Biocontrol bacterium capable of efficiently antagonizing valsa lilii bulb and application of biocontrol bacterium

Abstract

The invention relates to a biocontrol bacterium capable of efficiently antagonizing valsa ceratosperma lilii and application of the biocontrol bacterium. The biocontrol bacterium comprises bacillus velezensis capable of inhibiting pathogenic bacteria of bulbus lilii. The bacillus velezensis is a bacillus velezensis R12 strain, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M 20241785. The bacillus velezensis R12 strain can be used for preventing and treating the lily bulb rot disease, and experimental results show that thalli of the bacillus velezensis R12 strain and fermentation liquor of the bacillus velezensis R12 strain have a relatively strong inhibition effect on fusarium and penicillium, have a relatively good antagonistic effect and can be used for effectively inhibiting the growth of common pathogenic bacteria of lilies.

Description

Technical Field

[0001] The present application relates to the technical field of lily bulb rot prevention and control, and in particular to a biocontrol bacterium that is highly effective in antagonizing lily bulb rot pathogens and an application thereof. Background Art

[0002] Lily (Lilium brownii) is a medicinal and ornamental plant, and its bulbs have the effects of moistening the lungs and relieving coughs, and calming the mind and nerves. This plant is grown all over the world. With the continuous expansion of lily planting areas, the disease problem is becoming increasingly serious. At the same time, long-term continuous cropping causes a large number of plants to dry up and die, and underground bulbs to rot, resulting in a significant drop in yield and quality, which seriously affects the production and planting benefits of lilies. Lily bulb rot, also known as stem rot, is an important disease that causes continuous cropping problems, causing varying degrees of loss in yield and quality. Fusarium and Penicillium are among its main pathogens.

[0003] At present, the main method for preventing and controlling lily bulb rot is chemical control. This method is quick to take effect, but it has problems such as environmental pollution and the possibility of drug resistance of lily bulb rot. In order to overcome the defects of chemical methods, biological control methods can be used as an alternative. Biological control is an environmentally friendly and pollution-free measure. The microorganisms used to prevent and control diseases mainly include fungi, bacteria and actinomycetes, among which bacteria are the most widely used. Bacillus sp. is an important type of biocontrol bacteria, which has a good biocontrol effect on a variety of pathogenic fungi. For example, the inhibition rate of Bacillus amyloliquefaciens LZN01 on Fusarium oxysporum watermelon-specific type is 57.1%; Bacillus cereus MH778713 can effectively inhibit Fusarium, the pathogen of tomato bulb rot, reducing the incidence of tomato bulb rot from 96% to 12%, and promoting the growth of tomatoes; Fengycin produced by Bacillus atrophaeus NX-12 can effectively inhibit the spore germination of Fusarium cucumber-specific type and cause cell wall damage. Different biocontrol bacteria have differences in antagonistic range, so it is necessary to screen the corresponding biocontrol bacteria according to the specific pathogenic bacteria. However, there is no development and application of biocontrol bacteria for the pathogen of lily bulb rot.

[0004] It can be seen that bacterial preparations with low cost, fast reproduction and no pollution have become important biological control measures for antagonizing lily bulb rot pathogens at home and abroad. In view of the harm of lily bulb rot pathogens, screening strains with better and more stable antagonistic effects and preparing microbial preparations are crucial to the healthy development of the lily industry. Summary of the invention

[0005] The object of the present invention is to provide a biocontrol bacterium that is highly effective in antagonizing lily bulb rot pathogens and its application, which can effectively inhibit lily bulb rot pathogens, such as Fusarium and Penicillium. The strain can be applied by direct root irrigation of the bacteria or root irrigation of fermentation liquid.

[0006] The first technical solution adopted by the present invention is: a biocontrol bacterium that is highly effective in antagonizing lily bulb rot pathogens, wherein the biocontrol bacterium is a Bacillus velezensis that can inhibit the lily bulb rot pathogens; the Bacillus velezensis is a Bacillus velezensis R12 strain, and the strain collection number is CCTCC NO: M 20241785.

[0007]

[0008] Furthermore, the pathogens of lily bulb rot include Fusarium and Penicillium.

[0009] The second technical solution adopted by the present invention is: use of a biocontrol bacterium that is highly effective in antagonizing lily bulb rot pathogens as described in the first technical solution in preventing and controlling lily bulb rot disease.

[0010] Furthermore, the Bacillus velezensis R12 strain cell or the fermentation liquid of the Bacillus velezensis R12 strain is used to prepare a control preparation for controlling lily bulb rot.

[0011] Furthermore, the method of using the Bacillus velezensis R12 bacteria to prepare a control agent for controlling lily bulb rot is as follows:

[0012] S1: The activated Bacillus velezensis R12 strain was inoculated into beef extract peptone medium, and cultured at 37°C and 200 rpm for 24 h to obtain a fermentation broth;

[0013] S2: Use sterile physiological saline to dilute the bacterial suspension to a concentration of OD600 = 0.8, and apply it to the soil containing the pathogenic bacteria of lily bulb rot at a weight ratio of 0.5-1.5%.

[0014] Furthermore, the method of using the fermentation liquid of the Bacillus velezensis R12 strain to prepare a control preparation for the control of lily bulb rot disease is as follows: fermenting the Bacillus velezensis R12 strain in a culture medium containing 4g / L CaCl2, 12g / L corn flour and 15g / L soybean meal for 36 hours, removing the bacteria by centrifugation, filtering and sterilizing, and applying the filtrate to soil containing lily bulb rot pathogens at a weight ratio of 0.3-1%.

[0015] The beneficial effects of the present invention are as follows: the Bacillus velezensis R12 strain provided by the present invention has a wide spectrum of action, can effectively antagonize Fusarium and Penicillium, two major pathogens causing lily bulb rot, and has an inhibition rate of 59.62%, 55.3% and 54.39% for Fusarium foetens L1, Fusarium oxysporum and Penicillium janthinellum F7, respectively; the Bacillus velezensis R12 bacterial agent is environmentally friendly and safe, and can replace the use of chemical agents. The operation method is simple, and the Bacillus velezensis R12 bacterial body or the fermentation liquid of the Bacillus velezensis R12 strain can be used for root irrigation to antagonize the growth of the main pathogens of lily bulb rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 These are diagrams showing the antagonistic effect of the Bacillus velezensis R12 strain described in the embodiments of the present invention on three lily bulb rot pathogens, wherein (a) is a diagram showing the antagonistic effect of Bacillus velezensis R12 bacteria on Fusarium foetens L1 bacteria, (b) is a diagram showing the antagonistic effect of Bacillus velezensis R12 bacteria on Fusarium oxysporum bacteria, and (c) is a diagram showing the antagonistic effect of Bacillus velezensis R12 bacteria on Penicillium janthinellum F7 bacteria.

[0018] Figure 2 This is a colony characteristic diagram of the Bacillus velezensis R12 strain described in the embodiment of the present invention;

[0019] Figure 3 is a 16S rDNA phylogenetic tree of the Bacillus velezensis R12 strain described in the examples of the present invention;

[0020] Figure 4 The effect of carbon sources on the antibacterial effect of the Bacillus velezensis R12 strain described in the examples of the present invention;

[0021] Figure 5 The effect of nitrogen source on the antibacterial effect of Bacillus velezensis R12 strain described in the embodiment of the present invention;

[0022] Figure 6 The effect of inorganic salts on the antibacterial effect of the Bacillus velezensis R12 strain described in the embodiment of the present invention;

[0023] Figure 7 The diagrams are of the antibacterial effect of the fermentation filtrate of the Bacillus velezensis R12 strain described in the embodiments of the present invention on pathogenic bacteria, wherein (a) is the experimental result diagram of the control group CK, (b) is the antibacterial effect diagram of the A1B3C3D3 experimental group, and (c) is the antibacterial effect diagram of the A1B2C3D3 experimental group;

[0024] Figure 8 This is a diagram showing the inhibitory effect of Bacillus velezensis R12 bacteria on pathogenic bacteria in the soil as described in an embodiment of the present invention;

[0025] Fig. 9 This is a diagram showing the inhibitory effect of the fermentation filtrate of the Bacillus velezensis R12 strain described in an embodiment of the present invention on pathogenic bacteria in the soil. DETAILED DESCRIPTION

[0026] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and therefore, the present invention is not limited to the limitations of the specific embodiments disclosed below.

[0027] The embodiment of the present invention provides a biocontrol bacterium that is highly effective in antagonizing lily bulb rot pathogens. The biocontrol bacterium is a Bacillus velezensis that can inhibit the pathogens of lily bulb rot; the Bacillus velezensis is a Bacillus velezensis R12 strain, and the strain collection number is CCTCC NO: M 20241785. It was deposited in the China Center for Type Culture Collection on August 14, 2024, and the address of the depository unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0028] The Bacillus velezensis R12 strain, hereinafter referred to as the R12 strain, was isolated from the rhizosphere soil of healthy lilies in Wanzai, Jiangxi Province. The specific screening and identification process is as follows:

[0029] Example 1: Screening and identification of R12 strain

[0030] 1. Screening of strains

[0031] (1) Strain isolation and purification

[0032] Lily rhizosphere soil was collected from the lily planting base in Baishui Township, Wanzai, Yichun, Jiangxi Province, and the biocontrol bacteria were isolated by gradient dilution method. The specific method is as follows: First, the rhizosphere soil was sieved, 5.0g of soil was weighed and placed in a triangular flask containing 45mL of sterile water. Oscillate at 28℃ and 150r / min for 30min to mix thoroughly. Then, according to 10 -1 ~10 -6 Perform gradient dilution. Take 100 μL of soil dilutions from each gradient and spread them on beef extract peptone medium plates. The beef extract peptone medium includes 10 g of peptone, 5 g of yeast powder, 10 g of NaCl and 18 g of agar powder. Then, culture at 30°C for 12 to 36 hours. Pick strains with different colony morphology, purify the strains by plate streaking method, preserve them in glycerol, and then store them at -80°C.

[0033] To activate a single colony, place it in liquid beef extract peptone medium and culture it at 37°C and 200 r / min for 24 hours to obtain a bacterial solution of the strain for later use.

[0034] (2) Screening of biocontrol bacteria

[0035] The single colony bacteria isolated in the above process were screened for antibacterial activity using the plate confrontation method. Three lily bulb rot pathogens, Fusarium foetens L1 and Fusarium oxysporum, and Penicillium janthinellum F7, were selected as test strains. Plates without bacteria were used as controls. The strains were cultured at 28°C for 4 to 7 days, and the strains that produced inhibition zones against the three tested pathogens were selected, and the size of the inhibition zones was measured. Among them, Fusarium oxysporum was purchased from the China Microbiological Culture Center, and Fusarium foetensL1 and Penicillium janthinellum F7 were isolated and obtained in the laboratory. By comparing the size of the inhibition zones, a biocontrol bacterium with a strong inhibitory effect on the three tested pathogens was screened and numbered R12.

[0036] Repeat the antagonism experiment of R12 strain against three test pathogens to complete the rescreening and obtain the following Figure 1 And the experimental results are shown in Table 1. Figure 1As shown, the R12 strain can produce obvious inhibition zones against the three pathogens. As shown in Table 1, the inhibition diameters of the R12 strain against Fusarium foetens L1, Fusarium oxysporum and Penicillium janthinellum F7 are 2.96 mm, 2.61 mm and 2.84 mm, respectively.

[0037] Table 1 Inhibitory effect of strain R12 on three pathogens

[0038]

[0039] 2. Identification of R12 strain

[0040] (1) Morphological identification

[0041] The R12 strain was inoculated on a liquid beef extract peptone medium plate and cultured at 37°C for 12 hours. The characteristics of the colonies were observed and morphological identification was performed in combination with the Bergey Manual of Bacterial Identification. Figure 2 As shown, the colony surface is rough, dry, wrinkled, with irregular edges, and Gram staining is positive.

[0042] (2) Physiological and biochemical identification

[0043] Physiological and biochemical tests were performed on the R12 strain according to the methods in the Manual of Identification of Common Bacteria Systems. The results are shown in Table 2. R12 is a Gram-positive bacterium, and the starch hydrolysis, gelatin hydrolysis, methyl red, oxidase reaction, catalase, and indole reaction tests were all positive, while the nitrate reduction, VP reaction, and H2S production tests were negative.

[0044] Table 2 Analysis of physiological and biochemical reaction characteristics of R12 strain

[0045] Physiological and biochemical experiments result Gram's stain test + Starch hydrolysis + Gelatin hydrolysis + Methyl red test + Oxidase reaction + Hydrogen peroxidase reaction + Indole reaction + Nitrate utilization - VP reaction - <![CDATA[H2S产生H2S production]]> -

[0046] Among them: "+" represents positive, and "-" represents negative.

[0047] (3) Molecular biological identification of R12 strain

[0048]

[0049] The sequence was compared with the NCBI database by BLAST homology comparison, and it was found that the R12 strain had the highest homology with some strains of Bacillus velezensis. The phylogenetic tree was constructed using the neighbor-linking method in MEGA 7.0 software. The results are as follows Figure 3 As shown, the R12 strain clustered with Bacillus velezensis CBMB205.

[0050] Example 2: Optimization of fermentation conditions of R12 strain

[0051] (1) Preparation of R12 strain seed solution

[0052] Pick a single colony of the R12 strain, inoculate it into liquid beef extract peptone medium, and shake culture it at 37°C and 200r / min for 12h to obtain seed liquid for single-factor experiments and orthogonal optimization experiments. The mycelium growth rate method was used to optimize the conditions, and the Fusarium foetens L1 strain was selected as the test strain. The specific method is to mix 1mL of the fermentation filtrate under each condition with 19mL of PDA medium at about 50°C, and then pour it into the plate. After the plate solidifies, a Fusarium foetens L1 cake is inoculated in the center of the plate, with one side of the mycelium attached to the plate. The plate mixed with water and PDA medium was used as a control, and each treatment was repeated 3 times. Measure the growth diameter of the test bacteria and calculate the inhibition rate:

[0053]

[0054] (2) Single factor test

[0055] Screening of carbon source types: The carbon source in beef extract peptone medium was replaced with equal amounts of corn flour, soluble starch, and mannitol, while other fermentation conditions remained unchanged. The best carbon source was screened using the mycelium growth rate method. Figure 4 As shown in the figure, when corn flour is used as the carbon source, the inhibition rate is the highest, reaching 74.8%. Therefore, corn flour was selected as the carbon source for the fermentation medium of R12 strain.

[0056] Screening of nitrogen source types: The nitrogen source in beef extract peptone medium was replaced by equal amounts of ammonium sulfate, soybean meal, and soybean powder, respectively, while other fermentation conditions remained unchanged. The best nitrogen source was screened by the mycelium growth rate method. Figure 5 As shown in the figure, when soybean meal is used as nitrogen source, the inhibition rate is the highest, reaching 78.4%. Therefore, soybean meal was selected as the nitrogen source for the fermentation medium of R12 strain.

[0057] Screening of inorganic salts: Use equal amounts of CaCl2, MgSO4, and KCl to replace the inorganic salts in beef extract peptone medium, keep other fermentation conditions unchanged, and fermentation is carried out. The mycelium growth rate method is used to screen out the best inorganic salt. The experimental results are as follows Figure 6 As shown in the figure, when CaCl2 is used as inorganic salt, the inhibition rate is the highest, reaching 74.3%. Therefore, CaCl2 is selected as the inorganic salt of the fermentation medium of R12 strain.

[0058] (3) Orthogonal test of fermentation medium

[0059] The selected CaCl2 (A: 4g / L, 8g / L, 12g / L), corn flour (B: 8g / L, 12g / L, 16g / L), soybean meal (C: 5g / L, 10g / L, 15g / L) were used as inorganic salt, carbon source and nitrogen source respectively, and the three factors A, B, C and fermentation time (D: 12h, 24h, 36h) were adjusted according to L9 (3 4 ) Design the factors and levels of the orthogonal experiment as shown in Table 3. Each treatment was cultured at 37°C and 200 r / min for 24 h, and the fermentation broth was collected by centrifugation and sterilized by filtration. According to the mycelium growth rate method, the antibacterial rate of each treatment was checked to screen out the best culture medium formula.

[0060] Table 3 Orthogonal primers and levels

[0061]

[0062] As shown in Table 4, the order of factors affecting the inhibition rate is fermentation time>carbon source>nitrogen source>inorganic salt, and the optimal inhibition conditions are: D3>B2>C3>A1. However, this result is inconsistent with the results of the third group A1B3C3D3 experimental group with the highest inhibition rate in the experiment. The inhibition rates of the A1B3C3D3 experimental group and the A1B2C3D3 experimental group need to be tested again. The results of the verification test showed that the fermentation filtrates of the R12 strain in the A1B3C3D3 experimental group and the A1B2C3D3 experimental group could effectively inhibit the growth of the Fusarium foetens L1 strain. Figure 7 As shown, the inhibition rates of the A1B3C3D3 experimental group and the A1B2C3D3 experimental group were 81.7% and 84.8%, respectively, that is, the A1B2C3D3 experimental group was superior to the A1B3C3D3 experimental group. Therefore, the A1B2C3D3 experimental group was selected as the optimal fermentation condition, i.e., 4g / L CaCl2, 12g / L corn flour and 15g / L soybean meal, and fermented for 36h. The inhibition rates of the fermentation filtrate of this combination on the Fusariumoxysporum strain and the Penicillium janthinellum F7 strain were 78.5% and 83.6%, respectively.

[0063] Table 4 Orthogonal test results

[0064]

[0065]

[0066] Example 3: Antibacterial effect of strain R12 on lily bulb rot pathogen in soil

[0067] (1) Inhibitory effect of R12 strain on lily bulb rot pathogen in soil

[0068] Preparation of R12 strain seed liquid: The activated R12 strain was inoculated into beef extract peptone medium, and cultured at 37°C and 200 r / min for 24 h with shaking to obtain fermentation liquid, which was diluted with sterile saline to OD600 = 0.8 and set aside.

[0069] After sterilization, the soil was mixed with spores of Fusarium foetens L1, Fusarium oxysporum and Penicillium janthinellum F7 respectively, and the concentration of pathogens was adjusted to 1×10 7 CFU / g soil. The bacterial suspension of R12 strain was applied to the soil containing lily bulb rot pathogen at a weight ratio of 0.5-1.5%, and the soil without bacterial suspension was used as the control group. The number of three pathogens was detected respectively, and the inhibition rate of R12 bacteria on the three pathogens was calculated.

[0070] like Figure 8 As shown, the R12 strain reduced the spore counts of Fusarium foetens L1, Fusarium oxysporum and Penicillium janthinellum F7, and the inhibition rates of the three pathogens were 82.4%, 76.8% and 63.5%, respectively, indicating that the R12 strain has the effect of inhibiting and killing pathogens in the soil environment.

[0071] (2) Inhibitory effect of R12 fermentation filtrate on lily bulb rot pathogen in soil

[0072] Preparation of R12 strain fermentation filtrate: According to the optimized fermentation medium formula in Example 2, i.e. 4g / L CaCl2, 12g / L corn flour and 15g / L soybean meal, ferment for 36 hours, centrifuge to remove the bacteria, and filter to sterilize. The addition of lily bulb rot bacteria is prepared according to (1) in this Example 3. The R12 strain fermentation filtrate is applied to the soil containing lily bulb rot bacteria at a weight ratio of 0.3-1%. The soil without the addition of the R12 strain fermentation filtrate is used as the control group, and the number of the three pathogens is detected respectively, and the inhibition rate of the R12 strain fermentation filtrate on the three pathogens is calculated.

[0073] The fermentation filtrate of R12 strain reduced the spore counts of Fusarium foetens L1, Fusarium oxysporum and Penicillium janthinellum F7. Fig. 9 As shown, the inhibition rates of R12 against the three pathogenic bacteria were 87.5%, 74.5% and 77.3%, respectively, indicating that the fermentation filtrate of R12 strain has the effect of inhibiting and killing pathogens in the soil environment.

[0074] In summary, the R12 strain and its fermentation liquid have a strong inhibitory effect on the three lily bulb rot pathogens. The best conditions for the antagonistic effect of the R12 strain fermentation filtrate on pathogens were screened through single factor experiments and orthogonal optimization experiments. The strain culture method is simple and the antagonistic effect is good. It can effectively inhibit the growth of common lily pathogens. In particular, the R12 strain fermentation filtrate has the potential to be developed into an efficient preparation for the prevention and control of lily bulb rot pathogens.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A biocontrol bacterium that is highly effective in antagonizing lily bulb rot pathogens, characterized in that: The biocontrol bacteria is a Bacillus velezensis capable of inhibiting the pathogen of lily bulb rot; the Bacillus velezensis is a Bacillus velezensis R12 strain, and the strain collection number is CCTCC NO: M 20241785.

2. The biocontrol bacterium for effectively antagonizing lily bulb rot pathogen according to claim 1, characterized in that: The pathogens of lily bulb rot include Fusarium and Penicillium.

3. Use of the biocontrol bacterium highly effective in antagonizing lily bulb rot pathogens as claimed in claim 1 in preventing and controlling lily bulb rot disease.

4. The use according to claim 3, characterized in that: The Bacillus velezensis R12 strain cell or the fermentation liquid of the Bacillus velezensis R12 strain is used to prepare a control preparation for controlling lily bulb rot.

5. The use according to claim 4, characterized in that: The method of using the Bacillus velezensis R12 bacteria to prepare a control agent for controlling lily bulb rot is as follows: S1: The activated Bacillus velezensis R12 strain was inoculated into beef extract peptone medium, and cultured at 37°C and 200 r / min for 24 h to obtain a fermentation broth; S2: Dilute with sterile saline to a concentration of OD 600 =0.8 bacterial suspension is applied to the soil containing the pathogen of lily bulb rot at a weight ratio of 0.5~1.5%.

6. The use according to claim 4, characterized in that: The method of using the fermentation liquid of the Bacillus velezensis R12 strain to prepare a control preparation for controlling lily bulb rot disease is as follows: fermenting the Bacillus velezensis R12 strain in a culture medium containing 4 g / L CaCl2, 12 g / L corn flour and 15 g / L soybean meal for 36 hours, removing the bacteria by centrifugation, filtering and sterilizing, and applying the filtrate to soil containing lily bulb rot pathogens at a weight ratio of 0.3-1%.

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