Multifunctional synergistic bacillus velezensis byta99, microbial inoculum and application thereof in disease prevention and growth promotion of pepper

Abstract

The application discloses a multifunctional synergistic bacillus velezensis BYTA99, a microbial agent and application of the bacillus velezensis BYTA99 and the microbial agent in pepper disease prevention and growth promotion, and aims to solve the technical problems of single function dimension, unstable prevention effect and insufficient growth promotion of existing pepper anthracnose biocontrol bacteria. Bacillus velezensis A bacillus velezensis (BYTA99) is screened from pepper rhizosphere soil in the application, and the preservation number is CCTCC NO: M 2026848, which has multiple functions of nitrogen fixation, phosphorus solubilization, growth enzyme promotion, oxidation resistance and antagonism to anthracnose pathogenic fungi. The biocontrol agent prepared by using the strain can be applied in multiple ways, can effectively colonize in the rhizosphere or in vivo of plants, can significantly reduce the incidence of pepper diseases, improve the yield and quality of peppers through multiple mechanisms such as antagonism to pathogenic bacteria, promotion of plant growth and improvement of nitrogen utilization efficiency. Meanwhile, the microbial agent is also helpful for improving soil structure and fertility, replacing or reducing the use of chemical fertilizers and pesticides, and has good economic, ecological and social benefits.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to a multifunctional synergistic Bacillus belye (B. belye). Bacillus velezensis BYTA99, fungal agents and their application in disease prevention and growth promotion of chili peppers. Background Technology

[0002] Anthracnose in peppers is caused by the genus *Colletotrichum* (… Colletotrichum Anthracnose is a devastating disease in chili pepper production caused by various pathogenic fungi, including *S. spp.*. It spreads rapidly under high temperature and humidity conditions, often leading to leaf drop and fruit rot, resulting in a 20%-30% yield reduction in normal years, and even total crop failure in severe cases. Long-term reliance on chemical pesticides has led to increasingly prominent problems such as increased pathogen resistance, excessive pesticide residues, and ecological degradation. Biological control using beneficial microorganisms and their inoculants, due to their advantages of being environmentally friendly and less prone to inducing drug resistance, has become an important direction for the green control of chili pepper anthracnose.

[0003] For the biological control of anthracnose in peppers, existing technologies have disclosed various antagonistic microbial agents and their application methods. Regarding the selection of microbial strains, existing methods mostly employ Bacillus subtilis (…). Bacillus subtilis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) or fluorescent pseudomonas ( Pseudomonas fluorescens Common biocontrol strains, such as those mentioned above, work by directly inhibiting the growth of anthracnose fungi through the production of antibacterial active substances, or by reducing infection opportunities through competition for ecological niches. In terms of preparation, the target strains are typically subjected to deep liquid fermentation to produce wettable powders, suspensions, or water-dispersible granules. Regarding application, current technologies generally recommend foliar spraying before or at the early stage of anthracnose in peppers, using 50-100 grams per acre, diluted with water, and sprayed every 7-14 days for 2-3 consecutive applications. A few technical solutions attempt to combine microbial agents with substances such as humic acid and amino acids to enhance the survival ability of the bacteria on leaves.

[0004] While the aforementioned technical solutions have achieved some success in the biological control of anthracnose in peppers, they still have the following shortcomings: ① Their functional dimensions are relatively singular, focusing mainly on one or two functions such as direct antagonism or nutrient activation, failing to simultaneously address pathogen enzymatic degradation, plant hormone regulation, and soil microecological improvement, resulting in significant fluctuations in field control efficacy and growth-promoting effects; ② Their rhizosphere colonization capacity is limited, and the survival and function of the bacteria in the soil are easily affected by environmental stress, requiring frequent application and increasing costs; ③ They are difficult to achieve multi-target regulation of plant oxidative stress and systemic resistance, and are unable to effectively inhibit membrane lipid peroxidation damage during periods of high disease incidence, resulting in insufficient improvement in the overall health of plants. Therefore, the industry urgently needs a multifunctional synergistic strain that integrates pathogen antagonism, multi-enzyme synergistic degradation, nutrient activation, hormone and redox balance regulation, and soil improvement to fundamentally solve the problem of the disconnect between anthracnose control and growth-promoting effects in peppers.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] This invention addresses the technical problems of existing biocontrol bacteria for pepper anthracnose, such as limited functional dimensions, unstable efficacy, and insufficient growth promotion. It isolates a *Bacillus belye* strain from pepper rhizosphere soil that possesses both strong pathogen antagonism and highly efficient growth-promoting functions. Bacillus velezensis BYTA99, through research, has been found to antagonize not only *Bacillus anthracis* (black spot anthrax) Colletotrichum Capsici This fungus not only promotes healthy growth but also possesses multiple functions such as nitrogen fixation, phosphorus solubilization, enzyme production, antioxidant activity, and rhizosphere colonization. It can significantly reduce the incidence of pepper diseases and improve pepper yield and quality. Furthermore, biocontrol agents prepared from this strain can help improve soil structure and fertility, replace or reduce the use of chemical fertilizers and pesticides, and promote sustainable agricultural development.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: The first aspect of the present invention provides a Bacillus belesiensis (B. belesiensis) Bacillus velezensis BYTA99, deposited on April 29, 2026 at the China Center for Type Culture Collection (Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO: M 2026848; this strain exhibits antagonistic activity against anthracnose fungus and secretes... β -1,3-glucanase has the ability to act as a pectinase, solubilize phosphorus, fix nitrogen, and produce indoleacetic acid.

[0008] A second aspect of the present invention provides a microbial inoculant containing at least one of the following: live Bacillus vesiculus BYTA99, fermentation broth, bacterial suspension, lyophilized powder, or immobilized bacterial cells; wherein the effective live count of Bacillus vesiculus BYTA99 in the microbial inoculant is ≥1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0009] A third aspect of the present invention provides a method for preparing the aforementioned microbial inoculant, comprising the following steps: (1) The Bacillus belye BYTA99 was inoculated into modified LB medium and cultured with shaking at 30-37℃ and 160-200 r / min for 10-14 h, and the OD was adjusted. 600nm Adjust the value to 0.6–1.0 to obtain seed solution; (2) Inoculate the seed culture into the modified LB medium at a volume ratio of 1% to 5%, and culture it with shaking at 25 to 30°C and 160 to 200 r / min for 10 to 14 h to obtain the bacterial culture; (3) Optionally, the bacterial culture is made into freeze-dried powder or immobilized bacterial cells.

[0010] Furthermore, the modified LB medium formula is as follows: yeast extract 4~6 g / L, tryptone 8~12 g / L, sodium chloride 8~12 g / L, brown sugar 1~1.5 g / L, pH 6.9~7.1.

[0011] A fourth aspect of the present invention provides the application of the aforementioned Bacillus berreatus BYTA99 or the aforementioned microbial agent in the preparation of a product for preventing and controlling anthracnose in peppers and simultaneously promoting pepper growth. This application is achieved through the synergistic effects of antagonizing pathogens, enzymatically degrading the cell walls of pathogens, phosphorus solubilization, nitrogen fixation, and regulating the level of indoleacetic acid.

[0012] Furthermore, the promotion of chili pepper growth is manifested by an increase in at least one of the following: plant height, stem diameter, leaf area, leaf spread, number of leaves, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight.

[0013] A fifth aspect of the invention provides the use of the aforementioned Bacillus belye BYTA99 or the aforementioned microbial agent in the preparation of a product for degrading the cell wall of a rhizosphere pathogen in peppers, wherein the degradation is achieved through the simultaneous secretion of amylase, β This is achieved through 1,3-glucanase, cellulase, protease, and pectinase.

[0014] A sixth aspect of the invention provides the use of the aforementioned Bacillus berberis BYTA99 or the aforementioned microbial agent in the preparation of a product for enhancing the stress resistance of chili peppers, wherein the enhancement of stress resistance includes regulating hydrogen peroxide and / or antioxidant enzyme activity, and inhibiting malondialdehyde accumulation.

[0015] A seventh aspect of the invention provides the use of the aforementioned Bacillus berberis BYTA99 or the aforementioned microbial agent in the preparation of products for improving soil for chili cultivation, wherein the improvement includes increasing the available phosphorus content of the soil, increasing the soil organic matter level, and improving the soil aggregate structure.

[0016] Furthermore, the application methods of the product include seed treatment, soil application, foliar spraying, or root irrigation.

[0017] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Synergistic antibacterial effect through antagonism and enzymatic hydrolysis, providing stable and long-lasting protection: Strain BYTA99 directly inhibits the anthracnose pathogen of peppers by producing antibacterial substances, while also secreting amylase, β -1,3-glucanase, cellulase, protease, and pectinase. Especially... β -1,3-glucanase and pectinase hydrolyze glucan and pectin in the cell wall of pathogens, respectively, forming a synergistic effect of attack and defense, effectively inhibiting mycelial growth and conidial germination, and maintaining high control stability even under field environmental fluctuations.

[0018] 2. Synergistic growth promotion through nitrogen fixation, phosphorus solubilization, and hormone regulation, resulting in robust and stress-resistant plants: The nitrogen-fixing and phosphorus-solubilizing activities of strain BYTA99 can activate soil nutrients and improve nitrogen and phosphorus supply levels; simultaneously, the IAA it produces can directly promote root elongation and cell proliferation. This strain can also regulate the activity of hydrogen peroxide and antioxidant enzymes in peppers, effectively inhibiting malondialdehyde accumulation and reducing membrane lipid peroxidation damage. From the perspectives of nutrient supply, hormone signaling, and redox balance, it synergistically enhances plant growth and stress resistance, overcoming the deficiency of single growth-promoting bacteria that "promote growth but not robustness".

[0019] 3. Synergistic effect of rhizosphere colonization and soil improvement, with lasting ecological benefits: BYTA99 has a strong colonization ability in the rhizosphere of chili peppers. After application, it can maintain the population dominance for a long time. It also promotes the formation of soil aggregate structure by secreting metabolites such as extracellular polysaccharides, increases soil organic matter and available phosphorus content, improves the soil micro-ecological environment, fundamentally inhibits the reinfection of pathogens, and reduces the number of times and amount of repeated application of inoculants.

[0020] 4. Multifunctional integration in a single strain, simple application, green and sustainable: This invention integrates multiple functions such as biological control of diseases, plant growth promotion, stress resistance regulation and soil improvement into a single strain, avoiding the antagonism or functional conflict that may occur when multiple strains are combined. Moreover, the preparation process of the microbial agent is simple, and it can be flexibly applied through seed treatment, soil application, foliar spraying or root irrigation, which significantly reduces the amount of chemical pesticides and fertilizers used, in line with the development trend of green and low-carbon agriculture. Attached Figure Description

[0021] Figure 1 To illustrate the differences in rhizosphere soil physicochemical properties among different resistant pepper species in one embodiment of this application (n=3, ns: p >0.05; * p <0.05;** p <0.01; *** p <0.001; **** p <0.0001).

[0022] Figure 2 This is a heatmap showing the top 30 rhizosphere bacteria at the phylum and genus level relative abundance under anthrax stress in one embodiment of this application; where A represents the phylum level and B represents the genus level.

[0023] Figure 3 This is a heatmap showing the top 30 relative abundances of rhizosphere fungi at the phylum and genus level under anthrax stress in one embodiment of this application; where A represents the phylum level and B represents the genus level.

[0024] Figure 4 In one embodiment of this application, strain BYTA99 is used to... C. capsici The plate antagonistic effect and antibacterial rate were determined; where A is the plate antagonistic assay, B is the antibacterial rate assay; AT is the biocontrol group, and TJ is the disease group.

[0025] Figure 5 This invention relates to the detection of metabolites of strain BYTA99 in one embodiment of the present application; wherein, A is amylase, B is doleacetic acid, and C is... β -1,3-glucanase, D is cellulase, E is protease, F is pectinase, G is nitrogenase, and H is phosphate solubilizer.

[0026] Figure 6 This illustrates the motility of strain BYTA99 on LB medium with different agar concentrations in one embodiment of this application.

[0027] Figure 7 This document describes the changes in morphological indicators of chili seedlings during an embodiment of this application; where A is plant height, B is stem diameter, C is maximum leaf area, D is leaf spread, E is number of leaves, and F is the first internode spacing (n=3, ns:). p >0.05;* p <0.05;**p <0.0l;*** p <0.001).

[0028] Figure 8 This embodiment illustrates the change in capsaicin content; where A represents above-ground fresh weight, B represents above-ground dry weight, C represents underground fresh weight, and D represents underground dry weight (n=3, ns: p >0.05;* p <0.05;** p <0.0l;*** p <0.001).

[0029] Figure 9 This document illustrates the changes in photosynthetic pigments, MDA, and indoleacetic acid (IAA) content in chili pepper leaves in one embodiment of this application; where A represents chlorophyll a, B represents chlorophyll b, and C represents total chlorophyll (n=3, letters indicate significant differences analyzed using one-way statistical analysis). p <0.05); D is malondialdehyde, E is indoleacetic acid (n=3, * p <0.05; *** p <0.001).

[0030] Figure 10 This embodiment illustrates the change in antioxidant enzyme content in chili pepper leaves; wherein, A is phenylalanine ammonia-lyase, B is polyphenol oxidase, C is peroxidase, and D is hydrogen peroxide (n=3, ns: p >0.05;* p <0.05;** p <0.01;*** p <0.001).

[0031] Figure 11 This is a variation of chili plant morphology in one embodiment of this application (scale bar: 1 cm).

[0032] Figure 12 This is the genome information of strain BYTA99 in one embodiment of this application; wherein, A is the effective data base distribution map, B is the HiFi reads length distribution histogram, C is the coding gene nucleic acid length map, and D is the GC content distribution.

[0033] Figure 13 The above are the NR and COG database annotation results for strain BYTA99 in one embodiment of this application; where A is the distribution map of the Top 10 species in the NR database and B is the COG functional classification.

[0034] Figure 14 This is a phylogenetic tree of strain BYTA99 in one embodiment of this application. Detailed Implementation

[0035] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0036] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0037] Example 1: The effects of anthracnose on the rhizosphere environment of different resistant pepper varieties 1. Experimental Materials The “Sanying No. 9” chili pepper variety used in this experiment was cultivated at the Chili Pepper Breeding and Cultivation Experiment Base of Henan Academy of Agricultural Sciences. The samples were divided into three groups based on the disease incidence of the plants (Table 1).

[0038] Table 1. Sample Data Source Information

[0039] 2. Experimental Methods Rhizosphere soil samples were collected using a five-point random S-shaped sampling method. Five soil samples were collected at each sampling point along a pre-defined S-shaped trajectory, mixed thoroughly, and weighed out as one composite sample (5 g each). To meet the requirements for subsequent soil DNA extraction and physicochemical property determination, all collected soil samples were first flash-frozen in liquid nitrogen for about 1 hour, then transferred to a -80℃ freezer for storage, and finally sent to Henan Keyoushen Biotechnology Co., Ltd. for testing.

[0040] One-way ANOVA combined with Tukey's HSD test and Wilcoxon's rank-sum test was used to analyze the significance of differences between groups. The significance level was set at: p <0.01 indicates a highly significant difference (**). p <0.05 indicates a significant difference (*). p A value >0.05 indicates no significance (ns), ensuring the statistical rigor of the analysis results.

[0041] 3. Analysis of differences in rhizosphere soil physicochemical properties and bacterial and fungal community diversity. (1) Differences in rhizosphere soil physicochemical properties: Tukey's HSD test was used to perform multiple comparison analysis on the mean values ​​of 16 physicochemical properties of rhizosphere soil of peppers at different health levels. The results showed that ( Figure 1The contents of trace elements such as calcium, magnesium, zinc, and iron in the rhizosphere soil increased significantly with the enhancement of pepper resistance, and the activity of acid phosphatase in resistant soil was significantly higher than that in susceptible soil. However, the activities of readily available nutrients such as nitrogen, phosphorus, and potassium, as well as other enzymes, did not differ significantly among different resistance groups. How these factors affect the regulation of plant nutrient absorption in relation to anthracnose requires further investigation.

[0042] (2) The results of the rhizosphere bacterial community diversity analysis are shown ( Figure 2 Regarding the microbial community, the resistant rhizosphere soils exhibited higher α-diversity, and the relative abundance of Acidobacteria decreased with decreasing resistance. The abundance of Veillellae and other Bacteria in the resistant rhizosphere soils was higher. RB41 The relative abundance of the genera increased significantly, which may inhibit the development of anthrax by promoting the decomposition and transformation of nutrients such as nitrogen, phosphorus, and potassium.

[0043] (3) Analysis of rhizosphere fungal community diversity ( Figure 3 Fungal community analysis revealed that Ascomycota had a relatively low abundance in the rhizosphere soil of resistant plants, while the dominance of Basidiomycota may be related to their key roles in organic matter decomposition, lignin degradation, and nitrogen and phosphorus cycling.

[0044] At the functional level, there are significant differences between the disease-resistant group and the disease-susceptible group in metabolic pathways such as cell apoptosis and mycelial development, indicating that the metabolic activity of rhizosphere microorganisms is closely coupled with plant disease resistance.

[0045] Example 2: Evaluation of the control effect of strain BYTA99 on anthracnose in peppers 1. Experimental Materials (1) Test strains: Strain BYTA99 was isolated and preserved by the laboratory of the Vegetable Research Institute of Henan Academy of Agricultural Sciences (collector: Yang Fan; collection location: Hezhai Village, Guyang Town, Lankao County, Henan Province; collection time: June 2024), and was stored at -80℃ in 30% glycerol for a long period of time; the pathogen of pepper anthracnose was *Anthracnose glomeratus* (… Colletotrichum Capsici (Isolated from diseased leaves of chili peppers in Xinxiang City, Henan Province)

[0046] (2) Culture medium Prepare the basic nutrient culture medium as follows: Potato glucose agar (PDA) medium: potato extract 3 g / L; glucose 20.0 g / L; agar 14.0 g / L; pH 5.6 ± 0.2.

[0047] Potato broth (PDB) medium: 3 g / L potato extract powder; 20.0 g / L glucose; pH 5.6 ± 0.2.

[0048] LB solid medium: yeast extract 5 g / L; tryptone 10 g / L; NaCl 0.2 g / L; brown sugar 1.2 g / L; agar powder 15 g / L; pH 6.9-7.1.

[0049] LB liquid medium: yeast extract 5 g / L; tryptone 10 g / L; NaCl 0.2 g / L; brown sugar 1.2 g / L; pH 6.9-7.1.

[0050] (3) Preparation of seed culture of strain BYTA99: First, strain BYTA99 was activated. The strain preserved in glycerol was streaked onto LB solid medium plates and incubated upside down in a 30℃ incubator for 1 day. Single colonies were selected for secondary streaking purification, and the activated strains were stored at 4℃ and -20℃ for later use. Preparation of seed culture (SC): A single activated colony was picked and inoculated into 100 mL of LB liquid medium and cultured with shaking at 30℃ and 170 r / min for 12 h to obtain the seed culture. The seed culture was diluted to OD using LB medium. 600 =0.8, reserved.

[0051] (4) Preparation of pathogenic bacterial solution: First, the strain C.capsici Activation treatment was performed. A 5 mm diameter pathogen block was taken using a punch and inoculated into PDA medium, then placed in a 28℃ mold incubator. The plates were first incubated upright for 12 h, then inverted for 7 days. This activation step was repeated once. The activated plates were stored at 4℃ and -20℃ for later use. The preparation steps for pathogen suspension (PI) were as follows: A piece of mycelial cake was taken using a punch and inoculated into 100 mL of PDB liquid medium, and cultured with shaking at 30℃ and 170 r / min for 3 days. The pathogen spore concentration was adjusted to 1 x 10⁻⁶ using a hemocytometer. 6 Quantity / mL, for later use.

[0052] 2. Inhibitory effect of strain BYTA99 on pathogens First, strain BYTA99 and C. capsiciPlate confrontation culture was performed. Using a sterile punch, 5 mm diameter pathogenic bacterial pellets were inoculated into the center of PDA plates and pre-cultured at 28℃ for 3 days. Subsequently, single colonies of BYTA99 were picked using a sterile 10 μL pipette tip and symmetrically inoculated 2.5 cm from both sides of the bacterial pellet; this treatment group was designated AT (biocontrol group). PDA plates inoculated only with the pathogen served as a blank control, designated TJ (susceptible group). All confrontation plates were incubated at 28℃, initially upright for 24 h to promote biocontrol colonization, then inverted for 7 days, with daily photographs taken at fixed times. After 15 days of incubation at 28℃, when the pathogen growth rate significantly slowed and stabilized, the inhibition rate was determined. The diameter of the inhibition zone was measured using the cross-hatching method, with three measurements taken for each plate, and the average value was used as the final inhibition rate. The colony diameters of AT and TJ were measured, and the inhibition rate was calculated using the following formula: Antibacterial rate = [(control group diameter - treatment group diameter) / (control group diameter - mycelium cake diameter)] × 100%.

[0053] Plate confrontation experiments showed that strain BYTA99 could significantly inhibit [the virus] within a short period of time. C. capsici mycelial growth ( Figure 4 A). Day 5 after inoculation with strain BYTA99, C. capsici Mycelial growth began to be inhibited, and the proliferation rate of pathogenic mycelia near strain BYTA99 slowed significantly; after co-culturing for 7 days, C. capsici The colonies were irregularly elliptical, showing a significant difference in morphology from normally growing pathogen colonies. The results of the disease inhibition rate assay indicated that ( Figure 4 B) The diameter of a normally growing pathogen colony is 8.2 cm, while the diameter of an inhibited pathogen colony is 3.3 cm. Therefore, the diameter of strain BYTA99 can be calculated to be... C. capsici The disease inhibition rate was 63.63%. These results preliminarily confirm that strain BYTA99 has a significant inhibitory effect on the anthracnose pathogen of pepper.

[0054] 3. Analysis of metabolites from strain BYTA99 Selective culture medium screening, specific color development, and morphological observation confirmed that strain BYTA99 can produce multiple metabolites, and all metabolic activity tests were positive. Figure 5 Enzyme detection showed that strain BYTA99, when inoculated onto starch agar medium and stained with Lugol's iodine solution, exhibited a clear halo around the colony and could secrete amylase; inoculated onto... β After screening with 1,3-glucan agar, carboxymethyl cellulose agar, skim milk, and pectinase, the colonies all had a transparent halo around them and could secrete... β-1,3-glucanase, cellulase, protease, and pectinase were detected. IAA activity identification showed that after inoculation of the BYTA99 seed culture into NB medium with shaking, the supernatant reacted with Salkowski chromogenic reagent to produce a yellow color, indicating the synthesis of IAA. Physiological activity assays showed that strain BYTA99 could grow normally on Assumption medium and possessed nitrogen-fixing ability; it formed a clear zone on Monkina medium, indicating phosphate-solubilizing activity.

[0055] 4. Evaluation of the motility of strain BYTA99 Different concentrations of LB semi-solid medium were used for testing. 0.3% (3 g / L) agar was used to detect swimming ability, while 0.5% (5 g / L) and 0.7% (7 g / L) agar were used to detect swarming ability. Solid LB medium with 1.5% (15 g / L) agar was used as a control to eliminate interference from twitching movements. 2.5 μL of SC was inoculated into the center of each medium, ensuring consistent inoculation. The medium was incubated at 30℃, and photographs were taken every 4 hours to observe the growth and spread of the strain.

[0056] Experimental results using semi-solid culture media with different agar concentrations showed that ( Figure 6 The strain BYTA99 showed a certain migration ability on media with different agar concentrations and diffused growth inside the media, indicating that it has motility and can tend to colonize the rhizosphere of peppers.

[0057] 5. Evaluation of the efficacy of strain BYTA99 in alleviating anthracnose damage in peppers. The selected chili pepper seeds were "San Ying No. 9". Seeds of uniform size and color were chosen, soaked in warm water for 45 minutes, then removed and soaked in a 1% potassium permanganate solution at room temperature for 20 minutes. The seeds were then rinsed five times with clean water. The treated seeds were placed in petri dishes lined with filter paper and cultured at 28℃. Once the seeds showed signs of sprouting, they were transplanted into seedling trays. The cultivation substrate was sterilized coconut coir:vermiculite:perlite = 3:1:1 (volume ratio).

[0058] The test material was chili seedlings. Seedlings that had been cultured for 35 days, exhibited uniform growth, were free from pests and diseases, and showed consistent physiological condition were selected and individually transplanted into each culture pot. These seedlings were then placed in a constant-temperature environment at approximately 27±1℃, with alternating 16 hours of light and 8 hours of darkness, and a relative humidity of 80%±5%. After 60 days of conventional culture, the corresponding bacterial solution was applied to the roots of the seedlings. The experiment consisted of four treatment groups, with 20 replicates for each treatment, as detailed below: Control group (CK): Sterile water was added to the roots; Pathogen treatment group (TJ): 20 mL of pathogen solution (mixed with 10 mL PI + 10 mL sterile water) was added to the roots every 5 days, for a total of 4 times; Biocontrol group (AT): On day 40, apply 20 mL of pathogen solution to the roots, and then apply it once every 5 days for a total of 4 times. After the pathogen solution is applied, add 20 mL of biocontrol solution (mixed with 10 mL SC + 10 mL sterile water) to the roots every 5 days for a total of 4 times. Growth promotion group (BYTA99): Add 20 mL of biocontrol solution (mixture ratio of 10 mL SC + 10 mL sterile water) to the roots every 5 days, for a total of 4 times.

[0059] (1) Analysis of chili pepper growth The results of morphological index determination showed that ( Figure 7 The pathogen infection significantly inhibited the above-ground growth of pepper plants; the control group had a plant height of 17.27 cm and a maximum leaf area of ​​19.41 cm². 2 Compared with the CK group, the TJ group showed significant decreases in plant height, stem diameter, maximum leaf area, leaf spread, and number of leaves, with reductions ranging from 26.17% to 43.99%. Only the internode spacing showed no significant difference, with the CK group showing a slightly higher value than the TJ group. The AT group showed significantly or highly significantly improved morphological indicators compared to the TJ group, with improvements ranging from 36.99% to 93.69%, indicating that strain BYTA99 can effectively alleviate the damage to pepper plant growth caused by pathogen infection. All indicators of the BYTA99 group were significantly higher than those of the other groups. The treatment group showed significant improvements in all morphological indicators compared to the control group (CK). Plant height increased to 23.13 cm, with an increase ranging from approximately 28.30% to 67.92%, significantly better than the AT group. This indicates that strain BYTA99 has a significant growth-promoting effect on pepper plants. Overall, the morphological indicators showed a consistent trend across different treatment groups, with the overall trend being BYTA99 group > AT group > CK group > TJ group. This suggests that strain BYTA99 possesses the dual function of alleviating pathogen stress and promoting pepper plant growth.

[0060] The fresh and dry weights of the aboveground and underground parts of the plant are important indicators reflecting the plant's material accumulation level and growth status. They can reflect the degree of material loss caused by pathogen infection to pepper plants, as well as the mitigating effect of biocontrol strains on this loss. The results showed ( Figure 8 Significant differences were found in the fresh and dry weights of the aboveground parts among the treatment groups: the fresh and dry weights of the aboveground parts in the CK group were 28.63 g and 5.72 g, respectively, while those in the TJ group decreased by 49.91% and 49.48% compared to the CK group, respectively, indicating that... C. capsiciInfection significantly reduced the accumulation of aboveground substances in peppers, resulting in significant material loss. In the determination of fresh and dry weight of underground parts, there was no significant difference between the CK and TJ groups. Specifically, the fresh and dry weights of underground parts in the CK group were 6.35 g and 1.27 g, respectively, while those in the TJ group were 5.89 g and 1.18 g, respectively. However, there were significant differences between the TJ group and the AT group, and between the CK group and the BYTA99 group. The fresh and dry weights of underground parts in the AT group increased by 48.73% and 59.32% compared to the TJ group, respectively. The fresh and dry weights of underground parts in the BYTA99 group reached 11.24 g. Overall, the plant material volume in each treatment group was 2.53 g, representing increases of 77.01% and 99.21% compared to the CK group, respectively. The cumulative amount and morphological indicators showed a consistent ranking from high to low, namely BYTA99 group > AT group > CK group > TJ group, indicating that pathogen infection inhibits pepper growth and reduces material accumulation. However, strain BYTA99 can not only effectively alleviate the damage and material loss caused by pathogens, but also significantly promote pepper plant growth and material accumulation, thus possessing good dual functions of biocontrol and growth promotion.

[0061] (2) Analysis of the substance content of chili peppers Chlorophyll is a key substance for plant photosynthesis, and changes in its content can reflect the plant's photosynthetic capacity and growth health. Measurement results show that, regarding chlorophyll a content ( Figure 9 A) There was no significant difference between the AT group and the CK group. The chlorophyll a content in the CK group was 1.87 mg / g. The chlorophyll a content in both groups was higher than that in the BYTA99 group and approximately 16 times that in the TJ group, indicating that pathogen infection significantly reduced the chlorophyll a content in pepper leaves. Regarding chlorophyll b content ( Figure 9 (B) The groups showed a clear ranking from highest to lowest chlorophyll b content. The AT group had the highest chlorophyll b content, followed by the CK and BYTA99 groups. The chlorophyll b content in the CK group was 0.62 mg / g. The chlorophyll b content in all three groups was approximately 1.6-1.8 times that of the TJ group. Regarding total chlorophyll content ( Figure 9 C) The AT group had the highest total chlorophyll content, which was not significantly different from the CK and BYTA99 groups. The total chlorophyll content of the CK group was 2.49 mg / g. The total chlorophyll content of all three groups was about 1.5-1.7 times that of the TJ group, which further confirmed that pathogen infection would inhibit chlorophyll synthesis in pepper leaves, while strain BYTA99 could effectively alleviate this inhibition and ensure normal photosynthesis of the plant.

[0062] Regarding MDA content ( Figure 9D) The groups showed clear differences: Group T had the highest MDA content at 32.6 nmol / g, significantly higher than both the CK and AT groups. The MDA content in the CK group was 18.9 nmol / g, and in the AT group it was 23.4 nmol / g. Group TJ showed a 72.5% increase compared to the CK group. Meanwhile, the MDA content in the BYTA99 group was 21.7 nmol / g, significantly higher than the CK group, indicating... C. capsici Infection significantly increases MDA accumulation in leaves, while strain BYTA99 can reduce MDA content to some extent, alleviating plant damage. Regarding IAA content ( Figure 9 E) The differences between the groups and the MDA content showed an opposite trend: the IAA content in the TJ group was significantly lower than that in the CK and AT groups. The IAA content in the CK group was 12.8 μg / g, while that in the TJ group was only 5.3 mg / g, a decrease of 58.6% compared to the CK group, indicating that... C. capsici Infection significantly inhibited the synthesis and accumulation of IAA in pepper leaves; while the IAA content in the BYTA99 group was 16.2 μg / g, which was still higher than that in the CK group, further confirming that the biocontrol strain BYTA99 can promote the accumulation of IAA in pepper leaves, which is closely related to its growth-promoting effect.

[0063] (3) Analysis of antioxidant enzyme activity in chili peppers Antioxidant enzymes, as important protective substances in plant cells, have the core function of scavenging reactive oxygen species induced by stress, inhibiting membrane lipid peroxidation, and activating the plant's own defense mechanisms, thereby enhancing the plant's immunity to stress. The results of the assays showed that ( Figure 10 The activities of the four antioxidant enzymes showed a consistent trend: the activities of all four antioxidant enzymes in group T were significantly higher than those in groups AT and CK. Specifically, the CAT enzyme activity in group CK was 163.98 U / g, while that in group TJ was 52.1% higher than in group CK; the activities of POD, PPO, and PAI enzymes were 79.1%, 57.7%, and 44.6% higher than in group CK, respectively. The antioxidant enzyme activities in group BYTA99 were significantly higher than those in group CK. The CAT enzyme activity was not significantly different from that in group CK, while the activities of POD, PPO, and PAL enzymes were 13.2%, 17.8%, and 12.6% higher than in group CK, respectively. The overall activity ranking was TJ group > AT group > BYTA99 group > CK group (except for CAT enzyme). These changes indicate that... C. capsici Infection can significantly induce an increase in the activity of antioxidant enzymes in pepper leaves, stimulating the plant's own defense response; while treatment with strain BYTA99 can regulate the activity of antioxidant enzymes to a certain extent, keeping them at a reasonable level, which can both stimulate the plant's defense capabilities and avoid damage to the plant caused by excessive accumulation of antioxidant enzymes.

[0064] (4) Analysis of the phenotypic characteristics of chili peppers The results of the pot experiment showed that ( Figure 11 Significant differences in plant phenotypes were observed among the treatment groups: the BYTA99 group exhibited the best growth, with robust and upright stems, lush and dark green leaves, and a growth pattern significantly superior to the CK group; the TJ group showed extremely poor growth, characterized by stunted plants, pale and dull leaves, and overall stunted growth; the AT group showed growth similar to the CK group, without stunting, and its leaf color returned to normal, indicating a significant alleviation of the disease phenotype. Further detailed observation of individual chili plants in each treatment group revealed that the TI group had smaller overall plant size, wrinkled stems, a significantly reduced number of underground roots, and poor growth; while the BYTA99 group exhibited lush leaves, robust stems, and a well-developed root system, demonstrating vigorous growth in both the above-ground and underground parts.

[0065] Example 3: Whole genome sequencing of strain BYTA99 1. Experimental Materials Prepare SC for later use, using the same method as in Example 2.

[0066] 2. Whole genome information analysis of strain BYTA99 Centrifuge 10 mL of SC at 5000 r / min for 3 min, discard the supernatant, and collect the bacterial pellet (6 replicates). Store it on dry ice and send it to Henan Keyushen Biotechnology Co., Ltd. for subsequent experiments. Extract genomic DNA from strain BYTA99 according to the kit instructions. After extraction, use a Nanodrop detector (OD2000) to detect the DNA. 260 / OD 280 Purity was determined by ratio. DNA was randomly fragmented using a Covaris ultrasonic disruptor. Library construction was completed through end repair, A-tail addition, adapter ligation, purification, and PCR amplification. After library construction, the DNA was quantified using Qubit 2.0, the insert length was detected using an Agilent 2100, and quality control was performed using precise qPCR quantification. Sequencing was then performed using an Illumina NovaSeq 6000 platform.

[0067] The quality control results show that ( Figure 12 A total of 1,549,467,300 valid sequencing reads were obtained from the biocontrol strain BYTA99 (AB). The GC content accounted for 47.63%, and the AT content was slightly higher than the GC content. The sequencing base composition conformed to the normal genome characteristics of the strain. PacBio single-molecule circular sequencing technology was used for sequencing. The obtained subreads were converted into HiFi reads using the CCS program. Statistical results showed that the total length of HiFi reads for the biocontrol strain BYTA99 was 876,942.457. Systematic prediction of the basic genome components of strain BYTA99 showed that ( ) Figure 12 The total genome length of strain BYTA99 is 3,980,777 bp, with a total of 4,104 coding genes predicted. The average length of the coding genes is 865.3 bp. The GC content of the coding gene region is 47.24%, and the coding gene length is mainly concentrated between 50-100 bp. The genome structure is consistent with the typical characteristics of Bacillus strains.

[0068] The TRNA gene prediction results (Table 2) show that the BYTA99 genome contains 27 RNA genes with a total length of 41,229 bp, accounting for 1.04% of the total genome length. Among them, there are 9 copies of each of the 5S, 16S, and 23S rRNA genes, suggesting that there may be 9 RNA operon repeat units in its genome. Specifically, the average length of the 23S rRNA gene is 2928 bp, the average length of the 5S rRNA gene is 115 bp, and the average length of the 16S rRNA gene is 1538 bp. This length is consistent with the typical 1525-1545 bp 16S rRNA length range of Bacillus.

[0069] Table 2. Statistical Table of rRNA Prediction Results

[0070] The assembled genome sequence of strain BYTA99 was subjected to species homology comparison analysis in the NT database to clarify its species classification and phylogenetic relationships. Genome sequencing results showed that the full-length genome of strain BYTA99 was 3,980,777 bp, with a GC content of 47.24%, encoding a total of 4104 genes. The comparison results (Table 3) showed that strain BYTA99 matched three strains with the highest similarity, namely… Bacillus sp. LUNF1, Bacillus velezensis strain DR-08 and Bacillus velezensis strain LDO2, all three strains mentioned above belong to the genus *Bacillus belyssus*, strain BYTA99 and... Bacillus velezensis The highest similarity.

[0071] Table 3. NT library alignment results for assembled genomes

[0072] 3. Functional annotation analysis of strain BYTA99 Protein sequences were aligned to NR (NCBI Non-Redundant Protein Sequence Database) and COG (Homologous Protein Cluster Database) using Diamond v.2.0.9, with an E-value of 1e-5. The alignment results from the NR database showed that ( Figure 13A) A total of 4066 validly annotated predicted protein sequences for BYTA99 were obtained. Among them, Bacillus Genera comprise 75.95% (3088 sequences), clearly defining BYTA99 as belonging to the genus Bacillus; at the species level, Bacillus velezensis The highest percentage was 12.67% (515 items), followed by... B. velezensis and B. amyloliquefaciens The group members, with a cumulative percentage >15%, combined with the threshold of average nucleotide similarity >95%, can identify strain BYTA99 as... Bacillus velezensis The genome coding genes of strain BYTA99 were compared and annotated with the COG database, and the results showed that ( Figure 13 B): Metabolism-related genes are dominant, with 313 in class G, 301 in class C, and 287 in class E, totaling over 900, which are expected to ensure rhizosphere carbon and nitrogen utilization and rapid colonization; Genetic information processing and cell membrane-related genes are abundant, with 203 in class J, 167 in class M, and 181 in class L, which may enhance plant growth and stress resistance; There are numerous genes for signal transduction and defense mechanisms, with 50 in class T and 23 in class V; Secondary metabolism-related genes are sufficient, with 100 in class Q, consistent with the predicted results of 12 BGCs, confirming that strain BYTA99 has the potential for antibacterial activity and growth-promoting substance synthesis.

[0073] 4. Systematic Evolution of Strain BYTA99 To further clarify the species relationship of the biocontrol strain BYTA99 and verify its species classification, a comparative phylogenetic analysis was performed on the BYTA99 strain. The results showed ( Figure 14 ), strain BYTA99 and Bacillus The strain showed the highest similarity to sp. LUNF1, reaching 95%. Combined with the results of species homology comparison, this further confirmed that strain BYTA99 belongs to the same group. Bacillus velezensis Ultimately, BYTA99 was identified as Bacillus belye (B. belye). Bacillus velezensis The sample is currently deposited at the China Center for Type Culture Collection (Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO: M 2026848 and deposit date: April 29, 2026.

[0074] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

Claims

1. A type of Bacillus belesii ( Bacillus velezensis BYTA99, characterized in that, Its accession number is CCTCC NO: M 2026848; this strain exhibits antagonistic activity against pepper anthracnose and secretes... β -1,3-glucanase has the ability to act as a pectinase, solubilize phosphorus, fix nitrogen, and produce indoleacetic acid.

2. A microbial inoculant, characterized in that, Contains at least one of the following: live Bacillus vesiculus BYTA99 as described in claim 1, fermentation broth, bacterial suspension, lyophilized powder, or immobilized bacterial cells; wherein the effective live count of Bacillus vesiculus BYTA99 in the microbial agent is ≥1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

3. A method for preparing the microbial inoculant as described in claim 2 or 3, characterized in that, Includes the following steps: (1) The Bacillus belye BYTA99 of claim 1 was inoculated into modified LB medium and cultured with shaking at 30-37°C and 160-200 r / min for 10-14 h, and the OD was adjusted. 600nm Adjust the value to 0.6–1.0 to obtain seed solution; (2) Inoculate the seed culture into the modified LB medium at a volume ratio of 1% to 5%, and culture it with shaking at 25 to 30°C and 160 to 200 r / min for 10 to 14 h to obtain the bacterial culture; (3) Optionally, the bacterial culture is made into freeze-dried powder or immobilized bacterial cells.

4. The preparation method according to claim 3, characterized in that, The modified LB medium formula is as follows: yeast extract 4~6 g / L, tryptone 8~12 g / L, sodium chloride 8~12 g / L, brown sugar 1~1.5 g / L, pH 6.9~7.

1.

5. The application of Bacillus berberis BYTA99 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of a product for controlling anthracnose in peppers and simultaneously promoting pepper growth, characterized in that, This application achieves its effect through the synergistic action of antagonizing pathogens, enzymatically breaking down pathogen cell walls, solubilizing phosphorus, fixing nitrogen, and regulating indoleacetic acid levels.

6. The application according to claim 5, characterized in that, The promotion of chili pepper growth is manifested by an increase in at least one of the following: plant height, stem diameter, leaf area, leaf spread, number of leaves, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight.

7. The use of Bacillus belye BYTA99 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of a product for degrading the cell wall of a rhizosphere pathogen in peppers, characterized in that, The degradation is achieved through the simultaneous secretion of amylase, β This is achieved through 1,3-glucanase, cellulase, protease, and pectinase.

8. The application of Bacillus belye BYTA99 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products for enhancing the stress resistance of chili peppers, characterized in that, The enhancement of stress resistance includes regulating hydrogen peroxide and / or antioxidant enzyme activity, as well as inhibiting malondialdehyde accumulation.

9. The application of Bacillus belyssus BYTA99 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products for improving soil for chili cultivation, characterized in that, The improvements include increasing the available phosphorus content in the soil, improving the soil organic matter level, and improving the soil aggregate structure.

10. The application according to any one of claims 5-9, characterized in that, The product can be applied via seed treatment, soil application, foliar spraying, or root irrigation.

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