Bacillus belye A125 and its application in the control of bacterial fruit spot disease in melons

Abstract

This invention belongs to the field of microbiology and relates to Bacillus bellis A125 and its application in controlling bacterial fruit spot disease in melons. The Bacillus bellis A125 of this invention is salt-tolerant and exhibits good inhibitory effects against fruit spot disease. Furthermore, Bacillus bellis A125 of this invention has a strong ability to produce cellulase, amylase, protease, and indole-3-acetic acid (IAA), and has a good growth-promoting effect on melons. The inoculant prepared using this biocontrol bacterium will not pollute the ecological environment during use and is harmless. Using the biocontrol bacterium of this invention can reduce the amount of other chemical pesticides used, lower the cost of controlling melon diseases, and promote melon growth, thus having good promotional value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and in particular relates to Bacillus vesicles A125 and its application in the prevention and control of bacterial fruit spot disease in melons. Background Technology

[0002] Muskmelons are a widely cultivated and important economic crop globally, prized for their sweet taste and rich nutritional value. They hold a significant position in the agricultural economy, bringing considerable economic benefits to growers. However, the outbreak and spread of bacterial fruit blotch (BFB) in muskmelons has become a key bottleneck restricting the high-quality development of the muskmelon industry. Initially, infected leaves show small, round or polygonal water-soaked spots, often with a yellow halo around the edge. Later, the center of the lesions gradually thins and dries out; in severe cases, multiple lesions merge, causing the leaves to wither. When fruits are affected, small water-soaked lesions first appear on the skin, then rapidly expand and turn brown and sunken. Later, cracking often occurs, and the pathogen extends deep into the flesh, causing water-soaked rot and complete loss of edibility and commercial value. This disease spreads extremely rapidly and affects a wide area, causing significant economic losses to the muskmelon industry.

[0003] The prevalence and spread of bacterial fruit spot disease in muskmelons exhibit significant unique characteristics, with diverse primary sources of infection and complex transmission routes. As a typical seed-borne disease, the pathogen can adhere to the seed surface or invade the seed's internal tissues. Infected seeds remain infectious even after 3-8 years of storage. After germination, the pathogen can directly infect cotyledons and true leaves, becoming a significant source of reinfection in the field. Furthermore, the pathogen can overwinter in field-grown seedlings, wild cucurbitaceous hosts, and diseased plant debris. Pathogens on diseased plant debris can survive in the soil for up to two years, spreading the following year via wind, rainwater, irrigation water, and insects. In field production, wounds from grafting, contamination of clothing and equipment by agricultural workers, and transplanting of infected rootstocks can all contribute to the short-distance spread of the pathogen. High temperature and humidity, especially during hot seasons accompanied by storms, accelerate pathogen reproduction and infection, becoming a key factor in disease outbreaks. Meanwhile, cultivation factors such as overly dense planting, poor ventilation and light penetration, unbalanced soil fertility, and continuous cropping can further reduce the disease resistance of melon plants and increase the risk of disease occurrence.

[0004] Currently, the control of bacterial fruit spot disease in melons mainly relies on a comprehensive approach including agricultural cultivation management, chemical control, and seed disinfection. However, there are many limitations in practical application. For example, copper-based fungicides, kasugamycin, and agricultural streptomycin sulfate are commonly used in production, but the overall effect is generally unsatisfactory. On the one hand, pathogens easily develop resistance to chemical agents, and long-term, large-scale use leads to a gradual decline in efficacy. On the other hand, chemical agents cause environmental pollution such as soil and water bodies, harming non-target organisms, which contradicts the concept of green agriculture and cannot fundamentally solve the problems of seed-borne pathogens and residual inoculum in the soil. Cultivation measures such as crop rotation, removal of diseased plant debris, reasonable planting density, and optimization of soil physicochemical properties can reduce the pathogen population to some extent, but due to limitations such as regional conditions, planting patterns, and production costs, these measures alone are insufficient to cope with large-scale disease outbreaks, resulting in limited control effects.

[0005] Biological control, with its unique advantages such as environmental friendliness, low resistance to pesticides, and effective reduction of pathogen residues, has become another development direction for the control of bacterial fruit spot disease in muskmelons. However, existing biocontrol strains still have significant technical shortcomings: most strains have weak environmental adaptability, and their control efficacy decreases significantly under high temperature and humidity, high pathogen populations, or different soil conditions, making it difficult to meet the control needs of complex field environments; in addition, the development of biocontrol products is immature, and the fermentation process of strains is complex and costly, limiting large-scale promotion and application. Therefore, screening for new biocontrol strains with strong antibacterial activity, good environmental adaptability, and stable control efficacy, clarifying their disease control mechanisms, and applying them to disease control is of great practical significance and application value for reducing losses in the muskmelon industry, ensuring product quality and safety, and promoting the sustainable development of the muskmelon industry. Summary of the Invention

[0006] The purpose of this invention is to provide Bacillus berleis A125 and its application in the control of bacterial fruit spot disease in muskmelons. The Bacillus berleis A125 was isolated from the soil of a mango orchard in Damao Village, Yazhou District, Sanya City (109°12′5″E, 18°24′38″N) and can inhibit various pathogens and bacterial diseases of muskmelons.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] Bacillus velezensis A125 is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 66573) on June 23, 2025. It is classified as Bacillus velezensis and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The present invention also provides a fungal agent for preventing bacterial spot disease in melons, comprising a bacterial suspension of the aforementioned Bacillus velezensis A125.

[0010] Furthermore, the concentration of Bacillus velezensis A125 in the bacterial suspension is 1~9×10⁻⁶. 7 CFU / mL.

[0011] The present invention also provides a method for preparing the above-mentioned inoculant for controlling bacterial fruit spot disease of melon, wherein the above-mentioned Bacillus velezensis A125 is inoculated into LB liquid medium for fermentation culture to obtain the inoculant for controlling bacterial fruit spot disease of melon.

[0012] Furthermore, the fermentation culture temperature is 35~40℃, and the culture time is 1~3 days.

[0013] The present invention also provides the application of the above-mentioned Bacillus velezensis A125 in the prevention and control of bacterial fruit spot disease in melons.

[0014] The present invention also provides the application of the above-mentioned Bacillus velezensis A125 in promoting melon growth.

[0015] The present invention also provides the application of the above-mentioned Bacillus velezensis A125 in the production of extracellular hydrolases.

[0016] The present invention also provides a method for preventing and controlling bacterial fruit spot disease in melons, wherein the above-mentioned bacterial agent for preventing and controlling bacterial fruit spot disease in melons is sprayed on the melon leaves.

[0017] The present invention also provides a method for promoting the growth of melons by injecting the above-mentioned fungal agent for preventing bacterial fruit spot disease of melons into the roots of the melons.

[0018] Beneficial effects

[0019] The *Bacillus berleis* A125 of this invention is salt-tolerant and exhibits good inhibitory effects against fruit spot fungi, effectively controlling bacterial fruit spot disease in melons. *Bacillus berleis* A125 also possesses the ability to produce cellulase, amylase, protease, and indole-3-acetic acid (IAA), and has a good growth-promoting effect on melons. The inoculant prepared using this biocontrol bacterium will not pollute the ecological environment during use and is harmless. Using the biocontrol bacterium of this invention can reduce the amount of other chemical pesticides used, lower the cost of controlling melon diseases, and promote melon growth, thus having good promotional value. Attached Figure Description

[0020] Figure 1 Phylogenetic tree of Bacillus belyssus A125;

[0021] Figure 2 Bioinformatics analysis of the whole genome of Bacillus belyssus A125;

[0022] Figure 3 The inhibitory effect of Bacillus belyssus A125 on pathogens;

[0023] Figure 4 Figure showing the results of salt stress tolerance test for Bacillus belyssus A125;

[0024] Figure 5 Figure showing the results of acid and alkali stress resistance determination for Bacillus belyssus A125; Figure 5 The pH of A is 2. Figure 5 The pH of B is 3. Figure 5 The pH of C is 11. Figure 5 The pH of D is 12;

[0025] Figure 6 HPLC-MS identification results of secondary metabolites of Bacillus belyssus A125;

[0026] Figure 7 Figure showing the antibacterial effect of crude extract of secondary metabolites of Bacillus belyssus A125;

[0027] Figure 8 The image shows the detection results of protease, cellulase, amylase, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase produced by Bacillus belye A125. Figure 8 A in the middle is a protease producer. Figure 8 B is cellulase, Figure 8 C in the middle represents amylase, Figure 8 D in the image represents 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase.

[0028] Figure 9 Results of the assay for the ability of Bacillus belyssus A125 to produce indole-3-acetic acid (IAA);

[0029] Figure 10 The image shows the disease control effect of Bacillus vesicles A125 on fruit spot disease on melon plants;

[0030] Figure 11 The image shows the effect of Bacillus vesiculosus A125 on the growth promotion of melon plants. Figure 11 In the middle, A represents plant height. Figure 11 In the middle, B represents the root length. Figure 11 C represents the fresh weight of the above-ground portion. Figure 11 D represents the dry weight of the above-ground portion. Figure 11 E represents the fresh weight of the underground portion. Figure 11 F represents the dry weight of the underground portion. Figure 11 G represents the stem diameter.

[0031] Biological Preservation Instructions

[0032] The Bacillus velezensis A125 provided by this invention is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:66573, deposit date June 23, 2025, classified as Bacillus velezensis, and deposited at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Taxonomic analysis of Bacillus belyssae A125 based on its core genome

[0036] Bacillus belye A125 strain was inoculated into liquid LB medium and incubated at 37°C for 12 hours at 200 rpm. The cells were collected by centrifugation, and genomic DNA was extracted using a bacterial DNA extraction kit (Hunan Aikerui Biotechnology Co., Ltd.). The 16S rRNA gene was then amplified by PCR using forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 3) and reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 4). The PCR conditions were: 95°C for 5 min; 95°C for 15 s; 56°C for 1 min; 72°C for 1 min; 32 cycles; 72°C for 10 min; and stored at 4°C. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rRNA gene sequence of Bacillus belye A125 is shown in SEQ ID No. 1.

[0037] The 16S rRNA gene sequence of Bacillus belyssus A125:

[0038]

[0039] PCR amplification and sequencing of the gyrB gene:

[0040] Forward primer gyrB-F:

[0041] 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3' (SEQ ID No. 5);

[0042] Reverse primer gyrB-R:

[0043] 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3' (SEQ ID No. 6); the PCR reaction conditions for the gyrB gene are the same as above. The partial gyrB gene sequence is approximately 1200 bp in length, and the result is shown in SEQ ID No. 2 of the sequence listing.

[0044] gyrB gene sequence:

[0045]

[0046] 16S rRNA and gyrB identification results showed that strain A125 belongs to Bacillus belesii.

[0047] Whole-genome sequencing of Bacillus was performed using an Illumina Hiseq 4000 and PacBio SMRT sequencing system. The biosynthetic gene clusters (BGCs) of Bacillus belesi A125 were predicted using the online tool antiSMASH (https: / / dl.secondarymetabolites.org / releases / 4.0.2 / ). Three lipopeptide compounds and three polyketide compounds were predicted in the BGCs of Bacillus belesi A125 chromosome, responsible for the biosynthesis of Fengycin, Surfactin, Iturin, Bacillaene, Difficidin, and Macrolide H, respectively.

[0048] Example 2

[0049] Determination of the antibacterial activity of Bacillus belysin A125

[0050] The antibacterial ability of Bacillus belyi was preliminarily determined, and the selected pathogenic fungi included Colletotrichum fructicola.

[0051] Determination of the antibacterial activity of Bacillus belyss against pathogenic fungi: A certain number of mycelial cakes were punched from the outermost edge of the original pathogenic fungal culture plate using a sterile punch with a diameter of approximately 0.6 cm. The mycelial cakes (mycelial side) were then transferred to the center of a new potato dextrose agar (PDA) culture plate. A Bacillus belyss A125 mycelial block was then placed at the same distance from the mycelial cakes. The plate was incubated at 28 ℃, and the antibacterial effect was observed. The results are as follows: Figure 3 As shown, this indicates that Bacillus berleis A125 has a certain inhibitory effect on Anthracnose.

[0052] Example 3

[0053] Determination of stress resistance of Bacillus belyssus A125

[0054] The stress resistance of Bacillus belye is closely related to its disease resistance and growth-promoting ability. Therefore, the stress resistance ability of Bacillus belye A125 was determined.

[0055] 1. Determination of salt stress tolerance of Bacillus belyssus A125

[0056] The salt stress resistance of strain A125 and the model strain FZB42 was determined by simulating different salt stresses of 1%, 3%, 5%, 7%, 9%, 11%, 13%, 13.1%, and 13.2%. Single colonies of the tested strains were picked and placed in LB liquid medium and incubated overnight at 37 °C and 200 rpm for 24 h until the bacterial concentration reached OD200. 600 When the concentration of bacterial culture reached 2.0, 5 μL of bacterial culture was transferred to LB solid medium with different salt gradients. The medium was then placed in a 37 ℃ incubator. After 2 days, the colony growth of each strain under different salt gradients was observed. The results are as follows: Figure 4 As shown in the figure. The results indicate that Bacillus belye A125 can grow under salt stress conditions with a salt content of 13.1%, and its growth status is better than that of the model strain FZB42, indicating that it has a certain degree of salt stress tolerance.

[0057] 2. Determination of the acid and alkali stress tolerance of Bacillus belyssus A125

[0058] Different acid-base stresses (pH gradients: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) were simulated to determine the resistance of *Bacillus belyssae* A125 and the model strain FZB42 to acid-base stress. Single colonies of the tested strains were picked and placed in LB liquid medium, incubated overnight at 37℃ and 200 rpm for 24 h. 5 μL of the bacterial culture was then transferred to LB liquid medium with different alkaline gradients (pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). The medium was then placed in a 37℃ shaking incubator. After 2 days, 5 μL of the bacterial culture was transferred to LB solid medium to observe the colony growth of each strain under different pH conditions. Each treatment was repeated three times. The results are shown below. Figure 5 As shown in the figure. The results indicate that Bacillus belye A125 can grow under acidic stress of pH 2 and alkaline stress of pH 12, demonstrating a certain degree of tolerance to acid and alkaline stress.

[0059] Example 4

[0060] Extraction, identification and antibacterial activity determination of secondary metabolites of Bacillus belyssus A125

[0061] 1. Extraction and identification of secondary metabolites of Bacillus belyssus A125

[0062] Bacillus belyssus A125 was activated on LB solid medium and incubated at 37 ℃ until single colonies grew. A single colony was picked and transferred to 20 mL of LB liquid medium and cultured at 37 ℃ with shaking at 200 rpm for 12 h. The Bacillus belyssus A125 culture was then transferred at a 1% ratio to 200 mL of LB liquid medium and cultured at 37 ℃ with shaking at 200 rpm for 48 h. 6 mL of resin (XAD-16N) was added to each A125 culture, and the culture was continued with shaking for 24 h. The culture was centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were dissolved in 30 mL of methanol, and the suspension was cultured at 37 ℃ with shaking at 200 rpm for 4 h. The suspension was centrifuged at 8000 rpm for 10 min, the supernatant was collected and filtered through filter paper. The supernatant was concentrated to 2 mL using a rotary evaporator, and then... The crude extract was filtered through a μm filter membrane, and the formation of lipopeptide compounds was identified and detected by high-performance liquid chromatography (HPLC). The results are as follows: Figure 6 As shown, this indicates that Bacillus belye can produce Fengycin, Surfactin, and Iturin.

[0063] 2. Determination of the antibacterial effect of Bacillus belyssus A125 secondary metabolites

[0064] The pathogen of fruit spot disease was inoculated into LB liquid medium and incubated at 37 ℃ and 200 rpm for 48 h. LB plates were prepared, and 20 μL of the pathogen suspension was added to each plate. The suspension was spread evenly with a spreader, and a hole was punched in the center of each plate. 20 μL of Bacillus belyssae A125 secondary metabolite was then added. After thorough drying, the plates were inverted and incubated at 37 ℃ for 24 h. The antibacterial effect was observed, and the results are as follows: Figure 7 As shown, this indicates that the secondary metabolites of Bacillus belye A125 have a good inhibitory effect on bacterial fruit spot disease.

[0065] 3. Determination of the ability of Bacillus belyssus A125 to produce extracellular hydrolases

[0066] Qualitative detection of protease production by Bacillus belyssus A125 was performed on skim milk agar plates: The ability of Bacillus belyssus to produce protease was determined in agar medium containing skim milk powder (20 g skim milk powder, 20 g agar, pH 7.0, sterilized at 115°C for 10 min). Single colonies of Bacillus belyssus were picked and incubated in LB broth overnight at 37 °C and 200 rpm for 12 h; 5 μL (OD) of the culture medium was added dropwise to the medium. 600A bacterial suspension with a concentration of 1.0 g / cm³ was incubated at 37 °C for 24-36 h. Afterward, the presence of a clear zone around the colonies was observed. Results were as follows: Figure 8 As shown in Figure A.

[0067] The cellulase production capacity of *Bacillus belyssiensis* A125 was determined using sodium carboxymethyl cellulose (CMC-Na) medium: Single colonies of *Bacillus belyssiensis* were picked and cultured overnight at 37 °C and 200 rpm for 12 h. 5 μL of the bacterial suspension (OD) was then aspirated onto a CMC-Na agar plate. 600 =1.0), seal the plate and incubate it in a 37 °C incubator; after 2 days, remove the petri dish, pour Gram's iodine stain solution until it submerges the surface of the plate, let it stand for 4 minutes, then pour off the stain solution and observe whether a clear zone forms around the colonies. If a clear hydrolysis zone appears around the bacterial cells, it indicates that the strain can produce the corresponding enzyme; if no clear hydrolysis zone appears, it indicates that the strain cannot produce this type of enzyme. The results are as follows. Figure 8 As shown in B.

[0068] Detection of amylase activity in Bacillus belyssae A125 on starch agar medium: Colonies of Bacillus belyssae were picked and incubated in LB broth at 37 °C, 200 rpm overnight for 12 h. 5 μL of bacterial suspension (OD200) was then added. 600 =1.0) was added to starch-containing culture medium and incubated in a 37°C incubator; after 2 days, the petri dishes were removed, Gram's iodine stain was poured in to submerge the surface of the plates, and after standing for 4 minutes, the stain was poured off. The experimental results were observed and recorded as follows. Figure 8 As shown in C.

[0069] Colonies of Bacillus belye were picked and placed in LB broth, incubated overnight at 37 °C and 200 rpm for 12 h, and then 5 μL of bacterial suspension (OD) was added. 600 =1.0) was added to DF medium, DF (containing ammonium sulfate) medium, and ADF medium. The growth of the strain in the three different media was observed. When the strain grew well in ADF medium but poorly in DF medium, it indicated that the strain could grow with 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole nitrogen source, which also indicated that the strain had the ability to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. The results are as follows. Figure 8 As shown in D.

[0070] The results showed that strain A125 could produce large transparent hydrolysis zones on skim milk powder medium, sodium carboxymethyl cellulose (CMC-Na) medium, and amylase detection medium, and could grow on ADF medium, indicating that it could produce cellulase, protease, amylase, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase.

[0071] 4. Determination of the ability of Bacillus belyssus A125 to produce indole-3-acetic acid (IAA)

[0072] Preparation of Salkowski colorimetric solution: Weigh 0.81 g of FeCl3 and dissolve it in 10 mL of ddH2O. Ensure complete dissolution and mix thoroughly to obtain a 0.5 mol / L stock solution. Prepare a 35% perchloric acid solution. Then, take 50 mL of the perchloric acid solution and mix it thoroughly with 1 mL of the 0.5 mol / L FeCl3 stock solution.

[0073] First, a stock solution of the bacterial strain was obtained using LB medium at 37 ℃ and 200 rpm. Then, the stock solution was transferred to YMB liquid medium at a ratio of 1% and incubated at 37 ℃ and 200 rpm for 48 h. After incubation, 2 mL of the bacterial culture was centrifuged at 12000 rpm to collect the supernatant. An equal volume of Salkowski colorimetric solution was added to the supernatant, and the mixture was thoroughly mixed. The mixture was then reacted in the dark for 30 min. If the solution turned red, it indicated that the bacterium could produce indole-3-acetic acid (IAA). Using an equal volume of YMB liquid medium as a blank control, standard solutions of indole-3-acetic acid (IAA) at concentrations of 2.5 μg / mL, 5.0 μg / mL, 7.5 μg / mL, 10.0 μg / mL, and 12.5 μg / mL were prepared. The OD values ​​of the standard and sample solutions were measured using a spectrophotometer. 530 The value was used to obtain the regression equation = 0.0428x - 0.0257. The calculated indole-3-acetic acid (IAA) production capacity of *Bacillus belyssus* A125 was 9.27 μg / mL, and that of the model strain FZB42 was 7.59 μg / mL. The results are as follows: Figure 9 As shown, the results indicate that Bacillus berlesii A125 can produce indole-3-acetic acid (IAA), and the ability of strain A125 to produce indole-3-acetic acid (IAA) is higher than that of the model strain FZB42, indicating that it has great potential in promoting plant growth and development and has the potential to be developed into a biocontrol agent.

[0074] Example 5

[0075] Efficacy of Bacillus vesiculosus A125 in controlling bacterial fruit spot disease of melons

[0076] Sterilized seedling substrate was filled into 50-cell seedling trays. One healthy, plump, and similarly sized honeydew melon seed was placed in each cell, and planted root-side down in the soil using tweezers, then covered with a thin layer of soil. After two weeks of cultivation at 25℃, one uniformly growing honeydew melon seedling was retained from each cell in the tray and transplanted using 350 mL plastic cups. After 20 days of cultivation in a 25℃ greenhouse, honeydew melon seedlings with similar growth characteristics were selected for disease prevention experiments.

[0077] Preparation of pathogen and Bacillus vesiculus A125 suspension: The pathogen of fruit spot disease was inoculated into LB liquid medium, and Bacillus vesiculus A125 was inoculated into LB liquid medium. They were then simultaneously incubated at 37 ℃ and 200 r / min in a shaker for 48 h. The bacterial suspension concentration was then adjusted to 10⁻¹⁰ using sterile water. 7 CFU / mL. For pathogen inoculation, first, the pathogen suspension was placed in a spray bottle and then evenly sprayed onto the cantaloupe leaves, ensuring the water droplets covered the leaf surface without dripping. 24 hours after pathogen spraying, a Bacillus berberis suspension was sprayed using the same method. The control group was sprayed with sterile water. The inoculated cantaloupe seedlings were then placed in a greenhouse incubator under the following conditions: 28℃, 75% humidity, 12h light, 12h dark alternation. After 7-10 days of cultivation, the disease incidence on the cantaloupe leaves was recorded, and the results are as follows: Figure 10 As shown.

[0078] Grading standards for bacterial fruit spot disease in melons: Grade 0 (no lesions); Grade 1 (lesions cover less than 5% of the entire leaf); Grade 3 (lesions cover 5%~25% of the entire leaf); Grade 5 (lesions cover 26%~50% of the entire leaf); Grade 7 (lesions cover 51%~75% of the entire leaf); Grade 9 (lesions cover 76%~100% of the entire leaf or the leaf is completely withered); Disease index = [Σ(number of diseased leaves at each level × representative value of each level)] ÷ (total number of leaves surveyed × highest level) × 100; Relative control efficacy = (disease index of control group - disease index of treatment group) ÷ disease index of control group × 100%.

[0079] Table 1. Control efficacy of Bacillus vesiculosus A125 against bacterial fruit spot disease in melons.

[0080]

[0081] The experimental results show that treating melon seedlings with Bacillus vesicles A125 reduced the incidence of bacterial fruit spot disease. The disease index of melons treated with the bacterial solution was 11.11%, which was 24.82% lower than that of the blank control group. The control effect of Bacillus vesicles A125 on bacterial fruit spot disease in melons was 69.07%, while the control effect of the model strain FZB42 on bacterial fruit spot disease was 65.96%. A125 has a better control effect on bacterial fruit spot disease in melons than FZB42.

[0082] Example 6

[0083] Study on the growth-promoting effect of Bacillus vesiculosus A125 on melon plants

[0084] Disinfection and germination of melon seeds: Select intact and plump seeds, soak them in 30% sodium hypochlorite solution for 120 seconds, then soak them in 75% ethanol for 30 seconds, and then rinse them repeatedly with ddH2O 5 times until there is no irritating odor remaining on the surface of the melon seeds; place sterile filter paper in a 9 cm sterilized glass petri dish, add an appropriate amount of sterile water with a pipette so that the filter paper in the dish is completely wetted with water; use sterile tweezers to pick up the melon seeds and place them on the filter paper, making sure that there is a certain distance between the seeds so that they can germinate;

[0085] Planting melon seedlings: Mix nutrient soil and vermiculite in a 2:1 ratio, sterilize at 121 ℃ for 20 minutes under high temperature and pressure, and set aside. Use a punch with a diameter of about 2-3 cm to punch a hole in the center of a disposable plastic cup, then fill it with substrate soil. Use tweezers to pick up the melon seeds that have grown radicles, plant them in the soil with the roots facing down, cover them with another layer of soil, place the disposable plastic cup in a tray, add an appropriate amount of water to the tray, and place it in a greenhouse at 28 ℃ for about 20 days.

[0086] Root irrigation treatment with Bacillus vesicles Belesi A125 fermentation broth: After the melon seedlings have grown to the stage of two leaves and one bud, select plants with uniform growth and dilute the seed culture of Bacillus vesicles Belesi A125 and the model strain FZB42 with sterile water to a concentration of 10. 7 CFU / mL, inject 50 mL of the solution into the root of each melon to achieve a final concentration of 10. 7 A bacterial suspension of CFU / mL was used, with sterile water as a blank control. A second treatment was performed after 10 days. Growth promotion data were collected after 20 days of culture. Results are as follows: Figure 11 As shown.

[0087] The results showed that strain A125 had a good promoting effect on the growth of muskmelon plants. Among them, strain A125 had the best promoting effect on the fresh weight of the above-ground parts and the height of the plants, with promotion rates as high as 497.22% and 147.86%, respectively. Strain A125 also had a good promoting effect on the growth of muskmelon roots and stems, with promotion rates of 14.55% and 35.62%, respectively. The growth promotion rate of this strain on muskmelon was higher than that of the model strain FZB42, indicating that Bacillus vesiculosus A125 has a good growth-promoting effect and has certain development potential.

[0088] As can be seen from the above embodiments, the Bacillus berberis A125 provided by the present invention has the ability to tolerate salt and alkali and can effectively prevent and control melon spot disease. It has a good growth-promoting effect on melons and has great application potential in biological pesticides and biological fertilizers.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bacillus belye ( Bacillus velezensis The application of A125 in simultaneously controlling bacterial fruit spot disease in muskmelons and promoting muskmelon growth is characterized by, The bacillus velezensis is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 66573, the preservation date is June 23, 2025, and the classification name is Bacillus velezensis The promotion of the growth of the honeydew melon includes increasing the plant height, root length and stem diameter of the honeydew melon; the bacillus velezensis Bacillus velezensis A125 is used for producing extracellular hydrolases, which are any one of cellulases, proteases, amylases, 1-aminocyclopropane-1-carboxylic acid deaminase.

2. The application according to claim 1, characterized in that, Melon plants were treated with a bacterial suspension of Bacillus velezensis A125.

3. The application according to claim 2, characterized in that, Bacillus belye in the bacterial suspension Bacillus velezensis The bacterial concentration of A125 is 1×10⁻⁶. 7 ~9×10 7 CFU / mL.

4. The application according to claim 2, characterized in that, The method for preparing the bacterial suspension includes: [using Bacillus belye] Bacillus velezensis A125 was inoculated into LB liquid medium for fermentation culture to obtain a bacterial suspension.

5. The application according to claim 4, characterized in that, The fermentation temperature is 35~40℃, and the fermentation time is 1~3 days.

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