Bacillus siamensis ARF-SR1, biocontrol inoculant and application of bacillus siamensis ARF-SR1 and biocontrol inoculant

Abstract

The invention relates to the technical field of biocontrol bacteria, in particular to bacillus siamensis ARF-SR1, a biocontrol bacterial agent and application of the bacillus siamensis ARF-SR1 and the biocontrol bacterial agent. The preservation number of the bacillus siamensis ARF-SR1 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No. 33715. The bacillus siamensis ARF-SR1 provided by the invention is a biocontrol strain separated from a healthy alfalfa field in Ningxia, has a remarkable inhibition effect on alfalfa root rot pathogenic bacteria (particularly fusarium solani, fusarium acuminatum and phomopsis sp.), has an antagonism rate of 57.71-68.86%, can reduce the incidence rate of alfalfa root rot, enriches a biocontrol bacterium resource library, and has a good application prospect. The composition is suitable for green control of alfalfa root rot.

Description

Technical Field

[0001] The present invention relates to the technical field of biocontrol bacteria, and particularly to a Bacillus siamensis Bacillus siamensis ARF-SR1, a biocontrol bacterial agent and its application. Background Art

[0002] Alfalfa (Medicago sativa), as a high-quality forage crop with rich nutrition and strong adaptability, occupies a crucial position in livestock production. However, alfalfa root rot, as a common disease, is one of the main problems restricting its development. It has long troubled the development of the alfalfa industry, not only causing a significant decrease in alfalfa yield, but also seriously affecting the quality, bringing quite a challenge to livestock production.

[0003] At present, the prevention and control strategies for alfalfa root rot pathogens mainly include chemical control and agricultural control. Although chemical control has significant effects in the short term, long-term use will lead to the generation of pathogen drug resistance and have negative impacts on the environment and ecosystem. Agricultural control mainly improves the soil environment and enhances the disease resistance of plants through measures such as crop rotation, deep plowing of soil, and rational fertilization. However, these measures need to be implemented for a long time and have limited effects. Biological control is a method that can utilize microorganisms and their metabolites to antagonize pathogens to control harmful organisms. Compared with chemical pesticides and traditional agricultural control technologies, biological control has the advantages of environmental protection, mildness, safety, and sustainability, and has received much attention in recent years. However, there are limited biocontrol bacteria for alfalfa root rot at present, and there is an urgent need for a biocontrol bacterium that has an antagonistic effect on multiple alfalfa root rot pathogens to enrich the biocontrol bacteria resource library. Summary of the Invention

[0004] To solve the above problems, the present invention provides a Bacillus siamensis ARF-SR1, a biocontrol bacterial agent and its application. The Bacillus siamensis ARF-SR1 provided by the present invention has a significant inhibitory effect on alfalfa root rot pathogens (especially Fusarium solani, Fusarium acuminatum and Phomopsis spp.), and the antagonistic rate is 57.71 - 68.86%, which can reduce the incidence of alfalfa root rot and enrich the biocontrol bacteria resource library.

[0005] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a Bacillus siamensis ARF-SR1 with a preservation number of CGMCC No. 33715.

[0006] The present invention provides a biocontrol bacterial agent, and the active ingredient includes the Bacillus siamensis ARF-SR1 described in the above technical solution.

[0007] Preferably, the optical density (OD) of Bacillus siamensis ARF-SR1 in the biocontrol agent 600 is ≥ 0.5.

[0008] The present invention provides a method for preparing the biocontrol agent described in the above technical solution, comprising the following steps: Inoculate Bacillus siamensis ARF-SR1 described in the above technical solution into a culture medium, and culture to obtain the biocontrol agent; the culture medium includes LB medium, TSB medium or a formulated medium; the formulated medium includes components with the following concentrations: corn starch 15 - 25 g / L, peptone 20 - 30 g / L, and NaCl 5 - 10 g / L, and pH = 7 - 8.

[0009] Preferably, the temperature of the culture is 25 - 38°C; the rotation speed of the culture is 180 - 200 r / min.

[0010] The present invention provides the application of Bacillus siamensis ARF-SR1 described in the above technical solution, or the biocontrol agent described in the above technical solution, or the biocontrol agent prepared by using the preparation method described in the above technical solution in preventing and treating plant root rot and / or promoting plant growth.

[0011] Preferably, the pathogens of the plant root rot include: Fusarium solani ( Fusarium solani ), Fusarium acuminatum ( Fusarium acuminatum ), and Phoma exigua ( Paraphoma rhaphiolepidis ) one or more of them.

[0012] Preferably, the plant includes alfalfa.

[0013] The present invention provides a method for preventing and treating root rot and / or promoting plant growth, comprising the following steps: Apply the biocontrol agent to the soil where the plant is planted and / or apply it to the plant; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by using the preparation method described in the above technical solution.

[0014] Preferably, the plant includes alfalfa; the application method includes root irrigation and / or spraying.

[0015] Beneficial effects: The present invention provides a strain of Bacillus siamensis ARF-SR1 with a preservation number of CGMCC No. 33715. The Bacillus siamensis ARF-SR1 provided by the present invention is a biocontrol strain isolated from a healthy alfalfa field in Ningxia, which has a significant inhibitory effect on the pathogens of alfalfa root rot (especially Fusarium solani, Fusarium acuminatum and Phomopsis sp.), with an antagonistic rate of 57.71-68.86%. It can reduce the incidence of alfalfa root rot, enrich the biocontrol bacteria resource library, and is suitable for the green control of alfalfa root rot. Brief Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0017] Figure 1 It is the colony morphology diagram, Gram staining diagram and scanning electron microscopy diagram of the strain ARF-SR1 on the TSB solid medium; Figure 2 It is the phylogenetic tree constructed by the strain ARF-SR1 based on 16S rDNA; Figure 3 It is the determination results of the nitrogen fixation ability, phosphorus solubilization ability and potassium solubilization ability of the strain ARF-SR1; Figure 4 It is the determination results of the protease production ability, amylase production ability, cellulase production ability and siderophore production ability of the strain ARF-SR1; Figure 5 It is the determination result of the IAA production ability of the strain ARF-SR1; Figure 6 It is the determination result of the biofilm formation ability of the strain ARF-SR1; Figure 7 It is the antagonistic effect diagram of the strain ARF-SR1 against Fusarium solani, the pathogen of alfalfa root rot; Figure 8 It is the antagonistic effect diagram of the strain ARF-SR1 against Fusarium acuminatum, the pathogen of alfalfa root rot; Figure 9 It is the antagonistic effect diagram of the strain ARF-SR1 against Phomopsis sp., the pathogen of alfalfa root rot.

[0018] Biological Deposit Description Bacillus siamensis ARF-SR1, classified and named as Bacillus siamensis Bacillus siamensis was deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 4, 2025. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33715. Detailed Embodiments

[0019] The present invention provides a strain of Bacillus siamensis ARF-SR1, with the preservation number of CGMCC No. 33715.

[0020] The Bacillus siamensis ARF-SR1 provided by the present invention is isolated from the soil of alfalfa in a certain alfalfa field in Xingfu Village, Gaozhuang Township, Pingluo County, Shizuishan City, Ningxia Hui Autonomous Region. It grows rapidly on TSB solid medium, with convex colonies, milky white in color, surface wrinkled and dry, and opaque; Gram-positive, with a diameter of about 0.5 μm and a length of about 2-4 μm. Under the microscope, the bacteria are in the shape of long rods, having a strong nitrogen-fixing ability, and being able to decompose organic phosphorus, inorganic phosphorus and potassium elements. In addition, the bacterium also has the ability to produce protease, cellulase, amylase, siderophore and indole acetic acid (IAA). It not only has a significant inhibitory effect on the common pathogenic bacteria of alfalfa root rot, Fusarium solani and Fusarium acuminatum, but also has a strong inhibitory effect on the pathogenic bacteria of alfalfa root rot, Phomopsis sp. Moreover, it can promote the growth of host plants through nitrogen fixation, phosphorus solubilization and IAA. Compared with chemical pesticides, applying the biocontrol agent containing Bacillus siamensis ARF-SR1 has a better control effect on alfalfa root rot, can be used to control alfalfa root rot, has the advantages of safety, greenness and environmental protection, and also enriches the biocontrol bacteria resource library for alfalfa root rot, with broad application prospects and market value.

[0021] Based on the above advantages, the present invention provides a biocontrol agent, the active ingredient of which includes the Bacillus siamensis ARF-SR1 described in the above technical solution.

[0022] As an embodiment, the OD value of Bacillus siamensis ARF-SR1 in the biocontrol agent 600 ≥0.5. As another embodiment, the OD value of Bacillus siamensis ARF-SR1 in the biocontrol agent 600 ≥0.8. As another embodiment, the OD value of Bacillus siamensis ARF-SR1 in the biocontrol agent 600 is 0.8-1.5. The biocontrol agent provided by the present invention has a significant inhibitory effect on the pathogenic bacteria of alfalfa root rot (especially Fusarium solani, Fusarium acuminatum and Phomopsis sp.).

[0023] The present invention provides a preparation method of the biocontrol agent described in the above technical solution, including the following steps: Inoculate the Bacillus siamensis ARF-SR1 described in the above technical solution into a culture medium, and culture to obtain the biocontrol agent; the culture medium includes LB medium, TSB medium or a prepared medium; the prepared medium includes the following components in concentrations: corn starch 15-25 g / L, peptone 20-30 g / L and NaCl 5-10 g / L, pH = 7-8.

[0024] As one embodiment, the temperature of the cultivation is 25 - 38 °C; the rotation speed of the cultivation is 180 - 200 r / min. As another embodiment, the temperature of the cultivation is 35 - 37 °C; the rotation speed of the cultivation is 186 r / min. As one embodiment, the prepared culture medium comprises components with the following concentrations: 20 g / L of corn starch, 25 g / L of peptone, and 7.5 g / L of NaCl, pH = 7.5. The prepared culture medium provided by the present invention selects agricultural production and processing by-products with low price, rich nutrition, and good fermentation effect as the main nutrient substances, and optimizes the culture medium. Compared with the LB culture medium, it not only further reduces the cost of applying the microbial agent in agriculture, but also the viable bacteria count, spore count, and spore rate are increased by 1.21 times, 1.43 times, and 1.27 times respectively compared with the LB culture medium. It is a culture medium suitable for growth and provides technical support for the actual production of the microbial agent.

[0025] Based on the above advantages, the present invention provides the application of Bacillus siamensis ARF - SR1 described in the above technical solution, or the biocontrol agent described in the above technical solution, or the biocontrol agent prepared by using the preparation method described in the above technical solution in preventing and treating plant root rot and / or promoting plant growth.

[0026] As one embodiment, the pathogens of the plant root rot include one or more of Fusarium solani, Fusarium acuminatum, and Phomopsis sp. As one embodiment, the plant can be alfalfa.

[0027] Based on the above advantages, the present invention provides a method for preventing and treating root rot and / or promoting plant growth, comprising the following steps: Applying the biocontrol agent to the soil where the plant is planted and / or applying it to the plant; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by using the preparation method described in the above technical solution.

[0028] As one embodiment, the plant can be alfalfa; the application method includes root irrigation and / or spraying. The present invention can apply the biocontrol agent to the soil and / or the plant by root irrigation and / or spraying, so as to exert the efficacy of Bacillus siamensis ARF - SR1 in preventing and treating root rot and / or promoting plant growth, which is a safe, green, and environmentally friendly method.

[0029] To further illustrate the present invention, the following describes in detail a strain of Bacillus siamensis ARF - SR1, a biocontrol agent, and their applications provided by the present invention in combination with examples and drawings, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1 Screening and Isolation of Strain ARF-SR1 In this example, the soil sample was collected from the rhizosphere soil of healthy alfalfa in Xingfu Village, Gaozhuang Township, Pingluo County, Shizuishan City, Ningxia Hui Autonomous Region, and stored at 4°C. 10 g of the tested soil was weighed and placed into a triangular flask containing 90 mL of sterile water. It was incubated at a constant temperature of 25°C with shaking at 180 r / min for 30 min. After standing for 1 min for natural sedimentation, the supernatant was taken, and the 10-fold gradient dilution method was used to dilute the soil microbial suspension to 10 -3 、10 -4 、10 -5 、10 -6 . 100 μl of each concentration was taken and evenly spread on the TSB solid medium, and then incubated in a constant temperature incubator at 37°C for 1 - 2 d. According to the differences in colony morphology, single colonies were picked and purified by the streak plate method onto a new TSB solid medium. After purification, the single colonies were transferred into 50% glycerol tubes and stored at -20°C for later use.

[0031] Using the pathogenic bacteria screened from the soil where alfalfa root rot occurred in this area as the target bacteria for antagonistic screening. The target bacteria cake (diameter 8 mm) was inoculated in the center of a 90-mm diameter PDA plate, and the tested strains were inoculated at a distance of 2.5 cm from the periphery of the bacteria cake. The plate inoculated only with the target pathogenic bacteria was used as a control. It was incubated upside down in an incubator at 28°C, and the antibacterial effect was observed and recorded after 5 d. Finally, a strain with better antibacterial effect was selected. The colony was convex, milky white, with a wrinkled and dry surface and opaque; Gram-positive staining, with a diameter of 0.5 μm and a length of 2 - 4 μm. Under the microscope, the bacterial cells were long rod-shaped, as shown in Figure 1 ( Figure 1 a is the colony photo in Figure 1 b is the microscopic observation photo of Gram staining in Figure 1 c is the scanning electron microscope observation photo in Figure 1 ). This strain was named ARF-SR1. After shaking the single colony of strain ARF-SR1 in TSB liquid medium, 50% glycerol was added and stored at -80°C.

[0032] Example 2 Molecular Identification of Strain ARF-SR1 Genomic DNA of strain ARF-SR1 was extracted according to the operation manual of the bacterial DNA extraction kit, and a spectrophotometer was used to detect the concentration and quality of the extracted DNA.

[0033] The genomic DNA of strain ARF-SR1 was amplified by PCR using the 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTC-3', SEQ ID NO.1) and 1492R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID NO.2). The 25-μl PCR amplification system contained 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. The PCR amplification conditions were as follows: 95°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, with 30 cycles; 72°C for 10 min; and stored at -20°C for later use. It was sent to Beijing Bomed Gene Technology Co., Ltd. for sequencing and identified after NCBI blast alignment. The nucleotide sequence of 16S rDNA is shown in SEQ ID NO.3 as follows:

[0034] The sequencing results were analyzed and compared by BLAST in NCBI, and a phylogenetic tree was constructed using MEGA software, as shown in Figure 2 . Combining the physiological and biochemical characteristics of strain ARF-SR1 and phylogenetic tree analysis, strain ARF-SR1 was identified as Bacillus siamensis Bacillus siamensis. In the present invention, strain ARF-SR1 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC No. 33715.

[0035] Example 3 Determination of the biocontrol performance of Bacillus siamensis ARF-SR1 Activation of the strain: Take out the preserved strain ARF-SR1 from the glycerol tube, take 100 μL of the bacterial solution, inoculate it into 10 mL of LB liquid medium, and shake culture at 37 °C and 150 rpm for 24 h until OD 600 = 1.0 to obtain the activated biocontrol bacterial solution (denoted as Bacillus siamensis ARF-SR1). The following biocontrol performance tests were carried out on the activated biocontrol bacterial solution. In all cases, LB liquid medium was used to replace the activated biocontrol bacterial solution as a control (CK), and each treatment was repeated 3 times. The specific steps are as follows: Determination of the nitrogen fixation ability of strain ARF-SR1: Place sterilized filter paper on the nitrogen-free medium. Take 5 μL of the activated biocontrol bacterial solution respectively, inoculate the bacterial solution on the filter paper, and let the filter paper absorb the bacterial solution. Place the plate at 28 °C for 3 d, and observe whether the strain can grow in the nitrogen-free medium and whether there is a clear zone.

[0036] Determination of the phosphorus solubilization ability of strain ARF-SR1: Place sterilized filter paper on the surface of the Mengjinna inorganic phosphorus medium (PKO) and Mengjinna organic medium respectively. Take 5 μL of the activated biocontrol bacterial solution, inoculate the bacterial solution on the filter paper, and let the filter paper absorb the bacterial solution. Place the plate at 28 °C for 3 d, and observe whether the strain can produce a phosphorus solubilization zone on the Mengjinna inorganic phosphorus medium and Mengjinna organic medium.

[0037] Determination of the potassium solubilization ability of strain ARF-SR1: Place sterilized filter paper on the surface of the potassium solubilization medium. Take 5 μL of the activated biocontrol bacterial solution, inoculate the bacterial solution on the filter paper, and let the filter paper absorb the bacterial solution. Place the plate at 28 °C for 3 d, and observe whether the strain can produce a dissolution zone on the potassium solubilization medium (silicate bacteria medium).

[0038] Determination of protease production ability of strain ARF-SR1: Sterilized filter paper was placed on skim milk solid medium. 5 μL of the activated biocontrol bacterial solution was taken respectively and inoculated on the filter paper to absorb the bacterial solution. After culturing in an incubator at 28 °C for 3 d, the area around the inoculation hole was observed. If a clear zone appeared, it indicated that the strain produced protease, and the diameter of the clear zone represented the amount of protease produced by the strain. The skim milk solid medium was composed 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; the pH value was 7.2.

[0039] Determination of amylase production ability of strain ARF-SR1: Sterilized filter paper was placed on amylase detection medium. 5 μL of the activated biocontrol bacterial solution was taken respectively and inoculated on the filter paper to absorb the bacterial solution. After culturing in a constant temperature incubator at 28 °C for 3 d, it was stained with diluted Lugol's iodine solution for 15 s, and the surface floating color was washed with 70% ethanol. Whether a clear zone appeared was observed. If it appeared, it indicated that the bacteria had the ability to produce amylase. The amylase detection medium was composed 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.

[0040] Determination of cellulase production ability of strain ARF-SR1: Sterilized filter paper was placed on cellulose Congo red medium. 5 μL of the activated biocontrol bacterial solution was taken respectively and inoculated on the filter paper to absorb the bacterial solution. After culturing in an incubator at 28 °C for 3 d, the area around the inoculation hole was observed. If a clear zone appeared, it indicated that the strain produced cellulase, and the diameter of the clear zone represented the amount of cellulase produced by the strain.

[0041] Determination of siderophore production ability of strain ARF-SR1: The activated biocontrol bacterial solution was inoculated into LB liquid medium and cultured at 37 °C and 200 r / min for 24 h. 10 mL of the cultured bacterial solution was taken respectively and centrifuged at 10000 r / min for 10 min. 3 mL of the centrifuged supernatant was mixed with 3 mL of CAS detection solution, and left to stand in the dark for 30 min. The absorbance value measured at 630 nm using a spectrophotometer was (A1), zeroed with double-distilled water, and the absorbance value of the equal-volume mixture of LB liquid medium and CAS detection solution was (A0). It was calculated according to the formula (A0 - A1) / A0×100% to obtain the relative content of siderophore. At the same time, 5 μL of the bacterial solution was inoculated on a CAS solid plate and cultured at 28 °C to observe whether there was an orange halo.

[0042] Determination of IAA production ability of strain ARF-SR1: The viable cell count was 108 The Bacillus siamensis ARF-SR1 seed solution with a concentration of CFU / mL was inoculated into the LB liquid medium containing 100 mg / L tryptophan at a ratio of 1% (v / v), with 3 replicates set. The control group was the same volume of sterile water added to the tryptophan LB liquid medium; cultured at 30 °C with shaking at 180 rpm for 24 h; after culturing, the bacterial suspension was centrifuged at 10000 rpm for 10 min, 1 mL of the supernatant was taken and added to 2 mL of Salkowski colorimetric solution, and the reaction 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 and slightly red are both positive, and no color change is negative. The results show the ability to produce IAA ( Figure 5 ).

[0043] Determination of the biofilm-forming ability of strain ARF-SR1: The Bacillus siamensis ARF-SR1 strain was inoculated into the LB liquid medium for activation and cultured until the logarithmic phase (OD 600 =0.8). 100 μL of the activated ARF-SR1 strain was aspirated into a 96-well plate. The control group was an equal volume of LB liquid medium. After culturing for 24 h, the suspended culture medium was aspirated. 100 μL of 1% crystal violet solution was added to each well of the 96-well plate with attached biofilm, and the plate was left to stand at room temperature for 15 - 30 min for staining. The crystal violet solution was removed, and the excess crystal violet 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 plate was gently shaken at 100 rpm at room temperature for 3 min to dissolve the crystal violet; the absorbance of crystal violet was measured at OD 600 to relatively quantify the biofilm formation, and the inhibition rate was calculated according to the following formula.

[0044] Inhibition rate = (OD 实验组 – OD 对照组 ) / OD 对照组 ×100%.

[0045] The above measurement results are shown in Figures 3 - 6 .

[0046] It can be seen from Figure 3 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.

[0047] It can be seen from Figure 4It can be seen that the strain ARF-SR1 is significantly higher than the control group (CK) in terms of amylase, protease, cellulase (reflecting the potential of the strain to degrade plant residues or destroy the cellulose structure of pathogenic bacteria), and the activity of secreting siderophores, indicating that it can play a biocontrol function through dual mechanisms of direct antagonism (enzymatically degrading the structure of pathogenic bacteria) and indirect competition (inhibiting with siderophores) by secreting multifunctional extracellular enzymes and siderophores, and has the potential for efficient antagonism against pathogenic bacteria and environmental adaptation. It is a multifunctional biocontrol candidate strain.

[0048] It is known from Figure 5 that the strain ARF-SR1 has the ability to release the plant growth hormone indole-3-acetic acid (IAA). After quantification, its OD 600 was measured. The production of IAA in the treatment group was significantly higher than that in the CK group, indicating that the strain ARF-SR1 has a high ability to produce IAA, further demonstrating that this bacterium can not only resist diseases but also promote plant growth.

[0049] Biofilm is a complex community structure formed by microorganisms on solid surfaces, wrapped by extracellular polymeric substances (EPS), which can enhance the stress resistance of bacteria. It can prevent the penetration of chemical pesticides or antibacterial substances of host plants, extend the action time of the strain, and enhance its continuous prevention and control effect; it can also help the strain attach to the root system or leaf surface of plants, form stable microecological sites, and promote its mutualism with host plants (such as secreting growth-promoting substances). As Figure 6 shown, after the treatment group ( Bacillus siamensis ARF-SR1) was stained with crystal violet, compared with the control group CK, the color became darker. After quantitatively measuring its OD 600 with an enzyme-labeled instrument, the production of its biofilm was significantly higher than that of the control group, indicating that the strain ARF-SR1 has a high ability to produce biofilm, further enhancing its colonization ability and disease resistance ability in the soil.

[0050] Example 4 Determination of the antagonistic performance of Bacillus siamensis ARF-SR1 against alfalfa root rot pathogens Preparation of the fermentation broth of strain ARF-SR1: Inoculate Bacillus siamensis ARF-SR1 into LB liquid medium for activation and culture until the logarithmic phase (OD 600 = 0.8).

[0051] Activation of alfalfa root rot pathogens: Three strains of pathogens isolated and identified from alfalfa root rot soil samples: Fusarium solani ( Fusarium solani ), Fusarium acuminatum ( Fusarium acuminatum ), and a strain of Phoma Paraphoma rhaphiolepidis ). Stick the side with mycelium of the three pathogen agar discs onto fresh PDA medium and culture them statically at 28 °C for 2 - 3 days. After subculturing 3 times, they are used as target pathogen agar discs for standby.

[0052] Flat confrontation antagonistic performance test: Inoculate 3 target bacteria cakes (8 mm in diameter) in the center of a 9-cm-diameter PDA plate. Inoculate the fermentation broth of strain ARF-SR1 at a distance of 2.5 cm around the bacteria cakes. Use the plate inoculated only with the target pathogenic bacteria as a control. Incubate it upside down in an incubator at 28 °C. Observe and record the antibacterial effect when the control group fills the plate. The results are shown in Figures 7 - 9 .

[0053] The results showed that strain ARF-SR1 had significant inhibitory effects on Fusarium solani (antagonistic rate 67.22%), Fusarium acuminatum (antagonistic rate 57.71%), and Phomopsis sp. (antagonistic rate 68.86%). Applying it to plants can reduce the incidence of alfalfa root rot.

[0054] Example 5 Optimization of the culture medium Seed liquid preparation: Take Bacillus siamensis ARF-SR1 from a -80 °C refrigerator. After activating it 2 - 3 times in LB medium, inoculate it into LB liquid medium and incubate it at 37 °C with shaking at 200 r / min for 12 h to obtain a seed liquid with a viable cell count of 10 8 CFU / mL.

[0055] The culture medium before optimization was LB liquid medium: 10.0 g / L of tryptone, 5.0 g / L of yeast extract powder, and 5.0 g / L of sodium chloride. Culture conditions: pH = 7.0, liquid loading volume was 50% of the fermenter volume, rotation speed was 200 r / min, inoculation amount was 3% (v / v), temperature was 37 °C, and culture time was 24 h.

[0056] The optimized culture medium consisted of the following components at the following concentrations: 20 g / L of corn starch, 25 g / L of peptone, and 7.5 g / L of NaCl, pH = 7.4.

[0057] The optimized fermentation temperature was 32 °C, the liquid loading volume was 60% of the fermenter volume, the rotation speed was 186 r / min, the inoculation amount was 4% (v / v), the temperature was 37 °C, and the fermentation time was 24 h.

[0058] Detect the viable cell count, spore count, and spore rate after 24 h of fermentation culture before and after optimization. The method is as follows: 1) Viable cell number: Count by the dilution plating method. Serial gradient dilute the cultured bacterial liquid, successively dilute it to 10 -1 、10 -2 、10 -3 ……10 -8At different concentrations, use a sterile pipette to aspirate an appropriate amount of 100 μL of bacterial suspensions with different dilution factors, and drop them on the surface of the solidified culture medium plate. Then, use a sterile spreading rod to evenly spread the bacterial liquid on the LB solid plate. Invert the plate and place it in a constant temperature incubator, and culture it at 37°C for 24 h. After the culture is completed, select the plates with the number of colonies between 30 and 300 for counting. Count the number of colonies on the plate and multiply it by the corresponding dilution factor to obtain the number of viable bacteria in the sample.

[0059] 2) Number of spores: The dilution steps are the same as those for viable bacteria counting. Continue to treat the diluted bacterial suspension in a water bath at 80°C for 15 min to kill the bacteria that have not formed spores, and then perform plate counting.

[0060] 3) Spore rate = number of spores / number of viable bacteria × 100%.

[0061] The results are as follows: The number of viable bacteria, the number of spores, and the spore rate before optimization were 6.81×10 9 CFU / mL, 5.25×10 9 CFU / mL, and 71.98%, respectively.

[0062] After optimization, the number of viable bacteria, the number of spores, and the spore rate reached 8.24×10 9 CFU / mL, 7.52×10 9 CFU / mL, and 91.23%, respectively, which were increased by 21.05%, 43.21%, and 26.75% compared with those before optimization, and were 1.21 times, 1.43 times, and 1.27 times those before optimization.

[0063] The present invention selects agricultural production and processing by-products with low price, rich nutrition, and good fermentation effect as the main nutrient substances, optimizes the culture medium, further reduces the cost of applying the microbial agent in agriculture, and provides technical support for the actual production of the microbial agent.

[0064] In summary, the strain ARF-SR1 screened by the present invention is a new type of biocontrol strain for preventing alfalfa root rot, has a significant inhibitory effect on the pathogens of alfalfa root rot (Fusarium solani, Fusarium acuminatum, and Phomopsis sp.), can reduce the incidence of alfalfa root rot, and is suitable for the green prevention and control of alfalfa root rot.

[0065] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A strain of Bacillus siamensis ( Bacillus siamensis ), ARF-SR1, characterized in that The preservation number is CGMCC No. 33715.

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

3. The biocontrol agent according to claim 2, wherein The OD of Bacillus siamensis ARF-SR1 in the biocontrol agent 600 value ≥ 0.

5.

4. The preparation method of the biocontrol agent according to claim 2 or 3, characterized in that, It includes the following steps: Inoculate Bacillus siamensis ARF-SR1 described in claim 1 into a culture medium and culture to obtain the biocontrol agent; the culture medium includes LB medium, TSB medium or a formulated medium; the formulated 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 temperature of the culture is 25-38 °C; the rotation speed of the culture is 180-200 r / min.

6. The application of Bacillus siamensis ARF-SR1 described in claim 1 or the biocontrol agent described in claim 2 or 3 or the biocontrol agent prepared by using the preparation method described in claim 4 or 5 in preventing and treating plant root rot and / or promoting plant growth.

7. The application according to claim 6, characterized in that, The pathogenic bacteria of the plant root rot include: Fusarium solani Fusarium solani ), Fusarium acuminatum Fusarium acuminatum ), and one or more of the species of Phomopsis Paraphoma rhaphiolepidis ).

8. The application according to claim 6 or 7, characterized in that, The plant includes alfalfa.

9. A method for preventing and treating root rot and / or promoting plant growth, characterized in that, It includes the following steps: Apply the biocontrol agent to the soil where the plant is planted and / or apply it to the plant; the biocontrol agent is the biocontrol agent described in claim 2 or 3 or the biocontrol agent prepared by using the preparation method described in claim 4 or 5.

10. The method according to claim 9, wherein The plant includes alfalfa; the application method includes root irrigation and / or spraying.

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