Bacillus siamensis GD12 and application thereof in prevention and treatment of pepper root rot, anthracnose and blight

Abstract

The application discloses a bacillus siamensis GD12 and application thereof in prevention and treatment of pepper root rot, anthracnose and blight, and belongs to the technical field of microorganisms. The inhibition rates of 50% volume fraction of sterile fermentation filtrate of the bacillus siamensis GD12 on pepper root rot fungus fusarium oxysporum, anthracnose fungus colletotrichum gloeosporioides and blight fungus phytophthora capsici reach 67.88%, 88.92% and 100% respectively. The filtrate can destroy the cell membrane integrity of the pathogenic bacteria, cause mycelium to be deformed, concave, collapsed or broken, and effectively inhibit spore germination and sporangium production, and in addition, can promote pepper seed germination. The potting test shows that the fermentation liquor has a prevention effect of more than 80% on the three diseases, and can promote the growth of pepper plants. The application has multiple functions of bacteriostasis, disease prevention and growth promotion, is environment-friendly, and provides a sustainable biological control resource for green prevention and control of soil-borne and air-borne diseases of pepper.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus sicca ( Siamese Bacillus GD12 and its application in the prevention and control of root rot, anthracnose and blight in peppers. Background Technology

[0002] chili( Red pepper Chili peppers are an important vegetable and economic crop widely cultivated globally. However, chili peppers are often seriously threatened by various diseases during cultivation, among which Fusarium equisetifolium (L.) Fusarium horsetail Root rot caused by *Anthracnose* (a fungus), and root rot caused by *Anthracnose* Colletotrichum fructicola Anthrax caused by ) and by Phytophthora capsici ( Phytophthora capsicum Three of the most destructive diseases affecting peppers are blight, anthracnose, and root rot. These diseases are widespread and severe, often leading to large-scale yield reductions or even total crop failure, causing huge economic losses to agricultural production. Phytophthora capsici has a wide host range and spreads rapidly, infecting the roots, stems, leaves, and fruits of peppers, causing symptoms such as root rot, stem rot, and damping-off. Anthracnose has a wide range of incidence and a high morbidity rate, seriously harming the development of the pepper industry. Root rot is caused by soil-borne pathogenic fungi and is particularly severe under continuous cropping conditions.

[0003] Currently, chemical control remains the primary means of controlling these diseases, with commonly used chemical agents including dimethomorph, azoxystrobin, metalaxyl, and cymoxanil. However, the long-term and excessive use of chemical pesticides has brought about many problems: the mechanisms of action of chemical agents often target a single link in the pathogen's metabolism, and once the site of action mutates, pathogens can easily develop resistance, thus reducing the effectiveness of control; the excessive use of chemical pesticides has led to a series of problems such as pesticide residue exceeding standards, environmental pollution, and disruption of the ecological balance. Therefore, developing efficient, safe, and environmentally friendly biological control alternatives has become a current research hotspot.

[0004] Bacillus ( Bacillus *Bacillus sicca* (spp.) exhibits great potential in the field of biological control due to its ability to produce highly resistant spores, broad-spectrum antibacterial activity, and good plant growth-promoting effects. Bacillus Siamese As an effective species of the Bacillus genus, it has attracted much attention due to its strong tolerance, adhesion and stability. Existing research shows that Bacillus sicca has broad-spectrum antibacterial properties and other biological activities, and has good development potential.

[0005] Chinese invention patent (authorization announcement number CN119570656B) discloses a strain of *Bacillus sicca* SD23LJ1314, but it does not involve the inhibitory effect on *Fusarium equisetifolium* (root rot) and *Anthracnose spp.* (anthracnose), nor does it provide data on potted plant control efficacy. Chinese patent application (publication number CN113308392A) discloses strain QN2MO-1, whose main targets are wilt and anthracnose, but does not involve the integrated control of *Phytophthora capsici* (blight). Chinese patent application (publication number CN121518322A) discloses strain BsiaSC07, used for the control of kiwifruit leaf spot disease, with a target pathogen completely different from this application. In summary, there is currently no information regarding a single *Bacillus sicca* strain capable of simultaneously inhibiting *Fusarium equisetifolium* (root rot) in peppers. Fusarium horsetail Anthracnose disease caused by Anthracnose fungus ( Colletotrichum fructicola This report presents strains or technologies that exhibit in vitro antibacterial activity, potted plant-based control efficacy, and growth-promoting functions against three major diseases: Phytophthora capsici, Phytophthora cirrhosa, and Phytophthora capsici. Screening for multifunctional Bacillus siannais strains is of great significance for the green control of pepper diseases and sustainable production. Summary of the Invention

[0006] The current technology lacks a strain of *Bacillus sicca* capable of simultaneously and effectively inhibiting the pathogens of three major diseases affecting chili peppers: root rot (*Fusarium equisetifolium*), anthracnose (*Anthracnose spp.*), and blight (*Phytophthora capsici*), while also promoting seed germination and plant growth. This invention aims to provide a strain of *Bacillus sicca* GD12 and its application in the control of root rot, anthracnose, and blight in chili peppers.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a strain of Bacillus sicca ( Siamese Bacillus The strain GD12 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No: 68595 and accession date of July 10, 2026.

[0008] The Siamese Bacillus ( Siamese Bacillus The nucleotide sequence of the 16S rRNA of GD12 is shown in SEQ ID NO.1.

[0009] The present invention also provides a biological agent comprising the above-mentioned Bacillus sicca ( Bacillus Siamese GD12, its fermentation broth or its sterile fermentation filtrate, or at least one of them.

[0010] Furthermore, the biological agent is a liquid formulation, powder, or immobilized formulation; preferably, the immobilized formulation is a microcapsule, microsphere, or granule.

[0011] The present invention also provides the application of the above-mentioned Bacillus sicca GD12 or the above-mentioned biological agent in at least one of the following (i) to (vi): (i) In inhibiting Fusarium equisetifolium ( Fusarium horsetail ), fruit anthracnose ( Colletotrichum fruit-growing ) or Phytophthora capsici ( Phytophthora capsicum Applications in growth; (ii) Application in inhibiting the germination of conidia of Fusarium equisetifolium and Anthracnoseus spp. or the production of sporangia of Phytophthora capsici; (iii) Application in the colony morphology or mycelial structure of teratogenic Fusarium equisetifolium, Anthracnose spp. or Phytophthora capsici; (iv) Application in the preparation of inhibitors of Fusarium equisetifolium, Anthracnoseus spp. or Phytophthora capsici; (v) Application in the prevention and control of pepper root rot, pepper anthracnose or pepper blight; (vi) Application in promoting pepper seed germination; (vii) Application in promoting the growth of chili pepper plants.

[0012] Furthermore, the aseptic fermentation filtrate of *Bacillus sicca* GD12 inhibits the growth of *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici* through at least one of the following methods: (1) Teratogenic pathogenic mycelium, causing the mycelium to become deformed, sunken, collapsed or broken; (2) Disrupts the cell membrane integrity of pathogenic fungal hyphae; (3) Inhibits the germination of conidia of Fusarium equisetifolium and / or Anthracnoseus spp.; (4) Inhibit the production of Phytophthora capsici sporangia.

[0013] The present invention also provides a method for preventing and controlling pepper root rot, pepper anthracnose and / or pepper blight, by applying an effective amount of the above-mentioned Bacillus sicca GD12 or the above-mentioned biological agent to the test pepper plants.

[0014] Furthermore, the application method is root irrigation, seed soaking, spraying, or mixing with soil; preferably, the viable count of Bacillus sicca GD12 in the biological agent is 1×10⁻⁶. 8 ~1×10 10 CFU / mL, or the volume fraction of the sterile fermentation filtrate is 10%~50%.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a strain of Bacillus sicca ( Siamese Bacillus GD12 is a novel bacterial strain isolated and screened from the rhizosphere soil of healthy chili peppers in a chili orchard in Yulao Town, Fengkai County, Guangdong Province. Morphological identification, Gram staining, and 16S rRNA phylogenetic analysis revealed that after culturing on LB solid medium at 29°C for 48 h, the colonies were milky white, opaque, dry, wrinkled, with irregular edges, and rod-shaped, indicating Gram-positive bacteria. Its 16S rRNA sequence is consistent with that of strain MG778747.1 in GenBank. Siamese Bacillus The homology is 100%. This strain has a unique genetic background and biological characteristics, and can produce broad-spectrum antifungal metabolites, providing an excellent strain resource for the development of subsequent biological agents.

[0016] The aseptic fermentation filtrate of Bacillus sicca GD12 exerts its antibacterial effect through multiple pathways: it disrupts the cell membrane integrity of pathogenic mycelia, leading to irreversible ultrastructural damage such as mycelial deformities, depressions, collapses, or breaks; it effectively inhibits the germination of conidia of Fusarium equisetifolium and Anthracnose cylindrica; and it reduces the number of sporangia produced by Phytophthora capsici. These multiple mechanisms of action help delay the development of drug resistance in pathogens. It also promotes pepper seed germination, increasing germination potential, germination rate, and germination index, with the optimal effect observed at concentrations of 20%–30%, achieving a germination rate exceeding 90%. Root irrigation with the live bacteria fermentation filtrate achieves over 80% control efficacy against the aforementioned three diseases, while simultaneously promoting pepper plant growth, increasing root length, plant height, stem diameter, overall fresh weight, leaf length, and leaf width. This dual function of disease prevention and growth promotion aligns with the requirements of green agriculture and sustainable development.

[0017] The biological agent provided by this invention exhibits broad-spectrum antibacterial activity in both the live bacterial cells and their secreted extracellular active metabolites: a 50% (v / v) sterile fermentation filtrate inhibited the mycelial growth of *Fusarium equisetifolium*, *Anthracnose spp.*, and *Phytophthora capsici* by 67.88%, 88.92%, and 100%, respectively. Simultaneously, this biological agent also demonstrates good control efficacy against the aforementioned three diseases and promotes pepper seed germination and plant growth. The flexible configuration of live bacterial cells, fermentation broth, and sterile fermentation filtrate provides diverse options for formulation development in different application scenarios, exhibiting broad adaptability.

[0018] The application provided by this invention, which uses a strain and its biological agents derived from the natural ecological environment, is environmentally friendly and provides high-quality strain resources for the green control of pepper root rot, anthracnose and blight. It can reduce the use of chemical pesticides, reduce pesticide residues and environmental pollution risks, and has extremely high application value and broad application prospects in the field of green control and growth promotion of pepper diseases. Attached Figure Description

[0019] Figure 1The present invention is based on Bacillus sicca ( Siamese Bacillus Colony morphology of GD12 on LB medium; Figure 2 The present invention is based on Bacillus sicca ( Siamese Bacillus A phylogenetic tree of GD12 based on the 16S rDNA gene sequence; Figure 3 The present invention is based on Bacillus sicca ( Siamese Bacillus Gram staining microscopic image of GD12; Figure 4 This invention relates to Bacillus sicca (Bacillus subtilis). Siamese Bacillus ) Plate contrast plot of GD12; where the first row is the blank control plot of three pathogens, and the second row A is Fusarium equisetifolium ( Fusarium horsetail ) Confrontation effect diagram; B is fruit anthrax bacteria ( Colletotrichum fructicola ) Confrontational effect diagram; C represents Phytophthora capsici ( Phytophthora capsicum (Standoff illustration)

[0020] Figure 5 The present invention is based on Bacillus sicca ( Siamese Bacillus The plate inhibition effect of GD12 aseptic fermentation filtrate on three pathogens; where A is Fusarium equisetifolium (…). Fusarium horsetail B. anthracnose ( Colletotrichum fructicola C represents Phytophthora capsici ( ); Phytophthora capsicum Each row, from left to right, represents the control group (CK) and the sterile fermentation filtrate treatment groups with volume fractions of 10%, 20%, 30%, 40%, and 50%. Figure 6 The present invention is based on Bacillus sicca ( Siamese Bacillus Scanning electron microscopy (SEM) images of pathogenic mycelia before and after treatment with aseptic fermentation filtrate of GD12; where A represents Fusarium equisetifolium (…). Fusarium horsetail B. anthracnose ( Colletotrichum fructicola C represents Phytophthora capsici ( ); Phytophthora capsicum ), CK represents the mycelium of the control group; GD12 represents the mycelium of the group treated with 50% sterile fermentation filtrate; Figure 7 The present invention is based on Bacillus sicca ( Siamese Bacillus PI staining detection image of pathogenic fungal hyphae and cell membrane integrity after treatment with GD12 aseptic fermentation filtrate; where A represents Fusarium equisetifolium (… Fusarium horsetail B. anthracnose ( Colletotrichum fructicola C represents Phytophthora capsici ( ); Phytophthora capsicum Each row, from left to right, represents the fermentation filtrate treatment groups: 0%, 10%, 20%, 30%, 40%, and 50%. Figure 8 The present invention is based on Bacillus sicca ( Siamese BacillusThe graphs show the inhibitory effects of GD12 aseptic fermentation filtrate on the germination and production of pathogenic fungal spores. Among them, A is the inhibitory effect on the germination of Fusarium equisetifolium conidia; B is the inhibitory effect on the germination of Anthracnose conidia; and C is the inhibitory effect on the production of Phytophthora capsici sporangia.

[0021] Figure 9 The present invention is based on Bacillus sicca ( Siamese Bacillus The effect of GD12 aseptic fermentation filtrate on chili seed germination is shown in the figure. CK is the control, A is the statistical results of the number of germinating chili seeds and germination potential on day 5 of culture, and B is the statistical results of the number of germinating chili seeds and germination rate on day 10 of culture.

[0022] Figure 10 The present invention is based on Bacillus sicca ( Siamese Bacillus The following graphs show the disease control effects of GD12 biological agent against pepper root rot, anthracnose, and blight. CK represents the control group; A represents the effect of inoculation with *Fusarium equisetifolium*; B represents the effect of GD12 fermentation broth + *Fusarium equisetifolium* treatment; C represents the effect of inoculation with *Phytophthora capsici*; D represents the effect of GD12 fermentation broth + *Phytophthora capsici* treatment; E represents the effect of inoculation with *Anthracnose citrinum*; and F represents the effect of GD12 fermentation broth + *Anthracnose citrinum* treatment.

[0023] Figure 11 The present invention is based on Bacillus sicca ( Siamese Bacillus The graph shows the growth-promoting effect of GD12 biological agent on chili peppers. In the graph, A is a comparison of the overall growth of chili pepper plants (the left side is the control group CK, and the right side is the GD12 fermentation liquid treatment group); B is a comparison of the root growth of chili pepper plants (the left side is the control group CK, and the right side is the GD12 fermentation liquid treatment group). Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0025] The following examples use three plant pathogenic fungi—Fusarium equisetifolium (…). Fusarium horsetail ), fruit anthracnose ( Colletotrichum fructicola ) and Phytophthora capsici ( Phytophthora capsicum All of these are strains isolated and preserved in the inventor's laboratory, and can be purchased by the public from the China Agricultural Microbial Culture Collection Center (ACCC) or related professional institutions. Among them are *Fusarium equiseti* and *Phytophthora capsici*. Phytophthora capsicum These can be purchased from the China Agricultural Microbial Culture Collection Center (ACCC), under the trade names *Fusarium equiseti* and *Phytophthora capsici*. Phytophthora capsicum The accession numbers are ACCC 38083 and ACCC 37132, respectively. Purchase channel: China Agricultural Microbial Culture Collection Center, address: No. 12, Zhongguancun South Street, Haidian District, Beijing. *Anthracnose spp.* Colletotrichum fructicola Also known as *Discocephalus spp.*, it can be purchased from the China Forestry Microbial Culture Collection Center (CFCC); the strain number is CFCC 52885.

[0026] The Siamese Bacillus provided by this invention ( Siamese Bacillus GD12 was deposited on July 10, 2026, at the Guangdong Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 68595. Depository: Guangdong Microbial Culture Collection Center (GDMCC); Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences; Deposit Date: July 10, 2026; Proposed Taxonomic Name: Siamese Bacillus The biological material (strain) is preserved in a viable state.

[0027] Example 1: Isolation, purification and identification of Bacillus sicca strain GD12 1. Isolation and purification of bacterial strains (1) Preparation of LB medium: Weigh 10.0 g of tryptone, 5.0 g of yeast extract and 10.0 g of sodium chloride, add 1000 mL of water and stir well. For solid LB medium, add 20.0 g of agar. After heating and dissolving thoroughly, dispense into 250 mL Erlenmeyer flasks and sterilize by moist heat at 121 °C for 20 min. Store for later use.

[0028] (2) Soil samples were collected from the rhizosphere soil of healthy chili peppers in a chili pepper orchard in Yulao Town, Fengkai County, Guangdong Province (23°51'N.111°75'E). The collected healthy soil samples were mixed, air-dried, sieved, and 10 g of soil sample was weighed and placed in a 90 mL Erlenmeyer flask to prepare a 10% soil suspension. The suspension was shaken on a shaker for 20 min, allowed to stand for 5 min, and then serially diluted 10-fold to a final concentration.-1 ~10 -6 200 μL of the diluted solution was evenly spread on LB medium for incubation. Three replicates were set up for each dilution. Bacteria with obvious differences in size, shape, color, texture, surface structure, etc. were observed and selected for streak purification on LB solid medium plates. After purification was repeated three times, the purified strain was inoculated onto liquid LB medium and cultured at 29°C for 24 h. The strain was then numbered GD12 and stored in 60% glycerol at -80°C.

[0029] 2. Strain identification Morphological identification: After streaking strain GD12 on LB solid medium and culturing at 29℃ for 48 h, the colony morphology was observed. Figure 1 As shown, the colonies are milky white, opaque, dry, and wrinkled with irregular edges. After Gram staining, they are observed under a light microscope. Figure 3 The bacteria are rod-shaped and are Gram-positive.

[0030] Molecular biological identification: The strain was inoculated into liquid LB medium and cultured at 37 °C for 12 h at 200 rpm. The cells were collected by centrifugation. DNA was extracted from the purified strain using the Plant Genomics DE705-50 DNA Extraction Kit (Beijing Jinsha Technology Biotechnology Co., Ltd.) and used as a PCR template. The 16S rRNA gene sequence of the strain was amplified using 16S rRNA PCR primers 27F / 1492R. The nucleotide sequence of 27F was 5'-AGAGTTTGATCCTGGCTCAG-3' (as shown in SEQ ID NO. 2); the nucleotide sequence of 1492R was 5'-GGTTACCTTGTTACGACTT-3' (as shown in SEQ ID NO. 3). The PCR reaction system consisted of: 5 μL of 10×EasyTaq Buffer, 4 μL of dNTP Mixture (2.5 mM each), 1 μL of 27F (10 μM), 1 μL of 1492R (10 μM), 1 μL of DNA template, 0.5 μL of EasyTaq DNA Polymerase (5 U / μL), and ddH2O to a final volume of 50 μL.

[0031] The PCR conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s; 56℃ annealing for 1 min; 72℃ extension for 1 min; 32 cycles; final extension at 72℃ for 10 min to obtain the PCR amplification product, which was stored at 4℃. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0032] The 16S rDNA sequence of Bacillus sicca GD12 is as follows: (As shown in SEQ ID NO.1).

[0033] The 16S rDNA sequencing results of the strain were compared with those in the NCBI database using BLAST. The comparison results showed that the 16S rDNA sequence of strain GD12 was consistent with that in GenBank accession number PQ813672.1. Siamese BacillusThe homology reached 100.00%, and highly homologous sequences were downloaded as reference sequences. A phylogenetic tree was constructed using MEGA 6.0 software with the neighbor-joining method, as shown below. Figure 2 As shown. Strain GD12 and Siamese Bacillus Based on morphological characteristics, Gram staining results, and 16S rDNA phylogenetic analysis, belonging to the same evolutionary branch, this strain was classified and named *Bacillus sicca*. Bacillus siamensis ), further named: Bacillus sicca ( Bacillus siamensis The strain GD12 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on July 10, 2026, with accession number GDMCC NO: 68595.

[0034] Example 2: Preparation of sterile fermentation filtrate of strain GD12 Bacillus sicca GD12 was streaked onto LB agar plates and activated at 29°C for 24 h. Single colonies were then picked up with an inoculation needle and placed in LB liquid medium. The medium was then incubated in a shaker at 29°C and 200 r / min for 12 h to obtain a seed culture. This seed culture was then inoculated into fresh LB liquid medium at a 1% (v / v) inoculation rate and incubated in a shaker at 29°C for 72 h to complete the preparation of the antagonistic bacterial fermentation broth. The fermentation broth was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm sterile filter to remove contaminants, yielding a sterile fermentation filtrate for later use. This is the sterile fermentation filtrate of Bacillus sicca GD12.

[0035] Example 3: Inhibitory effect of strain GD12 on the mycelial growth of three pathogenic fungi 1. Flat Plate Standoff Experiment The plate confrontation method was used to determine the effect of GD12 on Fusarium equisetifolium ( Fusarium equiseti ), fruit anthracnose ( Colletotrichum fructicola ) and Phytophthora capsici ( Phytophthora capsici Inhibition effect on mycelial growth. Specific operation is as follows: Using a sterile punch (7 mm diameter), collect mycelial cakes from the edge of activated *Fusarium equisetifolium*, *Anthracis citrinum*, or *Phytophthora capsici* colonies. Inoculate the mycelial cakes onto fresh PDA medium, or inoculate *Phytophthora capsici* cakes onto the center of fresh V8 solid medium (90 mm diameter). Place sterile filter paper discs (7 mm diameter) at four symmetrical points approximately 27 mm from the pathogenic mycelial cakes. Use a pipette to add 10 μL of GD12 fermentation broth to each of the four sterile filter paper discs. A control group (CK) without any inoculum was used. Three replicates were prepared for each treatment. Incubate the plates at 26°C for 6 days. After incubation, measure the concentration of *Fusarium equisetifolium* (…) on each plate. Fusarium equiseti ), fruit anthracnose ( Colletotrichum fructicola ) or Phytophthora capsici ( Phytophthora capsici The growth radius of the colony is used to calculate the inhibition rate of the pathogen.

[0036] The formula for calculating the inhibition rate is as follows: Inhibition rate (%) = (radius of pathogen in control group - radius of pathogen in treatment group) / radius of pathogen in control group × 100%.

[0037] Experimental results show that: Bacillus sicca ( Bacillus siamensis GD12 showed significant inhibitory effects against all three pathogens, as shown in the results. Figure 4 As shown, the GD12 fermentation broth exhibited an inhibition rate of 79.52% against Fusarium equisetifolium, an inhibition rate of 86.58% against Anthracnose, and an inhibition rate of 88.54% against Phytophthora capsici.

[0038] 2. Preparation of drug-containing tablets PDA medium (for *Fusarium equisetifolium* and *Anthracis citrinum*) and V8 medium (for *Phytophthora capsici*) were prepared separately, sterilized at 121°C, and then cooled to approximately 50-60°C. Aseptic fermentation filtrate of *Bacillus sicca* GD12 was prepared according to the method in Example 2. This filtrate was added to the molten (±45°C) medium at volume ratios of 0% (control group, replaced by an equal volume of sterile PDA / V8 medium), 10%, 20%, 30%, 40%, and 50%, respectively. After thorough mixing, the filtrate was quickly poured into sterile petri dishes and allowed to solidify, yielding a series of plates containing different concentrations of fermentation filtrate. Each concentration treatment was repeated three times.

[0039] 3. Inoculation and Culture Using a sterile punch with a diameter of 7 mm, collect mycelial cakes from the edges of pre-activated colonies of three pathogens (Fusarium equisetifolium, Anthracnose spp., and Phytophthora capsici). Inoculate the mycelial cakes onto the centers of different concentration-graded agar plates, mycelial side down. Incubate the plates upside down in a 28°C constant temperature incubator (see [link to incubation instructions]). Figure 4 ).

[0040] 3. Calculation of antibacterial rate When the colonies in the control group (0%) had grown to cover approximately 2 / 3 of the petri dish, the colony radius of each treatment group was measured, and the inhibition rate of the sterile fermentation filtrate at different concentrations on the mycelial growth of each pathogen was calculated using the following formula: Inhibition rate (%) = [(Coronavirus radius of control group - Coronavirus radius of treatment group) / (Coronavirus radius of control group)] × 100% The results are as follows Figure 5 As shown, from top to bottom, the bacteria are *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici*. From left to right, the control groups (CK and CK) are treated with 10%–50% GD12 aseptic fermentation filtrate. The results showed that the aseptic fermentation filtrate of *Bacillus sicca* GD12 inhibited the mycelial growth of *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici*, with the inhibitory effect increasing with increasing fermentation filtrate concentration. As the concentration of the fermentation filtrate increased, the colony diameter of each pathogen gradually decreased, and the inhibition rate gradually increased. When the fermentation filtrate concentration was 50%, the inhibition rates against *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici* reached 67.88%, 88.92%, and 100%, respectively, confirming that the antibacterial active substances produced by strain GD12 have good broad-spectrum and high-efficiency characteristics.

[0041] Example 4: Effects of sterile fermentation filtrate on the ultrastructure of pathogenic fungal hyphae Mycelial cakes (7 mm in diameter) were collected from the edges of pathogen colonies cultured for 5 days at 28°C on PDA medium (Fusarium equisetifolium, Anthracnose spp.) or V8 medium (Phytophthora capsici). These cakes were then transferred to liquid medium containing 50% (v / v) sterile fermentation filtrate (with an equal volume of sterile PD liquid medium or V8 liquid medium as a control) and cultured at 29°C with shaking at 200 r / min for 24 h. Mycelial samples were collected, washed three times with 0.1 M phosphate-buffered saline (PBS, pH 7.2), and fixed overnight at 4°C with 2.5% glutaraldehyde fixative. The fixed samples were washed three times with PBS and then dehydrated sequentially with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, 15 min at each step. After dehydration, the samples were critically dried, sputter-coated with gold, and the ultrastructure of the mycelial surface was observed under a scanning electron microscope.

[0042] The results are as follows Figure 6 As shown, the images on the left, in order, represent the control groups of *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici*. The control group hyphae have smooth surfaces, clear outlines, are plump, and have intact structures. The image on the right shows the treatment group. In the treatment group, the hyphae become rough, wrinkled, and show obvious depressions, collapses, and even breaks. The cell wall and cell membrane structures are severely damaged, indicating that the extracellular active substances produced by strain GD12 can destroy the cell wall and cell membrane structures of the pathogenic hyphae, causing irreversible ultrastructural damage.

[0043] Example 5: Effect of sterile fermentation filtrate on the cell membrane integrity of pathogenic mycelia (PI staining) *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici* were inoculated onto PDA (the first two pathogens) or V8 (*Phytophthora capsici*) plates and incubated at 28°C for 5-7 days. Mycelial cakes were collected from the colony edges using a 7mm diameter sterile punch and transferred to 10mL centrifuge tubes containing 0%, 10%, 20%, 30%, 40%, and 50% of the sterile fermentation filtrate, respectively. The tubes were incubated at 29°C and 200 rpm for 12 hours with shaking. The liquid was discarded, and the mycelia were washed three times with PBS buffer. Propidium iodide (PI) staining solution at a final concentration of 10μg / mL was added, and staining was performed at room temperature in the dark for 15 minutes. After staining, the tubes were washed three times with PBS and observed under a fluorescence microscope. PI is a nucleic acid dye that cannot penetrate intact cell membranes; it can only enter cells with damaged membranes and bind to DNA, emitting red fluorescence.

[0044] The results are as follows Figure 7 As shown, the control group hyphae showed almost no red fluorescence, indicating that the cell membrane was intact. With increasing concentration of the sterile fermentation filtrate, the intensity of red fluorescence in the hyphae of the three pathogenic bacteria gradually increased. When the concentration reached 50%, all three pathogenic bacteria hyphae exhibited strong red fluorescence, indicating severe damage to cell membrane integrity and a significant increase in permeability. These results indicate that the extracellular active substances produced by Bacillus sicca GD12 disrupt the cell membrane structure of the pathogenic bacteria hyphae, which is one of the important factors contributing to its broad-spectrum antibacterial effect.

[0045] Example 6: Inhibition of pathogenic spore germination and sporangium production by aseptic fermentation filtrate Inhibition of conidial germination was achieved by preparing conidial suspensions of *Fusarium equisetifolium* and *Anthracis cirrhosa* (10⁻¹⁰). 6 (Spores / mL) An equal volume of spore suspension was mixed with 50% sterile fermentation filtrate prepared in Example 2, with an equal volume of sterile water mixed with the spore suspension as a control. After incubation at 28℃ for 12 h, spore germination was observed under an optical microscope. 100 spores were examined for each treatment. The spore germination rate and spore germination inhibition rate were calculated using the following formulas. Each treatment was repeated 3 times.

[0046] Spore germination rate (%) = number of germinating spores / total number of spores × 100; Spore germination inhibition rate (%) = (control spore germination rate - treatment spore germination rate) / control spore germination rate × 100.

[0047] Inhibition of sporangium production by Phytophthora capsici: Phytophthora capsici mycelium cakes were inoculated onto V8 plates and cultured for 3 days. The mycelium cakes were then removed from the edge of the colony and transferred to a liquid culture medium containing sterile fermentation filtrate. Sterile water was used as a control. After culturing at 28°C for 48 hours, the number of sporangia produced was counted under a microscope.

[0048] The results are as follows Figure 8 As shown, Figure 8 In the table, A, B, and C represent the spore germination inhibition rate or sporangium production number of *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici*, respectively. Compared with the control group, the aseptic fermentation filtrate of *Bacillus sicca* GD12 effectively inhibited the germination of conidia of *Fusarium equisetifolium* and *Anthracis citrinum*, and significantly reduced the sporangium production number of *Phytophthora capsici*.

[0049] Example 7: Effect of sterile fermentation filtrate on pepper seed germination Healthy, uniformly sized chili seeds were disinfected with 5% sodium hypochlorite for 5 minutes, rinsed thoroughly with sterile water, and drained. The disinfected seeds were then placed in a 5-fold dilution of GD12 sterile fermentation filtrate with sterile water and cultured at 28°C for 24 hours. An equal volume of sterile water was used as a control group. The treated seeds were then evenly placed in sterile petri dishes lined with double-layered sterile germination paper, 50 seeds per dish, with each treatment repeated three times. The seeds were then cultured at 28°C for 10 days. Using sterile water as a control group, seed germination potential, germination rate, fresh weight, hypocotyl and radicle length, and 5-day germination index were measured.

[0050] Germination rate (%) = Number of germinated seeds on day 10 / Total number of seeds tested × 100%; Germination potential (%) = Number of seeds germinated on day 5 / Total number of seeds tested × 100%; Germination index (GI) = ∑(Gt / Dt); where Gt is the number of germinated seeds on day t, and Dt is the number of germination days.

[0051] Depend on Figure 9 As can be seen, A represents the number of germinated seeds and germination potential on day 5 of chili seed culture, and B represents the number of germinated seeds and germination rate on day 10. Compared with the blank control group CK, all treatment groups of GD12 aseptic fermentation filtrate CFS can increase the number of germinated seeds and germination potential, and the seed germination speed is faster. Fermentation broth with a volume fraction of 10% to 50% all have the effect of promoting germination, and the effect is best in the concentration range of 20% to 30%.

[0052] In summary, the present invention relates to Bacillus sicca ( Bacillus siamensis GD12 aseptic fermentation filtrate can accelerate the early germination of chili seeds and improve the uniformity of germination. In chili seedling cultivation, it can shorten the emergence cycle and ensure uniform emergence, and has excellent seed germination promotion effect and safety of use.

[0053] Example 8: Pot experiment to determine the presence of Bacillus sicca ( Bacillus siamensis The efficacy of GD12 against three diseases of chili peppers The LB medium was prepared in the same way as in Example 1, without adding agar. After being fully dissolved, it was dispensed into Erlenmeyer flasks (100 mL of culture medium per flask), capped, wrapped, sterilized at 121°C for 20 min, cooled, and stored for later use.

[0054] Preparation of PD medium: Weigh 200 g of potatoes, boil the potatoes for 30 min, filter through 4 layers of gauze, add 20 g of glucose, add water to the filtrate to make up to 1000 mL, dispense into Erlenmeyer flasks, seal and sterilize at 121 ℃ for 20 min, and store for later use.

[0055] The fermentation broth of Bacillus sicca GD12 was prepared according to the method in Example 2. The viable count in the fermentation broth was approximately 1 × 10⁻⁶. 8 CFU / mL.

[0056] Cultures of Fusarium equisetifolium, Anthracnose spp., and Phytophthora capsici Preparation of *Fusarium equisetifolium* and *Anthracis cirrhosa* spore suspensions: *Fusarium equisetifolium* and *Anthracis cirrhosa* mycelial cakes were inoculated into PD medium and placed in a constant temperature shaker at 28 ℃ and 180 r / min for 5 days. After 5 days, the suspensions were filtered through sterile gauze and sterile water was added to adjust the concentration to obtain 1x10⁻⁶ spores. 6 CFU / mL spore suspensions were prepared to complete the preparation of *Fusarium equisetifolium* and *Anthracnose cirrhosa* suspensions. Preparation of *Phytophthora capsici* spore suspension: *Phytophthora capsici* cakes were inoculated into 10% V8 liquid medium and cultured in the dark for 3 days. After washing three times with sterile water, sterile water was added and the medium was incubated under light for 48 hours. After observing the production of a large number of sporangia, the medium was placed in a 4°C refrigerator for 30 minutes, then returned to room temperature to release zoospores. The concentration was adjusted with sterile water to obtain a concentration of 10 CFU / mL spores. 4 Spore suspension.

[0057] Disease-resistant pot experiment: Healthy six-leaf pepper seedlings with similar growth were selected. Pepper root rot and pepper blight were treated with bacterial solution drenching, while pepper anthracnose was treated by spraying. Root drenching treatment group: *Bacillus sicca* (…) was added to the solution. Bacillus siamensis 50 mL of GD12 fermentation broth was inoculated. Two days later, using the root-damage inoculation method, 50 mL of *Fusarium equisetifolium* spore suspension and *Phytophthora capsici* spore suspension were applied to the roots of each chili pepper plant. Chili seedlings inoculated only with liquid LB / V8 served as a negative control, while those inoculated only with *Fusarium equisetifolium* spore suspension and *Phytophthora capsici* spore suspension served as a positive control. Spraying treatment group: *Bacillus sicca* (…) was sprayed evenly… Bacillus siamensisTwo days after applying GD12 fermentation broth to pepper leaves, the leaf veins were punctured with a sterile syringe needle, and a suspension of *Anthracnose spores* was evenly sprayed onto the pepper leaves. Pepper seedlings sprayed only with liquid LB served as a negative control, while those inoculated only with the *Anthracnose spores* suspension served as a positive control. Each group consisted of 6 pots of plants, with 3 replicates. Fourteen days later, the disease incidence, disease index, and control effect of each treatment were observed and statistically analyzed. The calculation formula is as follows: Grading method for root rot and blight in chili seedlings: Grade 0: No obvious symptoms; Grade 1: A small number of brown lesions appear on the roots and base of the stem of the seedlings; Grade 2: Lesions on the roots and stems extend to the heart leaves or the lesions encircle the entire stem, and the plant slightly collapses; Grade 3: The plant collapses, and most leaves wilt or turn yellow; Grade 4: The plant collapses, and the whole plant wilts; Grade 5: The plant dies.

[0058] Incidence rate (%) = Number of infected plants / Total number of plants surveyed × 100; Disease index (%) = Σ (number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100; Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100; Anthracnose disease severity grading method for peppers: Grade 0: No lesions; Grade 1: Lesion area less than 2% of leaf area; Grade 3: Lesion area 3%–8% of leaf area; Grade 5: Lesion area 9%–15% of leaf area; Grade 7: Lesion area 16%–25% of leaf area; Grade 9: Lesion area more than 25% of leaf area. Disease index = [(Σ(number of diseased leaves at each level × relative grade) / (total number of leaves surveyed × 9)] × 100, control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0059] The results are as follows Figure 10 As shown, Bacillus sicca ( Bacillus siamensis GD12 treatment has a good effect on the occurrence of root rot, anthracnose and blight in peppers.

[0060] Table 1: Growth-promoting effect of GD12 on pepper plants

[0061] Note: Different lowercase letters in the same column indicate significant differences at the p<0.05 level (t-test). Data were compared using Duncan's new multiple range method.

[0062] The disease index and control effect are shown in Table 1. After root irrigation with Bacillus sicca GD12 fermentation broth, the disease index of the three diseases in pepper plants decreased significantly. The disease index of the treatment groups was all below 15%. The control effect of pepper root rot was 87.18%, anthracnose was 85.29%, and pepper blight was 87.05%, indicating that Bacillus sicca (… Bacillus siamensis GD12 reduces the occurrence of the above three pepper diseases to a certain extent and has disease resistance.

[0063] Example 9: Pot experiment to determine the growth-promoting effect of Bacillus sicca GD12 on pepper. 1. Preparation of fermentation broth The LB medium was prepared in the same manner as in Example 1, except that agar was not added. The prepared LB liquid medium was dispensed into Erlenmeyer flasks (100 mL of culture medium per flask), capped, sealed, and sterilized at 121°C for 20 min.

[0064] Preparation of PD medium: Weigh 200 g of potatoes, boil the potatoes for 30 min, filter through 4 layers of gauze, add 20 g of glucose, add water to the filtrate to make up to 1000 mL, dispense into Erlenmeyer flasks, seal and sterilize at 121 ℃ for 20 min, and store for later use.

[0065] The fermentation broth of Bacillus sicca GD12 was prepared according to the method in Example 2. The viable count in the fermentation broth was approximately 1~2×10⁻⁶. 8 CFU / mL.

[0066] 2. Potted plant experiment Healthy six-leaf pepper seedlings with similar growth were selected and treated with a bacterial solution drenching. 50 mL of Bacillus sicca GD12 fermentation broth was added, while the control group was drenched with an equal volume of LB liquid medium. Six plants were used in three replicates. Twenty-eight days after colonization, the pepper seedlings were harvested to observe their growth. The results are as follows: Figure 11 As shown in Table 2.

[0067] Table 2: Growth-promoting effect of GD12 on pepper plants

[0068] Note: Different lowercase letters in the same column indicate significant differences at the p<0.05 level (t-test).

[0069] Depend on Figure 11As shown in Table 2, after root irrigation with Bacillus sicca GD12 fermentation broth, the root length, plant height, stem diameter, whole plant fresh weight, leaf length, and leaf width of the pepper plants were all higher than those of the control group (p<0.05), with growth rates of 60.37%, 31.28%, 73.09%, 68.73%, 85.74%, and 87.12%, respectively. The results indicate that Bacillus sicca GD12 of the present invention has a significant growth-promoting effect on peppers.

[0070] The Siamese Bacillus provided by this invention ( Bacillus siamensisCompared with the existing Bacillus sicca strains SD23LJ1314 (CN119570656B), QN2MO-1 (CN113308392A) and BsiaSC07 (CN121518322A) disclosed in the technology, GD12 has better advantages. Regarding in vitro antibacterial activity, the 50% volume fraction sterile fermentation filtrate of Bacillus sicca GD12 showed inhibition rates of 67.88%, 88.92%, and 100% against the mycelial growth of Fusarium equisetifolium, Anthracnose cylindrica, and Phytophthora capsici, respectively, with complete inhibition against Phytophthora capsici. In contrast, the inhibitory spectrum of the control strain SD23LJ1314 did not include Fusarium equisetifolium or Anthracnose cylindrica. The inhibition rate of the control strain QN2MO-1 ranged from 60.78% to 84.51%, but no comprehensive inhibitory activity against these three pathogens was recorded. The control strain BsiaSC07 showed an inhibition rate of 76.40% against Fusarium graminearum, but its target was the causative agent of kiwifruit leaf spot disease, not the three pathogens of pepper. Regarding the efficacy in potted plants, the root irrigation / spraying treatment of the GD12 fermentation liquid of this invention achieved control effects of 87.18%, 87.05%, and 85.29% against pepper root rot, blight, and anthracnose, respectively, with all three diseases exceeding 85% efficacy. In contrast, the comparative strain SD23LJ1314 only recorded a field efficacy of 71.69% and a potted plant efficacy of 78.53% against pepper blight, with no recorded efficacy against pepper root rot and anthracnose. The comparative strains QN2MO-1 and BsiaSC07 did not provide any potted plant efficacy data for pepper diseases. In terms of disease control target coverage, the GD12 strain of this invention is the first to systematically screen pathogens of three major diseases of pepper: root rot (Fusarium equisetifolium), anthracnose (Anthracnose cylindrica), and blight (Phytophthora capsici). Its control efficacy against these three diseases was verified at both in vitro and in vivo levels, while the three comparative strains in the prior art failed to simultaneously cover all three diseases. Regarding growth-promoting functions, Bacillus sicca GD12 can promote pepper seed germination, with a germination rate exceeding 90%, and significantly increase the root length, plant height, stem diameter, whole plant fresh weight, leaf length, and leaf width of pepper plants (growth rates of 60.37%, 31.28%, 73.09%, 68.73%, 85.74%, and 87.12%, respectively). None of the three comparative strains have been documented to possess multiple functions of inhibition, disease prevention, and growth promotion. In summary, the Bacillus sicca provided by this invention (… Bacillus siamensis GD12 and its biological agents can simultaneously and effectively inhibit the root rot pathogen of pepper, Fusarium equisetifolium (…). Fusarium equiseti Anthracnose pathogen of pepper (Fructus anthracnose) Colletotrichum fructicola ) and the pathogen of pepper blight, Phytophthora capsici ( Phytophthora capsiciThis strain exerts a broad-spectrum antagonistic effect against three important chili pepper pathogens through multiple mechanisms, including disrupting the integrity of the pathogenic mycelial cell membrane, causing teratogenic hyphae ultrastructure, and inhibiting spore germination and production. It also promotes chili pepper seed germination, provides control over the three chili pepper diseases, and promotes chili pepper plant growth. This strain and its biological agents are broad-spectrum and environmentally friendly, suitable for the green control of chili pepper root rot, anthracnose, and blight, as well as for growth promotion during chili pepper seedling and cultivation. They possess good potential for industrial production and agricultural application, making them suitable for large-scale promotion and application.

[0071] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A strain of Bacillus sicca ( Bacillus siamensis GD12, characterized in that, The Siamese Bacillus ( Bacillus siamensis GD12 is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCCNo:68595 and deposit date of July 10, 2026.

2. The Bacillus sicca according to claim 1 ( Bacillus siamensis GD12, characterized in that, The Siamese Bacillus ( Bacillus siamensis The nucleotide sequence of the 16S rRNA of GD12 is shown in SEQ ID NO.

1.

3. A biological agent, characterized in that, The biological agent comprises the Bacillus sihamnium as described in claim 1 or 2. Bacillus siamensis GD12, its fermentation broth or its sterile fermentation filtrate, or at least one of them.

4. The biological agent according to claim 3, characterized in that, The biological agent is a liquid formulation, a powder, or an immobilized formulation.

5. The Bacillus sicca as described in claim 1 or 2 ( Bacillus siamensis GD12 or the biological agent according to claim 3 or 4 simultaneously inhibits Fusarium equisetifolium ( Fusarium equiseti ), fruit anthracnose ( Colletotrichum fructicola ) and Phytophthora capsici ( Phytophthora capsici Applications during growth.

6. The Bacillus sicca according to claim 1 or 2 ( Bacillus siamensis The application of GD12 or the biological agent described in claim 3 or 4 in the simultaneous preparation of biological control agents for the prevention and control of pepper root rot, pepper anthracnose and pepper blight.

7. The Bacillus sicca according to claim 1 or 2 ( Bacillus siamensis The application of GD12 or the biological agent described in claim 3 or 4 in promoting the germination of chili seeds and the growth of chili plants.

8. The application according to claim 6, characterized in that, The Siamese Bacillus ( Bacillus siamensis The aseptic fermentation filtrate of GD12 inhibits the growth of *Fusarium equisetifolium*, *Anthracis citrinum*, and *Phytophthora capsici* by at least one of the following methods: (1) Teratogenic pathogenic mycelium, causing the mycelium to become deformed, sunken, collapsed or broken; (2) Disrupts the cell membrane integrity of pathogenic fungal hyphae; (3) Inhibits the germination of conidia of Fusarium equisetifolium and Anthracnoseus spp.; (4) Inhibit the production of Phytophthora capsici sporangia.

9. A method for controlling pepper plants from root rot, anthracnose, and blight, characterized in that, Apply an effective amount of the Bacillus sicca described in claim 1 or 2 to the tested pepper plants. Bacillus siamensis GD12 or the biological agent as described in claim 3 or 4.

10. The method according to claim 9, characterized in that, The application methods are root irrigation, seed soaking, spraying, or mixing with soil.

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