

[0036] 1. Isolation of strain NEAU-ZGX24

[0037] The surface of honeysuckle from Linyi City, Shandong Province was disinfected using a seven-step disinfection method: (1) rinsed for 1 min with an aqueous solution containing actinomycete ketone (100 mg / L) and naphthiocarboxylic acid (20 mg / L); (2) rinsed 3 times with sterile water; (3) rinsed for 5 min with 5% NaOCl solution; (4) rinsed for 10 min with 2.5% Na2S2O3 solution; (5) rinsed for 5 min with 75% ethanol solution; (6) rinsed 3 times with sterile water; (7) rinsed for 10 min with 10% NaHCO3 solution. Each 100 mg sample was mixed with 1 mL of water, ground in a mortar, and the liquid was diluted to 10 mL.-1 10 -2 10 -3 200 μL of the diluted liquid was spread onto LB agar plates containing 100 mg / L actinomycete ketone, with three replicates for each treatment. The inoculated plates were sealed and incubated at 37°C for 1–3 days. Different single colonies were then streaked onto fresh LB agar plates for purification. A single bacterium was obtained after purification and cultured, designated NEAU-ZGX24, and stored in a -80°C cryogenic freezer with 20% glycerol.

[0038] 2. Identification of strain NEAU-ZGX24

[0039] (1) Morphological identification of strain NEAU-ZGX24

[0040] The colony characteristics of strain NEAU-ZGX24 on LB solid medium are milky white, opaque, nearly round in shape, and wrinkled on the surface, which is consistent with the morphological characteristics of Bacillus.

[0041] (2) Molecular biological identification of strain NEAU-ZGX24

[0042] Molecular biological identification of strain NEAU-ZGX24 was performed: the 16S rRNA sequence was obtained by PCR and submitted to GenBank, with accession number PQ270443. Gene sequence alignment was performed using the EZ website (https: / / www.ezbiocloud.net / ), and phylogenetic analysis was conducted using the neighbor-joining (NJ) method with MEGA7 software to construct a phylogenetic tree.

[0043] The results show (see) Figure 1 The 16S rRNA sequence of strain NEAU-ZGX24 is most similar to that of Bacillus siamensis KCTC 13613, with a similarity of 99.86%, and the two cluster on the same branch on the phylogenetic tree. Therefore, strain NEAU-ZGX24 can be identified as a member of Bacillus siamensis.

[0044] The strain NEAU-ZGX24 screened in this invention was deposited at the China Center for Type Culture Collection on October 14, 2024, with accession number CCTCC NO: M 20242176, and the address of the depository is Wuhan University, Wuhan, China.

[0045] Example 2: Screening for antagonistic activity of Bacillus thymus NEAU-ZGX24

[0046] This embodiment aims to investigate the antagonistic activity of strain NEAU-ZGX24 against Botrytis cinerea, which causes gray mold in tomatoes, and its broad-spectrum antibacterial activity.

[0047] The plant pathogens used in this study are 12 common plant pathogenic fungi in agricultural production. The species and their sources are as follows: *Botrytis cinerea*, *Fusarium moniliforme*, *Fusarium graminearum*, *Fusarium solani*, *Stagonosporopsis cucurbitacearum*, *Bipolaris sorokiniana*, *Fusarium oxysporum*, *Fusarium subglutinans*, *Fusarium verticillioides*, *Verticillium dahliae*, *Colletotrichum orbiculare*, and *Magnaporthe oryzae*, provided by Northeast Agricultural University.

[0048] Pathogen culture method: All pathogens were inoculated onto PDA medium and cultured in a constant temperature incubator at 28℃ for optimal results.

[0049] 1. Determination of the antagonistic activity of strain NEAU-ZGX24 against Botrytis cinerea

[0050] The antagonistic activity of strain NEAU-ZGX24 against Botrytis cinerea was determined using the plate confrontation method, and the specific steps are as follows:

[0051] In a sterile operating room, using a 6mm diameter punch, mycelial cakes were collected from the edge of pathogen colonies cultured for 7 days. Using a sterile toothpick, the mycelial cakes were inoculated onto one side of a PDA plate (with the mycelial side facing the culture medium). Using a sterile toothpick, purified strain NEAU-ZGX24 was streaked onto the other side of the PDA plate. The plate was then sealed. The control group was inoculated only with the pathogen. Three replicates were performed. All treatments were incubated at 28°C for 7 days, and the inhibition rate was calculated.

[0052] Inhibition rate % = (distance from bacteria to the front of the pathogen colony / distance from bacteria to the front of the pathogen cell disc) × 100.

[0053] The results of the antibacterial activity were as follows (see Figure 2 The strain NEAU-ZGX24 showed a significant inhibitory effect on Botrytis cinerea, with an inhibition rate of 54.10%.

[0054] 2. Detection of the antagonistic activity of lipopeptides produced by strain NEAU-ZGX24 against Botrytis cinerea.

[0055] The acid precipitation method was used to detect the lipopeptides produced by strain NEAU-ZGX24. The specific steps are as follows:

[0056] The NEAU-ZGX24 strain was fermented in LB medium for 3 days. The resulting fermentation broth was centrifuged at 10,000 rpm for 10 minutes at 4°C. Crude lipopeptides were obtained by acid precipitation. Specifically, the pH of the supernatant was adjusted to 2.0, and the sample was incubated overnight at 4°C to precipitate. The suspension containing the precipitate was centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, and the precipitate was extracted with methanol. Finally, the methanol containing the dissolved compound was concentrated by rotary evaporation, and the concentrate was dissolved in methanol. The antibacterial activity was verified by plate confrontation experiments.

[0057] The results show (see) Figure 3 The strain NEAU-ZGX24 can produce lipopeptides, which have a certain inhibitory effect on Botrytis cinerea.

[0058] 3. Broad-spectrum antibacterial experiment on volatile organic compounds (VOCs) produced by strain NEAU-ZGX24

[0059] The broad-spectrum antibacterial activity of volatile organic compounds (VOCs) produced by strain NEAU-ZGX24 was detected using the plate-on-plate method. The specific method is as follows:

[0060] In a sterile operating room, 200 μL of the purified NEAU-ZGX24 bacterial suspension was spread onto an LB agar plate, sealed, and incubated at 37°C for 8–12 hours. Using a 6 mm diameter punch, holes were made in a 7-day cultured plate of pathogenic bacteria. The perforated mycelial discs were placed in the center of a fresh PDA agar plate (with the mycelial side facing the culture medium), and then inverted onto the plate containing the cultured bacteria. The plate was sealed. The control group was inoculated with pathogenic bacteria and then inverted onto the LB agar plate. Three replicates were performed. All treatments were incubated at 28°C for 7 days. The diameter of the pathogenic colonies was measured, and the inhibition rate was calculated.

[0061] Inhibition rate % = (control colony area - treated colony area) / control colony area × 100.

[0062] The results show (see) Figure 4 , Figure 5The VOCs produced by strain NEAU-ZGX24 have a strong inhibitory effect on all 12 pathogens mentioned above. Among them, the inhibition rate against Botrytis cinerea, Fusarium moniliforme, Fusarium graminearum, and Stagonosporopsis cucurbitacearum reached 100%.

[0063] Example 3: Determination of plant growth-promoting characteristics of Bacillus NEAU-ZGX24

[0064] This embodiment mainly investigates the ability of strain NEAU-ZGX24 to produce indoleacetic acid, specifically using a colorimetric method: a single colony of strain NEAU-ZGX24 was transferred to a 5 mL LB liquid test tube containing 0.002 g / mL tryptophan, and cultured at 37°C for 24 h. After centrifugation, the supernatant was collected, and 2 times the volume of colorimetric solution was added to the supernatant. For the control group, 2 times the volume of colorimetric solution was added to the same liquid culture medium without bacteria. The mixture was shaken and then observed for any color change.

[0065] The results show (see) Figure 6 Compared with the control group CK, the experimental group showed a light red color, indicating that strain NEAU-ZGX24 can produce indoleacetic acid.

[0066] Example 4: Application of Bacillus thymus NEAU-ZGX24 in the control of tomato gray mold

[0067] This embodiment investigates the biocontrol effect of strain NEAU-ZGX24 on tomato fruit, detached tomato leaves, and tomato plants.

[0068] 1. Experiment on the control of postharvest gray mold of tomato fruit by strain NEAU-ZGX24

[0069] In a sterile operating room, 200 μL of the purified NEAU-ZGX24 bacterial suspension was spread onto an LB agar plate, sealed, and incubated at 37°C for 8–12 hours for later use. Tomato fruits were disinfected with 75% alcohol, then rinsed three times with sterile water and air-dried. Holes were made in the surface of the tomato fruits using a toothpick. Botrytis cinerea, which had grown on PDA plates for 15 days, was then punched with a 6 mm diameter punch. All mycelia from each mycelial cake were collected and filled into the punched tomato fruits, and the following treatment was performed:

[0070] CK: Tomato fruits are punctured and placed in a sealed container with LB plates for fumigation;

[0071] BCK: Puncture holes in tomato fruits, add pathogenic fungal hyphae to the wounds, and fumigate with LB plates in a sealed container;

[0072] Treatment: Puncture holes in the tomato fruit, add pathogenic fungal hyphae to the wounds, and fumigate with NEAU-ZGX24 fungal board in a sealed container.

[0073] Each treatment was repeated three times. After fumigation at 28℃ for 7 days, the disease incidence was recorded, and the incidence rate, disease index, marketable fruit rate, and edible fruit rate were calculated according to the tomato fruit grading standard.

[0074] The severity of diseased fruit is classified into 5 levels:

[0075] Grade 0: No lesions;

[0076] Grade 1: Lesions ≤ 5%;

[0077] Grade 2: 5% < lesions ≤ 10%;

[0078] Grade 3: 10% < lesions ≤ 20%;

[0079] Grade 4: 20% < lesions.

[0080] Disease index (%) = 100% × ∑(Disease level of diseased fruit × Number of fruits at that level) / (Highest disease level of diseased fruit × Total number of test fruits);

[0081] Marketable fruit rate (%) = 100% × [(Number of grade 0 fruits + Number of grade 1 fruits) / Total number of fruits];

[0082] Edible fruit rate (%) = 100% × [(Number of grade 0 fruits + Number of grade 1 fruits + Number of grade 2 fruits) / Total number of fruits];

[0083] Incidence rate (%) = 100% × (number of diseased fruits / total number of fruits).

[0084] In an experiment to control postharvest gray mold in tomatoes, the BCK group, which only had Botrytis cinerea mycelium added, showed obvious rotten lesions. However, the disease index of fruits fumigated with VOCs produced by strain NEAU-ZGX24 was significantly reduced (P<0.05), and the incidence of disease was extremely low, indicating a better control effect with a control efficacy approaching 82.73% (see Table 1 and 2). Figure 7 Therefore, the VOCs produced by strain NEAU-ZGX24 are a good biocontrol agent and can be used for postharvest preservation of fruits.

[0085] Table 1. Effect of fumigation with VOCs produced by strain NEAU-ZGX24 on the control of postharvest gray mold in tomatoes.

[0086]

[0087] 2. Control experiment of strain NEAU-ZGX24 on detached tomato leaves infected with tomato gray mold.

[0088] In a sterile operating room, 200 μL of the purified bacterial suspension of strain NEAU-ZGX24 was spread onto an LB agar plate, sealed, and incubated at 37°C for 8–12 hours for later use. Tomato leaves at the three- to four-leaf stage were picked, sprayed with 75% alcohol, rinsed three times with sterile water, and air-dried. Holes were made in the surface of the tomato leaves with a toothpick. Botrytis cinerea, which had grown on a PDA plate for 15 days, was then punched with a 6 mm diameter punch. The mycelial cake of the pathogen was applied to the wound (with the mycelial side facing the tomato leaf surface). The following treatment was performed:

[0089] CK: Puncture holes in the tomato leaves and attach them to the LB plate;

[0090] BCK: Puncture holes in tomato leaves, apply pathogenic fungal cakes to the wounds, and then place them face down on LB plates;

[0091] Treatment: Puncture holes in tomato leaves, apply pathogenic fungal cakes to the wounds, and then cover them with NEAU-ZGX24 fungal substrate.

[0092] Each treatment was repeated three times. After fumigation at 28℃ for 7 days, the incidence of disease was recorded, and the incidence rate and disease index were calculated according to the tomato leaf grading standard.

[0093] Grade 0, no symptoms;

[0094] Grade 1: Lesions appear in less than one-quarter of the leaf area;

[0095] Grade 2: Lesions are visible in one-quarter to one-half of the leaf area;

[0096] Grade 3: Lesions appear on half to three-quarters of the leaf area;

[0097] Grade 4: Lesions are visible over more than three-quarters of the leaf area.

[0098] Disease index = Σ(number of diseased leaves at each level × representative value at each level) / (total number of leaves × highest representative extreme value) × 100;

[0099] Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100.

[0100] In the detached leaf experiment, the BCK group, which only had *Botrytis cinerea* mycelium applied, showed extensive rot symptoms. However, the leaves fumigated with VOCs produced by strain NEAU-ZGX24 showed a significantly reduced disease index (P<0.05), and almost no disease was observed on the fumigated leaves, indicating a better control effect with a control efficacy close to 94.81% (see Table 2). Figure 8 ).

[0101] Table 2. The efficacy of VOCs produced by strain NEAU-ZGX24 against tomato leaves.

[0102]

[0103] 3. Identification of VOCs produced by strain NEAU-ZGX24 that exert disease resistance effects

[0104] VOCs were identified by HS-GC-IMS, resulting in 14 compounds. Then, in a sterile environment, holes were punched in a 7-day culture plate of *Botrytis cinerea* using a 6 mm diameter punch. The perforated fungal discs were placed in the center of a fresh PDA plate (with the hyphae side facing the culture medium), and a 6 mm diameter sterile filter paper was placed in the center of the plate lid. The 14 identified single pure VOCs were then added to a concentration of 100 μL / L. An equal volume of sterile water was added to the control group. All treatments were incubated at 28°C for 7 days, and the fungal colony diameter was measured to calculate the inhibition rate.

[0105] The results show (see) Figure 9 Among these 14 single pure VOCs, 3-methylbutyric acid, ethyl heptanoate, and valeric acid showed the most significant inhibitory effects on Botrytis cinerea, and these three substances achieved a 100% inhibition rate against Botrytis cinerea at a concentration of 100 μL / L.

[0106] 4. The effect of strain NEAU-ZGX24 on the control of tomato gray mold in pot experiments.

[0107] (1) Preparation of tomato plants

[0108] Disinfect tomato seeds with 75% alcohol for 3 minutes, then rinse 3 times with sterile water and soak in sterile water for 2 days. Add sterilized filter paper to a petri dish, evenly sprinkle the soaked tomato seeds on the filter paper, add 5 mL of sterile water, and germinate at 28℃ for 1 day. Then plant the seeds in a seedling tray. When they grow to the two-leaf or three-leaf stage, transplant them into small flower pots. After they grow to the four-leaf stage, potted plant experiments can be carried out.

[0109] (2) Preparation of pathogen spore suspension

[0110] Botrytis cinerea was inoculated onto fresh PDA plates and cultured at 28°C for 15 days. A suitable amount of sterile water was added to the mycelial surface, and spores were scraped off using a blue pipette tip. The resulting liquid was passed through four layers of gauze, and the spores were observed under an optical microscope. The concentration was adjusted to 10. 5 CFU / mL, store at 4℃ for later use.

[0111] (3) Preparation of VOCs produced by strain NEAU-ZGX24

[0112] In a sterile operating room, take 25 μL of the NEAU-ZGX24 strain frozen at -20℃ and place it in a test tube containing 5 mL of liquid LB medium. Incubate at 37℃ for 12 h to obtain bacterial suspension. Take 200 μL of the bacterial suspension and spread it on an LB plate. Seal the plate and incubate at 37℃ for 8–12 hours for later use.

[0113] (4) Potted Plant Prevention Experiment

[0114] Tomato plants with uniform growth at the four-leaf to six-leaf stages were selected and divided into three groups: CK, BCK, and Treatment. CK was sprayed with sterile water, while BCK and Treatment were sprayed with a pre-prepared concentration of 10. 5 The pathogen spore suspension of CFU / mL was applied until liquid flowed down the leaves. CK and BCK were immediately fumigated with LB plates and bags, while Treatment was fumigated with a plate coated with strain NEAU-ZGX24 and bags. All treatments were incubated in a 28℃ light incubator.

[0115] Each treatment was repeated three times. After 10 days, the incidence of disease was recorded, and the incidence rate and disease index were calculated according to the tomato leaf grading standard.

[0116] Grade 0, no symptoms;

[0117] Grade 1: Lesions appear in less than one-quarter of the leaf area;

[0118] Grade 2: Lesions are visible in one-quarter to one-half of the leaf area;

[0119] Grade 3: Lesions appear on half to three-quarters of the leaf area;

[0120] Grade 4: Lesions are visible over more than three-quarters of the leaf area.

[0121] Incidence rate = Number of infected plants / Total number of plants × 100%;

[0122] Disease index = Σ(number of diseased leaves at each level × representative value at each level) / (total number of leaves × highest representative extreme value) × 100;

[0123] Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100.

[0124] In the prevention experiment, the BCK group, which was only inoculated with the pathogen, showed large-area lesions and wilting on tomato leaves, and in severe cases, the entire plant wilted and turned brownish-yellow. In contrast, the Treatment group, due to the VOCs produced by the strain NEAU-ZGX24, showed a significant reduction in the gray mold disease index of tomato plants (P<0.05), a significant reduction in the area of ​​lesions on leaves, and no wilting of the entire plant. The control effect was 75.04% (see Table 3).

[0125] Table 3. Control efficacy of strain NEAU-ZGX24 against tomato gray mold in pot experiments.

[0126]

[0127] 5. Potted plant therapy experiment

[0128] Tomato plants with uniform growth at the four-leaf to six-leaf stages were selected and divided into three groups: CK, BCK, and Treatment. CK was sprayed with sterile water, while BCK and Treatment were sprayed with a pre-prepared concentration of 10. 5 The pathogen spore suspension was prepared at CFU / mL until liquid flowed down the leaves. After 48 hours, CK and BCK were fumigated with LB plates and bags, while Treatment was fumigated with a plate coated with strain NEAU-ZGX24 and bags. All treatments were incubated in a 28℃ light incubator.

[0129] Each treatment was repeated three times. After 10 days, the incidence of disease was recorded, and the incidence rate and disease index were calculated according to the tomato leaf grading standard.

[0130] Grade 0, no symptoms;

[0131] Grade 1: Lesions appear in less than one-quarter of the leaf area;

[0132] Grade 2: Lesions are visible in one-quarter to one-half of the leaf area;

[0133] Grade 3: Lesions appear on half to three-quarters of the leaf area;

[0134] Grade 4: Lesions are visible over more than three-quarters of the leaf area.

[0135] Incidence rate = Number of infected plants / Total number of plants × 100%;

[0136] Disease index = Σ(number of diseased leaves at each level × representative value at each level) / (total number of leaves × highest representative extreme value) × 100;

[0137] Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100.

[0138] In the treatment experiment, the BCK group, which was only inoculated with the pathogen, showed large areas of lesions and wilting on tomato leaves, with severe cases resulting in the entire plant wilting and turning brownish-yellow. In contrast, the Treatment group, due to the VOCs produced by strain NEAU-ZGX24, showed a significantly lower disease index of gray mold compared to the control group treated with LB board fumigation (P<0.05). The lesion area on tomato leaves was also significantly reduced, and no entire plant wilting occurred. The control efficacy was 71.08% (Table 4).

[0139] Table 4. The therapeutic effect of strain NEAU-ZGX24 on tomato gray mold in pot experiments.

[0140]

[0141] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A strain of Bacillus siamensis NEAU-ZGX24, characterized in that, The preservation number of the Bacillus NEAU-ZGX24 is CCTCC NO: M 20242176.

2. The Bacillus thymosus NEAU-ZGX24 according to claim 1, characterized in that, The Bacillus NEAU-ZGX24 of Siam can produce IAA, antimicrobial peptides and volatile antimicrobial substances; the volatile antimicrobial substances include 3-methylbutyric acid, ethyl heptanoate and valeric acid.

3. The application of Bacillus NEAU-ZGX24 of claim 1 in inhibiting the mycelial growth of Botrytis cinerea.

4. The application of the antimicrobial peptide produced by Bacillus NEAU-ZGX24 of claim 1 in inhibiting the mycelial growth of Botrytis cinerea.

5. The application of the volatile organic compounds produced by Bacillus NEAU-ZGX24 of claim 1 in inhibiting the growth of plant pathogenic fungi.

6. The application according to claim 5, characterized in that, The plant pathogenic fungi mentioned include *Botrytis cinerea*, *Fusarium moniliforme*, *Fusarium graminearum*, *Fusarium solani*, *Stagonosporopsis cucurbitacearum*, *Bipolaris sorokiniana*, *Fusarium oxysporum*, *Fusarium subglutinans*, *Fusarium verticillioides*, *Verticillium dahliae*, *Colletotrichum orbiculare*, and *Magnaporthe oryzae*.

7. The application according to claim 5, characterized in that, The volatile organic compounds include 14 compounds, among which 3-methylbutyric acid, ethyl heptaate, and valeric acid are the substances that play an anti-disease role.

8. The application of Bacillus neuraminidum NEAU-ZGX24 as described in claim 1 in the control of postharvest gray mold in tomatoes, characterized in that, The application method for Bacillus thymosus NEAU-ZGX24 in the aforementioned application is fumigation.

9. The application of Bacillus neuraminidum NEAU-ZGX24 as described in claim 1 in the prevention and control of gray mold infection during tomato growth, characterized in that, The application method for Bacillus thymosus NEAU-ZGX24 in the aforementioned application is fumigation.

10. The use of a fungicide containing Bacillus NEAU-ZGX24 of claim 1 in the prevention and control of postharvest gray mold of tomatoes or gray mold infection during tomato growth.

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