Bacillus siamensis arf-sr1, biocontrol agent and application thereof

Abstract

The present application relates to the technical field of biocontrol bacteria, and particularly relates to a bacillus siamensis (Bacillus siamensis) ARF-SR1, a biocontrol bacterium and application thereof. Bacillus siamensis The present application provides the preservation number of the bacillus siamensis ARF-SR1 as CGMCC No.33715. The bacillus siamensis ARF-SR1 provided by the present application is a biocontrol strain isolated from a healthy alfalfa field in Ningxia, has a significant inhibitory effect on alfalfa root rot pathogenic bacteria (especially Fusarium solani, Fusarium oxysporum and Phoma sp.), an antagonistic rate of 57.71-68.86%, can reduce the incidence of alfalfa root rot, enriches the biocontrol bacteria resource library, and is suitable for green control of alfalfa root rot.

Description

Technical Field

[0001] This invention relates to the field of biocontrol bacteria technology, and in particular to a strain of Bacillus sicca ( Bacillus siamensis ARF-SR1, biocontrol agents and their applications. Background Technology

[0002] Alfalfa (Medicago sativa), a high-quality forage crop rich in nutrients and highly adaptable, plays a crucial role in livestock production. However, alfalfa root rot, a common disease, is one of the main problems restricting its development. It has long plagued the alfalfa industry, leading to a significant decline in alfalfa yield and severely impacting quality, posing a considerable challenge to livestock production.

[0003] Currently, control strategies against alfalfa root rot pathogens mainly include chemical control and agricultural control. While chemical control is effective in the short term, long-term use leads to the development of pathogen resistance, negatively impacting the environment and ecosystem. Agricultural control primarily improves the soil environment and enhances plant resistance through crop rotation, deep tillage, and rational fertilization. However, these measures require long-term implementation and have limited effectiveness. Biological control utilizes microorganisms and their metabolites to antagonize pathogens, thereby controlling harmful organisms. Compared to chemical pesticides and traditional agricultural control techniques, biological control offers advantages such as environmental friendliness, gentleness, safety, and sustainability, and has received considerable attention in recent years. However, the number of biocontrol bacteria targeting alfalfa root rot is currently limited, necessitating the development of a biocontrol bacterium with antagonistic effects against multiple alfalfa root rot pathogens to enrich the biocontrol bacteria resource pool. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a strain of Bacillus sicca ARF-SR1, a biocontrol agent, and their applications. The Bacillus sicca ARF-SR1 provided by this invention exhibits significant inhibitory effects against alfalfa root rot pathogens (especially Fusarium solani, Fusarium scutellarioides, and Pyrophyllus spp.), with an antagonism rate of 57.71–68.86%, thus reducing the incidence of alfalfa root rot and enriching the biocontrol resource library.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a strain of Bacillus sicca ARF-SR1, with accession number CGMCC No. 33715.

[0007] This invention provides a biocontrol agent, the active ingredient of which includes Bacillus sicca ARF-SR1 as described in the above technical solution.

[0008] Preferably, the OD of Bacillus sicca ARF-SR1 in the biocontrol agent is... 600 Value ≥ 0.5.

[0009] This invention provides a method for preparing the biocontrol agent described in the above technical solution, comprising the following steps:

[0010] The *Bacillus sicca* ARF-SR1 described in the above technical solution is inoculated into a culture medium to obtain the biocontrol agent; the culture medium includes LB medium, TSB medium or a prepared culture medium; the prepared culture medium includes the following components at the following concentrations: corn starch 15~25 g / L, peptone 20~30 g / L and NaCl 5~10 g / L, pH=7~8.

[0011] Preferably, the culture temperature is 25~38℃; the culture rotation speed is 180~200 r / min.

[0012] This invention provides the application of Bacillus sicca ARF-SR1 or the biocontrol agent described in the above-described technical solutions, or the biocontrol agent prepared by the preparation method described in the above-described technical solutions, in the prevention and control of plant root rot and / or the promotion of plant growth.

[0013] Preferably, the pathogens causing plant root rot include: Fusarium solani (…). Fusarium solani Fusarium argentis ( ), Fusarium argentis ( Fusarium acuminatum ) and *Heterostylus* genus ( Paraphoma rhaphiolepidis One or more of the following.

[0014] Preferably, the plant includes alfalfa.

[0015] This invention provides a method for preventing and / or promoting plant growth from root rot, comprising the following steps:

[0016] The biocontrol agent is applied to the soil in which the plants are planted and / or applied to the plants; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.

[0017] Preferably, the plant includes alfalfa; the method of application includes root irrigation and / or spraying.

[0018] Beneficial effects:

[0019] This invention provides a strain of Bacillus sicca, ARF-SR1, with the preservation number CGMCC No. 33715. The Bacillus sicca ARF-SR1 provided by this invention is a biocontrol strain isolated from healthy alfalfa fields in Ningxia. It exhibits significant inhibitory effects on alfalfa root rot pathogens (especially Fusarium solani, Fusarium argentis, and Pyrophyllus), with an antagonism rate of 57.71–68.86%. This reduces the incidence of alfalfa root rot, enriches the biocontrol strain resource library, and is suitable for green control of alfalfa root rot. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 The images show the colony morphology, Gram staining, and scanning electron micrograph of strain ARF-SR1 on TSB solid medium.

[0022] Figure 2 A phylogenetic tree of strain ARF-SR1 constructed based on 16S rDNA;

[0023] Figure 3 The results show the determination of nitrogen fixation capacity, phosphorus solubilization capacity and potassium solubilization capacity of strain ARF-SR1;

[0024] Figure 4 The results show the protease-producing, amylase-producing, cellulase-producing, and heptaphilase-producing abilities of strain ARF-SR1.

[0025] Figure 5 The results show the IAA production capacity of strain ARF-SR1.

[0026] Figure 6 The results show the biofilm formation ability of strain ARF-SR1.

[0027] Figure 7 The image shows the antagonistic effect of strain ARF-SR1 against Fusarium solani, the pathogen of alfalfa root rot.

[0028] Figure 8 The image shows the antagonistic effect of strain ARF-SR1 against Fusarium argentis, the pathogen of alfalfa root rot.

[0029] Figure 9 This image shows the antagonistic effect of strain ARF-SR1 against *Heterostigma heterostemum*, the pathogen causing alfalfa root rot.

[0030] Biological Preservation Instructions

[0031] Bacillus sicca ARF-SR1, classified as Bacillus sicca Bacillus siamensisIt was deposited on March 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33715. Detailed Implementation

[0032] This invention provides a strain of Bacillus sicca ARF-SR1, with accession number CGMCC No. 33715.

[0033] The *Bacillus sicca* ARF-SR1 strain provided by this invention was isolated from alfalfa soil in Xingfu Village, Gaozhuang Township, Pingluo County, Shizuishan City, Ningxia Hui Autonomous Region. It grows rapidly on TSB solid medium, producing raised, milky-white colonies with a wrinkled, dry, and opaque surface. Gram-positive, it has a diameter of approximately 0.5 μm and a length of approximately 2-4 μm. Under a microscope, the bacteria appear as long rods and possess strong nitrogen-fixing capabilities. It can also decompose organic and inorganic phosphorus and potassium. Furthermore, this bacterium can produce proteases, cellulases, amylases, ferritins, and indoleacetic acid (IAA). It not only significantly inhibits common alfalfa root rot pathogens such as *Fusarium solani* and *Fusarium argentis*, but also has a strong inhibitory effect on the pathogen *Heterostilbene*. Moreover, it can promote host plant growth through nitrogen fixation, phosphorus solubilization, and IAA production. Compared with chemical pesticides, the application of biocontrol agents containing Bacillus sicca ARF-SR1 has a better control effect on alfalfa root rot. It can be used to control alfalfa root rot and has the advantages of being safe, green and environmentally friendly. It also enriches the biocontrol bacteria resource bank for alfalfa root rot and has broad application prospects and market value.

[0034] Based on the above advantages, the present invention provides a biocontrol agent, the active ingredient of which includes Bacillus sicca ARF-SR1 as described in the above technical solution.

[0035] As one implementation method, the OD of Bacillus sicca ARF-SR1 in the biocontrol agent... 600 Value ≥ 0.5. As another embodiment, the OD value of Bacillus sicca ARF-SR1 in the biocontrol agent... 600 Value ≥ 0.8. As another embodiment, the OD value of Bacillus sicca ARF-SR1 in the biocontrol agent... 600 The value is 0.8~1.5. The biocontrol agent provided by this invention has a significant inhibitory effect on alfalfa root rot pathogens (especially Fusarium rotii, Fusarium scutellarioides, and Pyrophyllus spp.).

[0036] This invention provides a method for preparing the biocontrol agent described in the above technical solution, comprising the following steps:

[0037] The *Bacillus sicca* ARF-SR1 described in the above technical solution is inoculated into a culture medium to obtain the biocontrol agent; the culture medium includes LB medium, TSB medium or a prepared culture medium; the prepared culture medium includes the following components at the following concentrations: corn starch 15~25 g / L, peptone 20~30 g / L and NaCl 5~10 g / L, pH=7~8.

[0038] In one embodiment, the culture temperature is 25-38℃; the culture rotation speed is 180-200 r / min. In another embodiment, the culture temperature is 35-37℃; the culture rotation speed is 186 r / min. In one embodiment, the culture medium comprises the following components at the following concentrations: corn starch 20 g / L, peptone 25 g / L, and NaCl 7.5 g / L, pH=7.5. The culture medium provided by this invention uses inexpensive, nutrient-rich, and well-fermented agricultural by-products as the main nutrients. The culture medium has been optimized, further reducing the cost of applying the microbial agent in agriculture compared to LB medium. Furthermore, the number of viable bacteria, spores, and spore rate are increased by 1.21 times, 1.43 times, and 1.27 times, respectively, making it a suitable culture medium for growth and providing technical support for the actual production of the microbial agent.

[0039] Based on the above advantages, the present invention provides the application of Bacillus sicca ARF-SR1 or the biocontrol agent or the preparation method described above in the above technical solution in the prevention and control of plant root rot and / or promotion of plant growth.

[0040] In one embodiment, the pathogen causing the plant root rot includes one or more of the following: *Fusarium solani*, *Fusarium argentea*, and *Heterostilbene*. In another embodiment, the plant may be alfalfa.

[0041] Based on the above advantages, the present invention provides a method for preventing and controlling root rot and / or promoting plant growth, comprising the following steps:

[0042] The biocontrol agent is applied to the soil in which the plants are planted and / or applied to the plants; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.

[0043] In one embodiment, the plant may be alfalfa; the application method includes root drenching and / or spraying. This invention allows the biocontrol agent to be applied to the soil and / or plants via root drenching and / or spraying, thereby exerting the efficacy of Bacillus sicca ARF-SR1 in preventing root rot and / or promoting plant growth. This is a safe, green, and environmentally friendly method.

[0044] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a strain of Bacillus sicca ARF-SR1, a biocontrol agent, and their applications provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1: Screening and isolation of strain ARF-SR1

[0046] In this embodiment, soil samples were collected from the rhizosphere soil of healthy alfalfa in Xingfu Village, Gaozhuang Township, Pingluo County, Shizuishan City, Ningxia Hui Autonomous Region. The soil was stored at 4℃. 10 g of the soil sample was weighed and placed in an Erlenmeyer flask containing 90 mL of sterile water. The flask was then incubated at 25℃ with constant shaking at 180 r / min for 30 min. After standing for 1 min to allow natural sedimentation, the supernatant was collected and diluted to 1000 μL using a 10-fold serial dilution method. -3 10 -4 10 -5 10 -6 For each concentration, 100 μl was evenly spread onto TSB solid medium and incubated at 37°C for 1–2 days. Based on the differences in colony morphology, single colonies were picked and purified by streak plating onto fresh TSB solid medium. After purification, the single colonies were transferred to 50% glycerol tubes and stored at -20°C for later use.

[0047] Antagonistic screening was conducted using pathogens screened from soil infected with alfalfa root rot in the affected area as target bacteria. Target bacterial discs (8 mm in diameter) were inoculated in the center of 90 mm diameter PDA plates, with the test strain inoculated 2.5 cm around the discs. Plates inoculated only with the target pathogen served as controls. The plates were incubated upside down at 28°C for 5 days, and the inhibitory effect was observed and recorded. A strain with good inhibitory effect was finally selected. The colonies were raised, milky white, wrinkled, dry, and opaque; Gram-positive, 0.5 μm in diameter, and 2–4 μm in length. Under a microscope, the bacteria appeared as long rods. Figure 1 As shown ( Figure 1 Image 'a' is a photograph of bacterial colonies. Figure 1 Image b is a microscopic photograph observed after Gram staining. Figure 1(c is a scanning electron microscope image). This strain was named ARF-SR1. After shaking a single colony of strain ARF-SR1 in TSB liquid medium, 50% glycerol was added, and the culture was stored at -80°C.

[0048] Example 2 Molecular identification of strain ARF-SR1

[0049] Genomic DNA of strain ARF-SR1 was extracted according to the instructions of the bacterial DNA extraction kit, and the concentration and quality of the extracted DNA were detected by spectrophotometer.

[0050] Genomic DNA of strain ARF-SR1 was amplified by PCR using universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTC-3', SEQ ID NO.1) and 1492R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID NO.2). The PCR amplification system consisted of 25 μl: 0.5 μl of each primer, 1 μl of template DNA, 10.5 μl of dd H2O, and 12.5 μl of 2×Taq PCR Master Mix. PCR amplification conditions were: 95℃ for 5 min; 94℃ for 1 min, 55℃ for 1 min, 72℃ for 1.5 min, 30 cycles; 72℃ for 10 min; stored at -20℃ for later use. The DNA was sent to Beijing Bomeide Gene Technology Co., Ltd. for sequencing and identified after alignment with NCBI BLAST. The nucleotide sequence of the 16S rDNA is shown in SEQ ID NO.3, as follows:

[0051]

[0052] The sequencing results were analyzed and compared using BLAST at NCBI, and a phylogenetic tree was constructed using MEGA software. (See [link]). Figure 2 Based on the physiological and biochemical characteristics and phylogenetic tree analysis of strain ARF-SR1, strain ARF-SR1 was identified as Bacillus sicca (Bacillus). Bacillus siamensis The strain ARF-SR1 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33715.

[0053] Example 3: Determination of the biocontrol performance of Bacillus sicca ARF-SR1

[0054] Strain activation: Take the preserved strain ARF-SR1 from the glycerol tube, take 100 μL of bacterial culture, and inoculate it into 10 mL of LB liquid medium. Incubate at 37℃ and 150 rpm for 24 h until OD (digesterone) is reached. 600 =1.0, to obtain the activated biocontrol bacterial solution (denoted as ). Bacillus siamensis ARF-SR1). The activated biocontrol bacterial solution was subjected to the following biocontrol performance tests. LB liquid medium was used as a control (CK) to replace the activated biocontrol bacterial solution in each treatment. Each treatment was repeated three times, as detailed below:

[0055] Nitrogen fixation capacity determination of strain ARF-SR1: Sterilized filter paper discs were placed on nitrogen-free medium. 5 μL of activated biocontrol bacterial solution was taken and inoculated onto the filter paper discs, allowing the filter paper discs to absorb the bacterial solution. The plates were placed at 28℃ for 3 days to observe whether the strain could grow in nitrogen-free medium and whether a clear zone was present.

[0056] Phosphorus solubilization capacity determination of strain ARF-SR1: Sterilized filter paper discs were placed on the surface of Mongkina inorganic phosphorus medium (PKO) and Mongkina organic phosphorus medium, respectively. 5 μL of activated biocontrol bacterial solution was taken and inoculated onto the filter paper discs, allowing the filter paper discs to absorb the bacterial solution. The plates were placed at 28℃ for 3 days to observe whether the strain could produce phosphorus-solubilizing zones on Mongkina inorganic phosphorus medium and Mongkina organic phosphorus medium.

[0057] Determination of potassium-solubilizing ability of strain ARF-SR1: Sterilized filter paper was placed on the surface of potassium-solubilizing medium. 5 μL of activated biocontrol bacterial solution was taken and inoculated onto the filter paper, allowing the filter paper to absorb the bacterial solution. The plate was placed at 28℃ for 3 days, and it was observed whether the strain could produce a lysis zone on the potassium-solubilizing medium (silicate bacteria medium).

[0058] Protease production capacity assay of strain ARF-SR1: Sterile filter paper discs were placed on skim milk solid medium. 5 μL of activated biocontrol bacterial solution was inoculated onto the filter paper discs, allowing the discs to absorb the solution. After incubation at 28℃ for 3 days, the area around the inoculation wells was observed. The formation of a clear zone indicated protease production by the strain, with the diameter representing the amount of protease produced. The skim milk solid medium consisted of the following components at the following concentrations: skim milk powder 10 g / L, peptone 10 g / L, sodium chloride 5 g / L, calcium chloride 0.1 g / L, and agar powder 18 g / L; pH 7.2.

[0059] Assay for the amylase production capacity of strain ARF-SR1: Sterile filter paper discs were placed on amylase detection medium. 5 μL of activated biocontrol bacterial solution was inoculated onto the filter paper discs, allowing the discs to absorb the solution. The discs were then incubated at 28℃ for 3 days. After staining with diluted Lugol's iodine solution for 15 seconds, the surface stain was washed with 70% ethanol. The presence of a clear zone was observed; if a clear zone was formed, it indicated that the bacteria had the ability to produce amylase. The amylase detection medium consisted of the following components at the following concentrations: starch 10 g / L, tryptone 10 g / L, glucose 5 g / L, sodium chloride 5 g / L, beef extract 5 g / L, and agar powder 18 g / L.

[0060] Assay for cellulase production capacity of strain ARF-SR1: Sterilized filter paper discs were placed on cellulose Congo red medium. 5 μL of activated biocontrol bacterial solution was inoculated onto the filter paper discs, allowing the discs to absorb the bacterial solution. After incubation at 28℃ for 3 days, the area around the inoculation wells was observed. If a clear zone was formed, it indicated that the strain produced cellulase. The diameter of the clear zone represented the amount of cellulase produced by the strain.

[0061] The ability of strain ARF-SR1 to produce ferophiles was determined: Activated biocontrol bacterial culture was inoculated into LB liquid medium and cultured at 37℃ and 200 r / min for 24 h. 10 mL of the cultured bacterial culture was centrifuged at 10000 r / min for 10 min. 3 mL of the supernatant was mixed with 3 mL of CAS detection solution and allowed to stand in the dark for 30 min. The absorbance (A1) was measured at 630 nm using a spectrophotometer. The absorbance (A0) was calculated by mixing equal volumes of LB liquid medium and CAS detection solution using double-distilled water. The relative ferophile content was calculated using the formula (A0-A1) / A0×100%. Simultaneously, 5 μL of the bacterial culture was inoculated onto a CAS solid plate and cultured at 28℃. The presence of an orange-yellow halo was observed.

[0062] IAA production capacity determination of strain ARF-SR1: A viable count of 10-1 was used.8 CFU / mL Bacillus sicca ARF-SR1 seed culture was inoculated at a ratio of 1% (v / v) into LB liquid medium containing 100 mg / L tryptophan, with three replicates. The control group consisted of the same volume of sterile water mixed with tryptophan in LB liquid medium. The culture was incubated at 30°C and 180 rpm for 24 h with shaking. After incubation, the bacterial suspension was centrifuged at 10,000 rpm for 10 min, and 1 mL of the supernatant was added to 2 mL of Salkowski colorimetric solution. The mixture was allowed to stand in the dark for 30 min, and the OD of the corresponding bacterial suspension was measured. 600 Observe the test results; red or slightly red indicates a positive result, while no color change indicates a negative result. The results show the ability to produce IAA (indicating a positive result). Figure 5 ).

[0063] Assay of biofilm formation ability of strain ARF-SR1: Bacillus sicca ARF-SR1 strain was inoculated into LB liquid medium for activation and cultured to the logarithmic growth phase (OD2). 600 =0.8), 100 μL of the activated ARF-SR1 strain was aspirated into a 96-well plate, with an equal volume of LB liquid medium as the control group. After culturing for 24 h, the suspension was aspirated, and 100 μL of 1% crystal violet solution was added to each well of the 96-well plate with a biofilm attached. The plate was stained by standing at room temperature for 15–30 min. The crystal violet solution was then removed, and excess solution was thoroughly rinsed with ddH2O and air-dried at room temperature. 100 μL of 95% ethanol solution was added to each well, and the crystal violet was dissolved by gentle shaking at 100 rpm for 3 min at room temperature. The solution was then added to the OD... 600 The absorbance of crystal violet was measured to relatively quantify biofilm formation, and the inhibition rate was calculated using the following formula.

[0064] Inhibition rate = (OD) 实验组 – OD 对照组 ) / OD 对照组 ×100%.

[0065] The above measurement results are shown in Figures 3-6 .

[0066] Depend on Figure 3 It can be seen that strain ARF-SR1 has obvious nitrogen fixation ability, inorganic phosphorus and organic phosphorus solubilization ability, and relatively weak potassium solubilization ability, indicating that ARF-SR1, as a biocontrol bacterium, can promote the growth of host plants through nitrogen fixation and phosphorus solubilization.

[0067] Depend on Figure 4It was found that strain ARF-SR1 showed significantly higher levels of amylase, protease, cellulase (reflecting the strain's potential to degrade plant residues or destroy the cellulose structure of pathogens), and secreted heptaphil activity than the control group (CK). This indicates that by secreting multifunctional extracellular enzymes and siderophores, it can exert biocontrol functions through a dual mechanism of direct antagonism (enzymatic decomposition of pathogen structures) and indirect competition (siderophore inhibition). It has the potential to efficiently antagonize pathogens and adapt to the environment, making it a multifunctional biocontrol candidate strain.

[0068] Depend on Figure 5 It was found that strain ARF-SR1 has the ability to release the plant growth hormone indoleacetic acid (IAA), and its OD value was determined after quantification. 600 The amount of IAA produced in the treatment group was significantly higher than that in the control group, indicating that strain ARF-SR1 has a high IAA production capacity, further demonstrating that this bacterium can not only resist disease but also promote plant growth.

[0069] Biofilms are complex community structures formed by microorganisms on solid surfaces, encapsulated by extracellular polymeric substances (EPS), which enhance the resilience of the microorganisms. They can prevent the penetration of chemical pesticides or antimicrobial substances from host plants, prolonging the duration of action and enhancing their sustained control effect; they can also help microorganisms attach to plant roots or leaf surfaces, forming stable microecological sites and promoting mutually beneficial symbiosis with the host plant (such as secreting growth-promoting substances). Figure 6 As shown, the processing group ( Bacillus siamensis After staining with crystal violet, ARF-SR1 showed a darker color compared to the control group (CK). Its OD value was quantitatively measured using a microplate reader. 600 Afterwards, the amount of biofilm produced was significantly higher than that of the control group, indicating that strain ARF-SR1 has the ability to produce biofilm efficiently, further enhancing its colonization ability and disease resistance in the soil.

[0070] Example 4: Determination of the antagonistic effect of Bacillus sicca ARF-SR1 against alfalfa root rot pathogens

[0071] Preparation of fermentation broth for strain ARF-SR1: Bacillus sicca ARF-SR1 was inoculated into LB liquid medium for activation and cultured until the logarithmic growth phase (OD2). 600 =0.8).

[0072] Activation of alfalfa root rot pathogens: Three pathogens isolated and identified from alfalfa root rot soil samples: Fusarium solani (… Fusarium solani Fusarium argentis ( ), Fusarium argentis ( Fusarium acuminatum ) and one plant of the genus *Heterostylus* ( Paraphoma rhaphiolepidis The mycelial side of the fungal discs of the three pathogens was attached to fresh PDA medium and incubated at 28°C for 2-3 days. After three subcultures, the fungal discs were used as target bacteria for later use.

[0073] Plate antagonistic performance test: Three target bacteria mycelia (8 mm in diameter) were inoculated in the center of a 9 cm diameter PDA plate. Fermentation broth of strain ARF-SR1 was inoculated around the mycelia at a distance of 2.5 cm from the periphery. A plate inoculated only with the target pathogen served as a control. The plates were incubated upside down at 28°C. The antibacterial effect was observed and recorded when the control plate fully colonized. Results are shown below. Figures 7-9 .

[0074] The results showed that strain ARF-SR1 had significant inhibitory effects on Fusarium rot (antagonism rate 67.22%), Fusarium raffins (antagonism rate 57.71%), and Pyrophyllus heterostemum (antagonism rate 68.86%), and its application to plants could reduce the incidence of alfalfa root rot.

[0075] Example 5 Optimization of Culture Medium

[0076] Seed culture preparation: Bacillus sicca ARF-SR1 was taken from a -80℃ freezer, activated 2-3 times in LB medium, and then inoculated into LB liquid medium. The culture was carried out at 37℃ with shaking at 200 r / min for 12 h, yielding a viable count of 102. 8 Seed culture of CFU / mL.

[0077] The culture medium before optimization was LB liquid medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 5.0 g / L sodium chloride. Culture conditions: pH=7.0, fermenter volume 50%, rotation speed 200 r / min, inoculum size 3% (v / v), temperature 37℃, and culture time 24 h.

[0078] The optimized culture medium consists of the following components at the following concentrations: corn starch 20 g / L, peptone 25 g / L, and NaCl 7.5 g / L, pH=7.4.

[0079] The optimized fermentation temperature was 32℃, the liquid volume was 60% of the fermenter volume, the rotation speed was 186 r / min, the inoculum size was 4% (v / v), the temperature was 37℃, and the fermentation time was 24 h.

[0080] The number of viable cells, the number of spores, and the spore rate were detected after 24 hours of fermentation before and after optimization, using the following methods:

[0081] 1) Viable cell count: The dilution plating method was used for counting. The cultured bacterial suspension was serially diluted to 10⁻⁶ each time. -1 10 -2 10 -3 ...10 -8Different concentrations of bacterial suspension were prepared by using a sterile pipette to draw 100 μL of each dilution and adding it to the surface of a solidified culture medium plate. The bacterial suspension was then evenly spread onto the LB agar plate using a sterile spreader. The plates were inverted and placed in a constant temperature incubator at 37°C for 24 h. After incubation, plates with colony counts between 30 and 300 were selected for colony counting. The number of colonies on the plates was counted and multiplied by the corresponding dilution factor to obtain the number of viable bacteria in the sample.

[0082] 2) Spore count: The dilution procedure is the same as for viable bacteria counting. The diluted bacterial suspension is then treated in an 80°C water bath for 15 minutes to kill bacteria that have not formed spores before plate counting.

[0083] 3) Spore rate = number of spores / number of viable bacteria × 100%.

[0084] The results are as follows:

[0085] Before optimization, the number of viable bacteria, the number of spores, and the spore rate were 6.81 × 10⁻⁶. 9 CFU / mL, 5.25×10 9 CFU / mL and 71.98%.

[0086] The optimized count of viable bacteria, spore count, and spore rate reached 8.24 × 10⁻⁶. 9 CFU / mL, 7.52×10 9 The CFU / mL and 91.23% were 21.05%, 43.21%, and 26.75% higher than before optimization, respectively. These figures were 1.21 times, 1.43 times, and 1.27 times higher than before optimization.

[0087] This invention selects agricultural production and processing by-products that are inexpensive, nutritious, and have good fermentation effects as the main nutrient substances, optimizes the culture medium, further reduces the cost of applying microbial agents in agriculture, and provides technical support for the actual production of microbial agents.

[0088] In summary, the strain ARF-SR1 obtained by this invention is a novel biocontrol strain for controlling alfalfa root rot. It has a significant inhibitory effect on alfalfa root rot pathogens (Fusarium rotii, Fusarium scutellarioides, and Pyrophyllus spp.), which can reduce the incidence of alfalfa root rot and is suitable for green control of alfalfa root rot.

[0089] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus sicca ( Bacillus siamensis ARF-SR1, characterized in that, The accession number is CGMCCNo.33715.

2. A biocontrol agent, characterized in that, The active ingredient includes Bacillus sicca ARF-SR1 as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, The OD of Bacillus sicca ARF-SR1 in the biocontrol agent 600 Value ≥ 0.

5.

4. The method for preparing the biocontrol agent according to claim 2 or 3, characterized in that, Includes the following steps: The Bacillus sicca ARF-SR1 of claim 1 was inoculated into a culture medium and cultured to obtain the biocontrol agent; The culture medium includes LB medium, TSB medium, or a prepared medium; the prepared medium includes the following components at the following concentrations: corn starch 15-25 g / L, peptone 20-30 g / L, and NaCl 5-10 g / L, pH=7-8.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 25~38℃; the culture rotation speed is 180~200 r / min.

6. The application of the Bacillus sicca ARF-SR1 of claim 1, or the biocontrol agent of claim 2 or 3, or the biocontrol agent prepared by the preparation method of claim 4 or 5, in the prevention and control of alfalfa root rot and / or the promotion of alfalfa growth; The pathogen causing alfalfa root rot is Fusarium solani (…). Fusarium solani Fusarium argentis ( ), Fusarium argentis ( Fusarium acuminatum )and Paraphoma rhaphiolepidis .

7. A method for preventing and / or promoting alfalfa root rot, characterized in that, Includes the following steps: The biocontrol agent is applied to the soil in which alfalfa is planted and / or applied to the alfalfa itself; the biocontrol agent is the biocontrol agent according to claim 2 or 3 or the biocontrol agent prepared by the preparation method according to claim 4 or 5; The pathogen causing alfalfa root rot is Fusarium solani (…). Fusarium solani Fusarium argentis ( ), Fusarium argentis ( Fusarium acuminatum )and Paraphoma rhaphiolepidis .

8. The method according to claim 7, characterized in that, The application methods include root irrigation and / or spraying.

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