Bacillus velezensis, biocontrol agent, preparation method and application thereof

Abstract

The application discloses Bacillus velezensis, a biocontrol agent, and a preparation method and application thereof. A Bacillus velezensis strain of the application is numbered as RHB-1, and the preservation number is CCTCC NO: M 2024586. The RHB-1 strain and secondary metabolites thereof, such as extracellular and intracellular secretions, extracellular crude protein and crude ester peptides, all have the effect of inhibiting the growth of Gloeocercospora sorghi and Colletotrichum sublineola, and the bacteriostatic effect of the extracellular secretions is better than that of the intracellular secretions, and the bacteriostatic effect of the extracellular crude protein is better than that of the extracellular crude ester peptide. The Bacillus velezensis RHB-1 and the secondary metabolites thereof can be used for preventing and treating main leaf diseases of sorghum, such as sorghum panthera disease and anthracnose disease, and provide high-quality resources for the preparation and development of biocontrol agents.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sorghum biocontrol, and particularly relates to Bacillus velezensis, a biocontrol agent, and a preparation method and application thereof. BACKGROUND

[0002] Sorghum bicolor is an annual herbaceous plant of the family Poaceae, which has the characteristics of high amylopectin content, low fat content, and moderate tannin content, and thus has unique advantages in brewing. In Guizhou, sorghum is an important raw material for the production of dozens of famous wines such as Maotai, Xijiu, and Huaijiu. With the gradual increase of sorghum planting area in Guizhou Province, various diseases occur frequently, especially large-area leaf diseases, which seriously affect the yield and quality of sorghum. Among them, sorghum anthracnose and sorghum leopard disease are the high-incidence diseases of sorghum for wine in Guizhou Province in recent years.

[0003] Sorghum anthracnose mainly damages leaves, and can also infect stems, spike branches, and grains. On the leaves, the lesion often expands from the leaf tip to the entire leaf. When the disease just occurs, the lesion is small, and is fusiform, circular or elliptical, with a central red-brown color and an orange-yellow or purple-red halo at the edge. In the later stage, small black conidial discs are formed on the lesion, and under the conditions of high temperature and rain, the number of lesions increases and merges into patches, and in severe cases, the leaves or the whole plant can die. Sorghum anthracnose has become one of the main diseases restricting the increase of sorghum yield in recent years. The field incidence of sorghum anthracnose varies at home and abroad, and can reach 10% to 100%. In severe disease years, susceptible varieties can cause significant grain yield loss, and the yield loss of susceptible sorghum varieties without chemical spraying treatment can reach 55% to 67%, of which the grain loss is 18% to 36%. In highly susceptible varieties, the yield loss caused by anthracnose can reach 86% to 100%.

[0004] Sorghum leopard disease mainly damages leaves. The lesion on the leaf surface is nearly circular or elliptical, purple-red, with 2 to 8 very obvious concentric rings, and the size of the lesion varies greatly, with a diameter of 1 to 2 cm in the early stage and 3 to 7 cm in the later stage. The lesion occurring on the leaf margin is semicircular. When the disease is severe, multiple lesions merge and connect, like "leopard spots", with a diameter of up to the width of the entire leaf, often causing leaf death. Under the conditions of humidity and cloudy weather, the strip-shaped leaf spot disease causes up to 85% of the photosynthetic area to be damaged, and the yield loss caused by leaf disease is from 32% to 60%.

[0005] In 2019, the Department of Agriculture and Rural Affairs of Guizhou Province issued the "Guizhou Province Wine Sorghum Raw Material Planting Plan (2020-2022)", which clearly proposes that by 2022, the sorghum planting area in the province will reach 4 million mu, of which a large part needs to be produced according to the standards of organic raw materials. Since wine sorghum is the main raw material for brewing Maotai and other high-grade Maotai-flavor liquor, the main variety 'Hongyingzi' in Guizhou is a special organic sorghum variety for National Liquor Maotai, which needs to avoid the use of pesticides, so in recent years, research on sorghum diseases has mostly been on seedling breeding and agricultural control, and research on biological control is needed to prevent and control the high incidence of sorghum diseases in Guizhou Province in recent years.

[0006] Bacillus is a gram-positive bacterium that can produce spores and is widely present in natural environments such as soil, animal intestines, and plant bodies. Because it not only has a broad-spectrum inhibitory effect on pathogenic bacteria, but also is harmless to humans and animals, and has the advantages of being able to promote plant development and improve plant immunity, many bacteria in the genus Bacillus are used for biological control. Endophytic Bacillus can produce plant growth regulators, induce plant resistance, and inhibit the growth of pathogens, and has been developed as an important resource for biological pesticides.

[0007] In summary, the prior art still lacks a green and efficient probiotic for preventing major leaf diseases of sorghum such as anthracnose and high-kernel disease. SUMMARY

[0008] In view of this, one of the purposes of the present application is to provide a Bacillus velezensis, characterized in that the strain number is RHB-1, the accession number is CCTCC NO: M 2024586, and it was preserved in the China Center for Type Culture Collection on March 29, 2024, and the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The RHB-1 strain of the present application can inhibit the growth of Gloeocercospora sorghi or Colletotrichum sublineola, and can prevent and control high-kernel disease and anthracnose of sorghum.

[0009] The second object of the present application is to provide a biocontrol agent, wherein the active ingredient comprises Bacillus velezensis RHB-1 according to claim 1, or a fermentation broth of the Bacillus velezensis RHB-1, or a supernatant of the fermentation broth of the Bacillus velezensis RHB-1, or extracellular volatile substances of the Bacillus velezensis RHB-1, or intracellular substances of the Bacillus velezensis RHB-1, or extracellular crude protein of the Bacillus velezensis RHB-1, or extracellular crude lipopeptide of the Bacillus velezensis RHB-1. The Bacillus velezensis RHB-1 and its secondary metabolites such as extracellular secretory substances, intracellular substances, extracellular crude protein and extracellular crude lipopeptide all have the effect of inhibiting the growth of X. vasicola or C. sorghi, and the extracellular secretory substances are better than the intracellular secretory substances, the extracellular crude protein is better than the extracellular crude lipopeptide, which indicates that the main bacteriostatic substance is in the extracellular crude protein.

[0010] The third object of the present application is to provide a preparation method of the biocontrol agent, comprising the following steps:

[0011] (1) preparing a sterile LB liquid medium;

[0012] (2) adding the Bacillus velezensis RHB-1 according to claim 1 to the LB liquid medium to prepare a seed liquid;

[0013] (3) after 12 hours, adding the seed liquid to a new sterile LB liquid medium for shock culture to obtain the biocontrol agent.

[0014] Preferably, the following steps are included:

[0015] (1) filling the LB liquid medium into a sterile Erlenmeyer flask with a capacity of 250 mL and a gas-permeable hole, adding 50 mL of the LB liquid medium to each flask, and sterilizing the LB liquid medium at 121 DEG C for 20 minutes to obtain a sterile LB liquid medium; inoculating the Bacillus velezensis RHB-1 into a 9 cm diameter NA medium containing 15 mL, and culturing at 25 DEG C in the dark for 24 hours to obtain a single colony;

[0016] (2) under sterile conditions, using a inoculating loop to inoculate the single colony into the sterile LB liquid medium, inoculating one single colony in one Erlenmeyer flask, and then shock culturing at 28 DEG C in a shaker at 180 r·min-1 for 12 hours to obtain a seed liquid;

[0017] (3) inoculating the seed liquid into a new sterile LB liquid medium at an inoculation amount of 2% (v / v) for shock culture for 24 hours to obtain the biocontrol agent.

[0018] The fourth object of the present application provides an application of the biocontrol agent in inhibiting the growth of G. sorghi or C. sublineola, wherein the active ingredient of the biocontrol agent comprises the B. velezensis RHB-1, the fermentation liquor of the B. velezensis RHB-1, the supernatant of the fermentation liquor of the B. velezensis RHB-1, the extracellular volatile substance of the B. velezensis RHB-1, the intracellular substance of the B. velezensis RHB-1, the extracellular crude protein of the B. velezensis RHB-1 or the extracellular crude lipopeptide of the B. velezensis RHB-1.

[0019] The fifth object of the present application provides an application of the biocontrol agent in preventing and treating the leopard skin disease of sorghum or / and the anthracnose disease of sorghum, wherein the active ingredient of the biocontrol agent comprises the B. velezensis RHB-1, the fermentation liquor of the B. velezensis RHB-1, the supernatant of the fermentation liquor of the B. velezensis RHB-1, the extracellular volatile substance of the B. velezensis RHB-1, the intracellular substance of the B. velezensis RHB-1, the extracellular crude protein of the B. velezensis RHB-1 or the extracellular crude lipopeptide of the B. velezensis RHB-1.

[0020] Preferably, the biocontrol agent is sprayed on the leaves of sorghum or on the sorghum plants by a drone to prevent and treat the leopard skin disease of sorghum or the anthracnose disease of sorghum.

[0021] Preferably, the biocontrol agent is the fermentation liquor of the B. velezensis RHB-1, and the spraying concentration is 10 8 cfu·mL -1 . Generally, the prepared fermentation liquor needs to be diluted to a concentration of 10 8 cfu·mL -1 , for example, the fermentation liquor concentration is 5×10 9 cfu·mL -1 , and needs to be diluted by 50 times to a concentration of 10 8 cfu·mL -1 .

[0022] Preferably, the spraying amount is 3L per mu, and the concentration is 10 8 cfu·mL -1 .

[0023] Preferably, the spraying is performed three times, and the spraying time is as follows: the first spraying is performed at the early stage of the disease, the second spraying is performed after 4 weeks, and the third spraying is performed after another 4 weeks. The spraying amount is 3L per mu each time.

[0024] The Bacillus velezensis RHB-1 strain and secondary metabolites thereof, such as extracellular secretions, intracellular substances, extracellular crude proteins and extracellular crude ester peptides, all have the effect of inhibiting the growth of X. viminalis or C. sorghi, and the bacteriostatic effect of extracellular secretions is better than that of intracellular secretions, the bacteriostatic effect of extracellular crude proteins is better than that of extracellular crude ester peptides, which indicates that the main bacteriostatic substance is in the extracellular crude protein. Large-area spraying of the fermentation liquor of the strain in the field can achieve a field control effect of 74.6% on sorghum anthracnose and 56.2% on sorghum leopard pattern disease, and can improve the yield of sorghum. The Bacillus velezensis RHB-1 and secondary metabolites thereof can be used for preventing and treating sorghum leopard pattern disease and anthracnose and other main leaf diseases of sorghum, and provide high-quality resources for the preparation and development of biocontrol agents.

[0025] Biological preservation instructions:

[0026] The Bacillus velezensis RHB-1, Latin name Bacillus velezensis, is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2024586, a preservation date of March 29, 2024, and an address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The column chart of the prevention and control effect of the biocontrol agent of the present application;

[0028] Figure 2 The growth curve chart of the RHB-1 strain of the present application;

[0029] Figure 3 The related phenotype chart of the RHB-1 strain of the present application;

[0030] Figure 4 The phylogenetic tree of the RHB-1 strain of the present application based on 16S rRNA;

[0031] Figure 5 The transfer stability of the RHB-1 strain of the present application to different pathogenic bacteria;

[0032] Figure 6 The bacteriostatic effect of the volatile substances produced by the RHB-1 strain of the present application on different pathogenic bacteria;

[0033] Figure 7 The bacteriostatic effect of the intracellular substances (sterile precipitate) and extracellular substances (sterile supernatant) of the RHB-1 strain of the present application on different pathogenic bacteria;

[0034] Figure 8The bacteriostatic effect of the extracellular crude protein and the extracellular crude lipopeptide of the strain RHB-1 of the application on different pathogenic bacteria;

[0035] Figure 9 The pictures of the sorghum plot and the sorghum ear of the artificial spraying and the unmanned aerial vehicle spraying of the fermentation liquor of the RHB-1 of the application. DETAILED DESCRIPTION

[0036] The application will be further described below in conjunction with the examples, but the application is not limited to these examples. Without departing from the principles of the application, a number of variations and improvements can also be made, which should also be considered to belong to the protection scope of the application.

[0037] Example 1: Isolation and identification of the RHB-1 strain

[0038] 1.1 Isolation of the biocontrol strain

[0039] In June 2022, the sorghum was randomly collected from Changgang Town, Zunyi City, Guizhou Province, and the biocontrol strain was isolated by tissue separation method. Several 3mm x 3mm tissue blocks of sorghum leaves were cut with sterile scissors, soaked in 75% ethanol solution for 1min, and then soaked in sterile water for 30s, and the process was repeated for 3 times. The tissue blocks were placed in a culture dish containing absorbent paper, and then dried in a sterile operation table for 2h. After that, the tissue blocks were placed on PDA culture medium and cultured in a 25℃ constant temperature incubator for 48h. Then, the bacterial colonies around the leaves were picked up with an inoculation ring, and the process was repeated for 3 times to obtain single strains. The obtained strains were suspended in 80% glycerol and stored in a-20℃ refrigerator for standby.

[0040] 1.2 Screening of the biocontrol strain

[0041] The obtained strains were screened against Gloeocercospora sorghi and Colletotrichum sublineola preserved in the laboratory by the plate confrontation method. The pathogenic fungi were activated, and the mycelium blocks were attached to the PDA plate and then placed in a 25℃ incubator for dark culture for 4 days. The antagonistic strains were streaked on NA medium and cultured for 24h. Single colonies were picked up and placed in LB liquid medium, and then cultured at 28℃ with 180r shaking for 12h to prepare the bacterial liquid. A 6mm mycelium block was taken from the edge of the fresh pathogenic fungus and placed in the center of the PDA plate. Sterile filter paper discs with a diameter of 6mm were attached to the plate 3cm away from the mycelium block on both sides. 10-15μL of bacterial liquid was added to the filter paper discs using a pipette, and sterile water was used as a control. Each treatment was repeated three times. The colony diameters of the treatment group and the CK were measured when the colony diameter of the control group reached 2 / 3 of the diameter of the culture dish, and the inhibition rate was calculated.

[0042] Bacteriostatic rate (%) = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group x 100% Figure 1 The inhibition rates of some isolated strains (such as: RHB-1, GL8, GL7, XZD6-B1, XZD6-B2) are shown in Table 1, wherein RHB-1 has good inhibitory effect on both Colletotrichum gossypii and Gluconectria sorghi, so the RHB-1 strain is selected for subsequent experiments.

[0043] 1.3 Identification of RHB-1 strain

[0044] (1) Growth curve determination: the growth rate of Bacillus was determined by turbidimetry, and a single colony was inoculated in a 300 mL triangular flask containing 50 mL of LB liquid medium, and incubated at 28°C, 180 r·min -1 constant temperature shaker overnight, and inoculated into a 300 mL triangular flask containing 50 mL of LB liquid medium at a ratio of 1:100, and incubated at 28°C, 180 r·min -1 constant temperature shaker, and the OD value of the bacterial solution was measured every 2 h at a wavelength of 600 nm on a spectrophotometer, for a total of 36 h. The experiment was repeated 3 times, the average value was calculated and the growth curve was plotted, and the results are shown in Table 2. Figure 2 The growth rate of the strain is relatively fast, and it conforms to the growth rule of Bacillus vallismortis.

[0045] (2) Colony morphology observation: the strain was diluted and plated on NA medium, incubated at 28°C for 48 h, and the morphology of single colonies was observed and recorded. After overnight incubation at 30°C, the colony morphology was observed using a stereoscopic mirror, and the results are shown in Table 3. Figure 3 (A, B are the colony pictures after 10 5 times dilution of the bacterial solution, and C is the result of Gram staining) shows that the single colony of RHB-1 strain is milky white and opaque, initially round in shape with neat edges, then wrinkles appear on the colony, with a convex middle, and there is mucous secretion after the colony is picked up. The result of Gram staining shows that the spores are purple, indicating that it is a Gram-positive bacterium.

[0046] (3) Molecular biological identification of the strain

[0047] The specific steps of extracting Bacillus DNA refer to the operation instruction of bacterial genomic DNA extraction kit (Beijing Solabio Science and Technology Co., Ltd.). The genomic DNA was used as a template for PCR amplification of the genome of the strain using 16S rRNA gene universal primers. The length of the PCR amplification product was detected by 1% agarose gel electrophoresis, and the amplification product was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing in time. The sequences in the database were analyzed by BLAST website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for homology comparison, and then the model strain sequence was searched and downloaded in the GenBank database. The Maximum-Likelihood method of MEGAX software was used to construct a phylogenetic tree. The phylogenetic tree is shown in Figure 4 According to the comparison results, it can be concluded that RHB-1 is B. velezensis.

[0048] Example 2: Subculture stability of RHB-1 strain

[0049] The strain RHB-1 was subcultured on NA slant medium for 7 times, subcultured every 24 h, and cultured at 30°C for 24 h. The stability of its antibacterial activity against Colletotrichum gloeosporioides and C. sorgihii was measured by the plate confrontation method. The results are shown in Figure 5 (A is the antibacterial effect against C. gloeosporioides, and B is the antibacterial effect against C. sorgihii) shows that after 7 generations of transformation, the inhibition effect of the strain RHB-1 is still more than 70%, which has good stability.

[0050] Example 3: Inhibition effect of volatile substances of RHB-1 strain

[0051] Activated RHB-1 colonies were taken and evenly and densely streaked on NA plates, so that the bacteria covered the plates. Plant pathogenic bacteria discs (Φ = 6 mm) were inoculated in the center of PDA plates, and then paired with NA plates covered with RHB-1 bacteria. The surrounding was sealed with sealing film, and incubated at 28°C in the dark. The blank NA plate was used as a control, and the colony diameters of the treated and control plates were observed and measured when the mycelium of the control plate was full. The diameter of the pathogenic bacteria was measured by cross method, the inhibition rate was calculated, and the significance was analyzed by SPSS software. The results are shown in Figure 6 (A is the prevention effect column chart, B is the antibacterial effect against C. gloeosporioides, and C is the antibacterial effect against C. sorgihii) shows that the volatile substances produced by RHB-1 have a certain effect on the inhibition of C. sorgihii, and the inhibition effect on C. gloeosporioides is very significant.

[0052] Example 4: Inhibition effect of extracellular and intracellular secretions of RHB-1 strain

[0053] Preparation of fermentation broth of Bacillus velezensis RHB-1: (1) LB liquid medium was filled into a 250 mL capacity triangular flask with a gas permeable hole, 50 mL of LB liquid medium was added to each flask, and high temperature sterilization was carried out at 121 ℃ for 20 min; the preserved Bacillus velezensis RHB-1 strain was streak inoculated into a 9 cm diameter NA medium containing 15 mL, and was cultured at 25 ℃ in the dark for 24 h to obtain a single colony; (2) under sterile conditions, the single colony was inoculated into the high-temperature sterilized LB liquid medium with an inoculation loop (one single colony was inoculated into one triangular flask), and was cultured at 28 ℃ in a shaker at 180 r·min-1for 12 h to prepare a seed liquid; (3) the seed liquid was inoculated into new LB liquid medium at an inoculation amount of 2% (v / v) and was shaken for 24 h to prepare a fermentation broth. -1 Preparation of fermentation broth of Bacillus velezensis RHB-1: (1) LB liquid medium was filled into a 250 mL capacity triangular flask with a gas permeable hole, 50 mL of LB liquid medium was added to each flask, and high temperature sterilization was carried out at 121 ℃ for 20 min; the preserved Bacillus velezensis RHB-1 strain was streak inoculated into a 9 cm diameter NA medium containing 15 mL, and was cultured at 25 ℃ in the dark for 24 h to obtain a single colony; (2) under sterile conditions, the single colony was inoculated into the high-temperature sterilized LB liquid medium with an inoculation loop (one single colony was inoculated into one triangular flask), and was cultured at 28 ℃ in a shaker at 180 r·min-1for 12 h to prepare a seed liquid; (3) the seed liquid was inoculated into new LB liquid medium at an inoculation amount of 2% (v / v) and was shaken for 24 h to prepare a fermentation broth.

[0054] 4.1 Inhibitory test of intracellular substances

[0055] 100 mL of the fermentation broth prepared above was taken, 10000 r·min-1 -1 , 30 min centrifugation, the precipitated bacterial cells were taken, 10 mL of cell lysis solution was added, and the mixture was placed in a 4 ℃ refrigerator for 1 h. After taking out, the cells were broken by using an ultrasonic cell crusher for 15 min until the solution was clear, and then the mixture was centrifuged at 4 ℃ at 12000 rpm for 5 min in a high-speed refrigerated centrifuge. Then the supernatant intracellular substance was taken into another tube for use.

[0056] After passing through a 0.22 μm bacterial filter, the plate confrontation method was used to detect the bacteriostatic effect. The pathogenic fungi were activated, and the mycelial block was pasted on a PDA plate and placed in a 25 ℃ incubator for dark culture for 4 days for use. Fresh pathogenic fungi with a mycelial block of 6 mm in diameter were taken and placed in the center of a PDA plate, and a 8 mm diameter hole was punched on both sides of the plate 3 cm away from the mycelial block. 20 μL of the supernatant intracellular substance was taken by a pipette and added to the hole, and the cell lysis solution was used as a control. Each treatment was repeated three times, and the culture was incubated in a constant temperature incubator. When the colony diameter of the control group reached 2 / 3 of the diameter of the culture dish, the colony diameters of the treatment group and the CK were measured, and the bacteriostatic rate was calculated.

[0057] 4.2 Inhibitory test of extracellular substances

[0058] 100 mL of the fermentation broth prepared above was taken, 10000 r·min-1 -1 , 30 min centrifugation, the precipitated bacterial cells were taken, 10 mL of cell lysis solution was added, and the mixture was placed in a 4 ℃ refrigerator for 1 h. After taking out, the cells were broken by using an ultrasonic cell crusher for 15 min until the solution was clear, and then the mixture was centrifuged at 4 ℃ at 12000 rpm for 5 min in a high-speed refrigerated centrifuge. Then the supernatant intracellular substance was taken into another tube for use.

[0059] After passing through a 0.22 μm bacterial filter, 20 μL of the extracellular substance was taken and the plate confrontation method was used to detect the bacteriostatic effect. The blank LB medium was used as a control, and the bacteriostatic rate was calculated.

[0060] 4.3 Results

[0061] Results are shown in Table 1 and Figure 7 As shown in Table 1 and Table 2 (A is the antibacterial effect on C. sorghi, B is the antibacterial effect on C. sorghi), the extracellular substance and intracellular substance of RHB-1 strain have inhibitory effect on C. sorghi and C. sorghi, and the extracellular substance is better than the intracellular substance.

[0062] Table 1 Inhibitory effect of intracellular substance (sterile precipitate) and extracellular substance (sterile supernatant) of strain RHB-1 on different pathogenic bacteria

[0063]

[0064] Example 5: Inhibitory effect of extracellular crude protein and crude lipopeptide of RHB-1 strain

[0065] 5.1 Extraction of extracellular crude protein and antibacterial experiment

[0066] Ammonium sulfate fractionation precipitation method is the main method for protein separation and extraction. Take 100 mL of fermentation broth of strain RHB-1 (the fermentation broth is prepared according to the method of Example 4), centrifuge at 10,000 r·min -1 for 30 min, remove the bacteria and reserve the supernatant, then add ammonium sulfate powder (slowly add to completely dissolve and not to produce precipitation) to a saturation of 20%, and stand at 4°C overnight; centrifuge at 10,000 r·min -1 for 30 min, remove the supernatant, collect the precipitate, and dissolve the precipitate with 10 mL of PBS buffer, and filter sterilize through a 0.22 μm bacterial filter to obtain a crude protein solution. Take the fresh pathogenic fungi with a mycelium block of 6 mm in diameter and place it in the center of a PDA plate, punch a hole of 8 mm in diameter on both sides of the plate 3 cm away from the mycelium block, and use a pipette to suck 15 μL of crude protein solution and 15 μL of PBS buffer into the hole and mix well, with PBS buffer as a control, and repeat each treatment 3 times.

[0067] 5.2 Extraction of extracellular crude lipopeptide and antibacterial experiment

[0068] Acid precipitation method is used to extract lipopeptide substances. Take 100 mL of fermentation broth of strain RHB-1, centrifuge at 10,000 r·min -1 for 30 min, remove the bacteria and reserve the supernatant, then adjust the pH to 2.0 with 6 mol·L -1 HCl, and place it in a 4°C refrigerator overnight, centrifuge at 8,000 r·min -1 for 10 min, collect the precipitate, extract with 20 mL of methanol, adjust the pH to 7 with 1 mol·L -1 of sodium hydroxide, and stand at 4°C refrigerator for 8 h, then centrifuge at 8,000 r·min -1The supernatant was obtained by centrifugation for 10 minutes, and the supernatant was filtered to remove bacteria to obtain a crude ester peptide solution of RHB-1. The inhibition activity of the crude ester peptide solution was determined by using a plate confrontation method, 30 μL of the crude ester peptide solution was added to each well, and methanol was used as a control, and each treatment was repeated 3 times.

[0069] 5.3 Results

[0070] The results are shown in Table 2 and Figure 8 (A is the inhibition effect of crude protein on C. sorghi, B is the inhibition effect of crude ester peptide on C. sorghi, C is the inhibition effect of crude protein on C. sorghi, and D is the inhibition effect of crude ester peptide on C. sorghi) shows that the extracellular crude protein and extracellular crude ester peptide of RHB-1 strain have inhibition effect on C. sorghi and C. sorghi, and the crude protein is better than the crude ester peptide.

[0071] Table 2 Inhibition effect of extracellular crude protein and crude ester peptide of RHB-1 strain on different pathogenic bacteria

[0072]

[0073] Example 6: Field test of RHB-1 strain

[0074] The fermentation broth of the biocontrol bacteria RHB-1 strain was prepared by the same method as in Example 4.

[0075] The field experiment was carried out in the "Hongyangzi" sorghum planting field in Shishan Town, Zunyi City, Guizhou Province. The appropriate time was selected, and the sorghum was planted in a 15 m x 15 m plot. Two treatments and one control were set up, which were: A, the fermentation broth diluted with artificial spraying (the concentration after dilution was 10 8 cfu·mL -1 )( Figure 9 biocontrol strain RHB-1 (treatment)); B, the fermentation broth diluted by unmanned aerial vehicle spraying (the concentration after dilution was 10 8 cfu·mL -1 )( Figure 9 biocontrol demonstration field); C, water as a blank control. Each treatment was repeated 3 times. The spraying amount of each plot was 1 L. The disease index was investigated at the early stage of disease, and then the first spraying was carried out. About 4 weeks later, the treatment was sprayed once, and about 4 weeks later, the treatment was sprayed again, a total of 3 times, each time 1 L. The disease index was investigated 3 weeks after the last spraying. The five-point sampling method was used to investigate the leaves, 12 plants were investigated at each point, and 6 leaves were investigated from top to bottom for each plant. The disease classification and data recording were carried out according to the method guideline (GB / T 17980.112-2004), and the control effect was calculated. The yield was measured by the lengthened meter method.

[0076] Disease classification method:

[0077] Each cell randomly take five points survey, each point take 5 strains survey all leaves, according to the following classification method, with leaf unit investigation. Corn big and small lesion classification method: 0 level: no disease; 1 level: lesion area accounts for 5% or less of leaf area; 3 level: lesion area accounts for 6% to 10% of leaf area; 5 level: lesion area accounts for 11% to 25% of leaf area; 7 level: lesion area accounts for 26% to 50% of leaf area; 9 level: lesion area accounts for 51% or more of leaf area.

[0078] Control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group x 100%

[0079] The results are shown in Tables 3, 4, 5 and Figure 9 The RHB-1 strain fermentation liquor has a control effect of more than 50% on the highland brome leopard and the highland brome anthracnose, and the hair weight yield is increased by more than 25%.

[0080] Table 3 Control effect of biocontrol bacteria RHB-1 on highland brome leopard

[0081]

[0082] Table 4 Control effect of biocontrol bacteria RHB-1 on highland brome anthracnose

[0083]

[0084] Table 5 Effect of biocontrol bacteria RHB-1 on yield

[0085]

[0086] The reagents, kits and instruments used in the above examples are commercially available; the conventional techniques and protocols not described in detail in the above examples are well known in the art, and therefore will not be described in detail here. The above examples and / or experimental examples describe preferred embodiments of the present application, however, the present application is not limited to the specific details in the above embodiments, within the technical concept range of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A Bacillus velezensis (B. velezensis) strain, characterized in that, Bacillus velezensis The strain number is RHB-1, and the preservation number is CCTCC NO: M 2024586, which was preserved in the China Center for Type Culture Collection on March 29, 2024, and the address is No. 299, Bajiyi Road, Wuchang District, Wuhan City, Hubei Province. ​ 2. A biocontrol agent, characterized in that, The active ingredient of the biocontrol agent includes Bacillus velezensis RHB-1 as claimed in claim 1, or a fermentation broth of the Bacillus velezensis RHB-1, or a supernatant of the fermentation broth of the Bacillus velezensis RHB-1.

3. A method for preparing a biocontrol agent, characterized by, The method comprises the following steps: (1) preparing a sterile LB liquid medium; (2) adding Bacillus velezensis RHB-1 as claimed in claim 1 to the LB liquid medium to prepare a seed liquid; (3) after 12 h, adding the seed liquid to a new sterile LB liquid medium for shaking culture to obtain the biocontrol agent.

4. The preparation method of the biocontrol agent according to claim 3, characterized in that, The method comprises the following steps: (1) filling LB liquid medium into a 250 mL capacity triangular flask with air holes, adding 50 mL of LB liquid medium to each flask, and sterilizing the LB liquid medium at 121 ℃ for 20 min to obtain sterile LB liquid medium; inoculating Bacillus velezensis RHB-1 into a 9 cm diameter NA medium containing 15 mL, and culturing at 25 ℃ in the dark for 24 h to obtain a single colony; (2) under sterile conditions, using a inoculation ring to inoculate the single colony into the sterile LB liquid medium, and shaking culture at 28 ℃ in a shaking table at 180 r·min-1 for 12 h to obtain a seed liquid; (3) inoculating the seed liquid into a new sterile LB liquid medium at an inoculation amount of 2% (v / v) for shaking culture for 24 h to obtain the biocontrol agent.

5. The use of a biocontrol agent in inhibiting the growth of Cercospora kikuchii (Kawamura) Sacc. et Sydow Gloeocercospora sorghi ) or / and Colletotrichum gossypii (Pass.) Wold. et Cunningh. Colletotrichum sublineola ), characterized in that, The active ingredient of the biocontrol agent includes Bacillus velezensis RHB-1 as claimed in claim 1, or a fermentation broth of the Bacillus velezensis RHB-1, or a supernatant of the fermentation broth of the Bacillus velezensis RHB-1.

6. The use of a biocontrol agent in the control of Bipolaris sorokiniana or / and Colletotrichum truncatum, characterized in that, The active ingredient of the biocontrol agent includes Bacillus velezensis RHB-1 as claimed in claim 1, or a fermentation broth of the Bacillus velezensis RHB-1, or a supernatant of the fermentation broth of the Bacillus velezensis RHB-1.

7. Use according to claim 6, wherein the compound is ###0003### The biocontrol agent is sprayed onto the leaves of sorghum or the plants of sorghum by artificial spraying or unmanned aerial vehicle spraying to prevent and treat sorghum tiger stripe disease or / and sorghum anthracnose.

8. Use according to claim 7, wherein the compound is ###0002### The biocontrol agent is the fermentation broth of Bacillus velezensis RHB-1, and the concentration of the spraying is 10 8 cfu·mL -1 .

9. Use according to claim 8, characterized in that, The spraying amount is 3 L per mu each time.

10. Use according to claim 9, wherein the compound is ###0002### The spraying frequency is three times, and the spraying time is the first spraying at the early stage of disease, the second spraying after 4 weeks, and the third spraying after another 4 weeks.

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