A high-efficiency biocontrol bacterium against lilium squama rot fungus and application thereof

Abstract

The application relates to a high-efficiency biocontrol bacterium for antagonizing lily bulb rot fungus and application thereof, which comprises a Bacillus velezensis capable of inhibiting the lily bulb rot pathogen; the Bacillus velezensis is a Bacillus velezensis R12 strain, and the strain preservation number is CCTCC NO: M 20241785. The Bacillus velezensis R12 strain can prevent and treat lily bulb rot, and experimental results show that the bacterial body and fermentation liquor of the Bacillus velezensis R12 strain have strong inhibiting effects on fusarium and penicillium, have good antagonistic effects, and can effectively inhibit the growth of common pathogenic bacteria of lilies.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lily bulb rot disease prevention and treatment, in particular to a biocontrol bacterium highly antagonizing lily bulb rot pathogen and application thereof. BACKGROUND

[0002] Lilium brownii is a medicinal and ornamental plant, and its bulb has the effects of moistening the lung to relieve cough and soothing the heart to tranquilize the mind. The plant is planted all over the world, and as the planting area of lily is continuously expanding, the disease problem is becoming increasingly serious. At the same time, continuous cropping for many years leads to a large number of plants withering and death and underground bulb rot, resulting in a substantial decline in yield and quality, which seriously affects the production and planting benefits of lily. Lily bulb rot, also known as stem rot, is an important disease causing continuous cropping obstacles, causing different degrees of loss to yield and quality. Fusarium and Penicillium are one of the main pathogenic bacteria.

[0003] At present, the main method to prevent and control lily bulb rot pathogen is chemical control. This method has a quick effect, but has problems such as environmental pollution and possible development of drug resistance of lily bulb rot pathogen. In order to overcome the defects of chemical methods, biological control methods can be used instead. Biological control is an environmentally friendly and pollution-free measure. Microorganisms used for disease control mainly include fungi, bacteria and actinomycetes, among which the application of bacteria is the most extensive. Bacillus sp. is an important biocontrol bacterium, which has good biocontrol effect on many pathogenic fungi. For example, Bacillus amyloliquefaciens LZN01 has an inhibition rate of 57.1% on Fusarium oxysporum f. sp. cucumeris; Bacillus cereus MH778713 can efficiently inhibit Fusarium, the pathogenic bacterium of tomato bulb rot, reducing the incidence of tomato bulb rot from 96% to 12%, and promoting the growth of tomato; Bacillus atrophaeus NX-12 produces fengycin, which can efficiently inhibit the spore germination of Fusarium cucumber special form and cause cell wall damage. Different biocontrol bacteria have differences in antagonistic range, so it is necessary to select the corresponding biocontrol bacteria according to the specific pathogenic bacteria. However, there is no development and application of biocontrol bacteria against lily bulb rot pathogen.

[0004] Therefore, bacterial preparations with the characteristics of low cost, rapid propagation and no pollution have become an important biological control measure for antagonizing lily bulb rot pathogen at home and abroad. In view of the harm of lily bulb rot pathogen, it is of great importance to the healthy development of lily industry to screen strains with better and more stable antagonistic effect and to prepare microbial preparations. SUMMARY

[0005] The application aims to provide a high-efficiency antagonistic lily bulb rot pathogenic bacteria and application thereof, which can efficiently inhibit lily bulb rot pathogenic bacteria such as fusarium and penicillium, and the strain can be applied by directly pouring root with the bacteria or pouring root with fermentation liquor.

[0006] The first technical solution adopted by the application is a high-efficiency antagonistic lily bulb rot pathogenic bacteria, which is a bacillus velezensis capable of inhibiting lily bulb rot pathogenic bacteria.

[0007]

[0008] Further, the lily bulb rot pathogen includes fusarium and penicillium.

[0009] The second technical solution adopted by the present application is: the application of the high-efficiency antagonistic lily bulb rot pathogen bacteria in the prevention and treatment of lily bulb rot.

[0010] Further, the Bacillus velezensis R12 strain is used to prepare a prevention and treatment preparation, and the lily bulb rot is prevented and treated.

[0011] Further, the method for preventing and treating the lily bulb rot by using the Bacillus velezensis R12 strain to prepare a prevention and treatment preparation is:

[0012] S1: inoculate the activated Bacillus velezensis R12 strain into a beef extract peptone culture medium, and culture at 37°C and 200r / min for 24h to obtain a fermentation liquor;

[0013] S2: dilute the Bacillus velezensis R12 strain into a bacterial suspension with a concentration of OD600=0.8 using sterile normal saline, and apply the bacterial suspension to the soil containing the lily bulb rot pathogen according to a weight ratio of 0.5-1.5%.

[0014] Further, the method for preventing and treating the lily bulb rot by using the Bacillus velezensis R12 strain to prepare a prevention and treatment preparation is: ferment the Bacillus velezensis R12 strain in a culture medium containing 4g / L CaCl2, 12g / L corn powder and 15g / L soybean meal for 36h, remove the bacterial bodies by centrifugation, filter the bacteria, and apply the filtrate to the soil containing the lily bulb rot pathogen according to a weight ratio of 0.3-1%.

[0015] The Bacillus velezensis R12 strain provided by the application has a wide action spectrum, can efficiently antagonize the two main pathogenic bacteria of lily bulb rot disease, Fusarium and Penicillium, and the inhibition rates of the Bacillus velezensis R12 strain on Fusarium foetens L1, Fusarium oxysporum and Penicillium janthinellum F7 are 59.62%, 55.3% and 54.39% 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 strain or the Bacillus velezensis R12 strain fermentation liquor can be used for root irrigation to antagonize the growth of the main pathogenic bacteria of lily bulb rot disease. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] Figure 1 The figure is the antagonistic effect of the Bacillus velezensis R12 strain on three lily bulb rot pathogens, wherein (a) is the antagonistic effect of the Bacillus velezensis R12 strain on Fusarium foetens L1, (b) is the antagonistic effect of the Bacillus velezensis R12 strain on Fusarium oxysporum, and (c) is the antagonistic effect of the Bacillus velezensis R12 strain on Penicillium janthinellum F7.

[0018] Figure 2 The figure is the colony characteristics of the Bacillus velezensis R12 strain;

[0019] Figure 3 The figure is the 16S rDNA phylogenetic tree of the Bacillus velezensis R12 strain;

[0020] Figure 4 The figure is the influence of carbon source on the antibacterial effect of the Bacillus velezensis R12 strain.

[0021] Figure 5 Effects of nitrogen sources on the antibacterial effect of the Bacillus velezensis R12 strain described in the embodiments of the present application;

[0022] Figure 6 Effects of inorganic salts on the antibacterial effect of the Bacillus velezensis R12 strain described in the embodiments of the present application;

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

[0024] Figure 8 Figures of the inhibitory effect of the Bacillus velezensis R12 strain described in the embodiments of the present application on pathogenic bacteria in soil;

[0025] Figure 9 Figures of the inhibitory effect of the fermentation filtrate of the Bacillus velezensis R12 strain described in the embodiments of the present application on pathogenic bacteria in soil. DETAILED DESCRIPTION

[0026] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the present application will be further described in detail below in combination with the drawings and specific embodiments. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, and therefore, the present application is not limited to the specific embodiments disclosed below.

[0027] The embodiments of the present application provide a biocontrol bacterium highly antagonizing Lily Bulb Rot Pathogen, which is a Bacillus velezensis capable of inhibiting Lily Bulb Rot Pathogen; the Bacillus velezensis is a Bacillus velezensis R12 strain, the strain preservation number is CCTCC NO: M 20241785, and it was preserved in the China Center for Type Culture Collection on August 14, 2024, and the address of the preservation unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0028] The Bacillus velezensis R12 strain, hereinafter referred to as R12 strain, is isolated from the rhizosphere soil of healthy lilies in Wanzai, Jiangxi, and 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) Isolation and purification of strains

[0032] Rhizosphere soil of lily was collected from Baihui planting base in Wanzai, Yichun, Jiangxi. The biocontrol bacteria were isolated by gradient dilution method. Specifically, 5.0 g of the soil was weighed into a triangular flask containing 45 mL of sterile water after the soil was sieved. The mixture was shaken at 28°C and 150 r / min for 30 min to mix thoroughly. Then, gradient dilution was performed according to 10 -1 ~ 10 -6 Each 100 μL of the soil dilution liquid at each gradient was taken and spread on a beef extract peptone medium plate. The beef extract peptone medium included 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, and 18 g of agar powder. Then, the plate was incubated at 30°C for 12-36 h. The strains with different colony morphologies were picked and purified by plate streaking method, and preserved in glycerol, followed by storage at -80°C.

[0033] To activate the single colonies, the single colonies were placed in liquid beef extract peptone medium and incubated at 37°C and 200 r / min for 24 h to obtain bacterial liquid for standby.

[0034] (2) Screening of biocontrol bacteria

[0035] The single colony bacteria isolated in the above process were subjected to preliminary screening of antibacterial activity by plate confrontation method. Fusarium foetens L1 and Fusarium oxysporum, which are lily bulb rot pathogens, and Penicillium janthinellum F7 were selected as test strains. The plates without inoculation of bacteria were used as controls, and the plates were incubated at 28°C for 4-7 d. The strains producing inhibition zones against the three test pathogenic bacteria were selected, and the sizes of the inhibition zones were measured. The Fusarium oxysporum was purchased from China Microbial Culture Collection Center, and the Fusarium foetens L1 and Penicillium janthinellum F7 were isolated in the laboratory. By comparing the sizes of the inhibition zones, the biocontrol bacteria with strong inhibitory effect on the three test pathogenic bacteria were screened, and numbered as R12.

[0036] The antagonistic experiment of R12 strain against the three test pathogenic bacteria was repeated to complete the rescreening, and the experimental results were shown in Table 1. Figure 1 Figure 1 ​As shown, R12 strain can produce obvious inhibition zone to three kinds of pathogenic bacteria, as shown in Table 1, the inhibition diameters of R12 strain to Fusarium foetens L1 strain, Fusarium oxysporum strain and Penicillium janthinellum F7 strain are 2.96mm, 2.61mm and 2.84mm respectively.

[0037] Table 1 Inhibition effect of R12 strain to three kinds of pathogenic bacteria

[0038]

[0039] 2. Identification of R12 strain

[0040] (1) Morphological identification

[0041] R12 strain was inoculated on liquid beef extract peptone medium plate and cultured at 37℃ for 12h, and the colony characteristics were observed, and morphological identification was carried out in combination with "Berger Bacteria Identification Manual". As shown in Table 3, the colony surface was rough, dry, wrinkled, and the edge was irregular, and gram staining was positive. Figure 2

[0042] (2) Physiological and biochemical identification

[0043] According to the method in "Common Bacteria System Identification Manual", physiological and biochemical determination was carried out on R12 strain. As shown in Table 2, R12 is a gram-positive bacterium, and the starch hydrolysis, gelatin hydrolysis, methyl red, oxidase reaction, peroxidase, indole reaction test are positive, while the nitrate reduction, V-P reaction and H2S production test are negative.

[0044] Table 2 Physiological and biochemical reaction characteristics analysis of R12 strain

[0045] Physiological and biochemical tests Results Gram’s stain test + Starch bydrolysis + Gelatin hydrolysis + Methyl red test + Oxidase reaction + Hydrogen peroxidase reaction + Indole reaction + Nitrate utilization - V-P reaction - <![CDATA[H2S产生H2S production]]> -

[0046] Among them, "+" is positive, "-" is negative.

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

[0048] ​

[0049] BLAST homology comparison of the sequence with the NCBI database found that the R12 strain had the highest homology with some strain sequences of Bacillus velezensis. The phylogenetic tree was constructed by using the adjacent linkage method in MEGA 7.0 software, and the results are shown in Figure 3

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

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

[0052] A single colony of R12 strain was picked and inoculated into liquid beef extract protein peptone medium and cultured at 37°C and 200 r / min for 12 h to obtain a seed solution for single factor experiment and orthogonal optimization experiment. Fusarium foetens L1 strain was used as the test strain to optimize the conditions by mycelial growth rate method. Specifically, 1 mL of fermentation filtrate under each condition was mixed with about 19 mL of PDA medium at about 50°C, and then poured into a flat plate. After the flat plate solidified, a Fusarium foetens L1 fungus cake was inoculated in the center of the flat plate, and the mycelium was attached to the flat plate. The flat plate mixed with water and PDA medium was used as a control, and each treatment was repeated 3 times. The growth diameter of the test fungus was measured, and the inhibition rate was calculated:

[0053]

[0054] (2) Single factor experiment

[0055] Screening of carbon source types: equal amounts of corn powder, soluble starch, and mannitol were used to replace the carbon source in the beef extract protein peptone medium, and other fermentation conditions remained unchanged. The best carbon source was screened by mycelial growth rate method. The experimental results are shown in Figure 4 When corn powder was used as the carbon source, the inhibition rate was the largest, which could reach 74.8%. Therefore, corn powder was selected as the carbon source for fermentation medium of R12 strain.

[0056] Screening of nitrogen source types: equal amounts of ammonium sulfate, soybean meal, and soybean powder were used to replace the nitrogen source in the beef extract protein peptone medium, and other fermentation conditions remained unchanged. The best nitrogen source was screened by mycelial growth rate method. The experimental results are shown in Figure 5 When soybean meal was used as the nitrogen source, the inhibition rate was the largest, which could reach 78.4%. Therefore, soybean meal was selected as the nitrogen source for fermentation medium of R12 strain.

[0057] ​Screening of inorganic salts: Equal amounts of CaCl2, MgSO4, and KCl were used to replace the inorganic salts in the beef extract peptone medium, while other fermentation conditions remained unchanged. Fermentation was then carried out, and the optimal inorganic salts were screened using the mycelial growth rate method. Experimental results are as follows: Figure 6 As shown, the inhibition rate was highest, reaching 74.3%, when CaCl2 was used as the inorganic salt. Therefore, CaCl2 was chosen as the inorganic salt for the fermentation medium of strain R12.

[0058] (3) Orthogonal experiment of fermentation medium

[0059] The selected CaCl2 (A: 4 g / L, 8 g / L, 12 g / L), corn flour (B: 8 g / L, 12 g / L, 16 g / L), and soybean meal (C: 5 g / L, 10 g / L, 15 g / L) were used as inorganic salts, carbon sources, and nitrogen sources, respectively. The three factors A, B, and C, along with the fermentation time (D: 12 h, 24 h, 36 h), were then used according to L9(3)... 4 The factors and levels of the orthogonal experiment were designed as shown in Table 3. Each treatment was cultured at 37℃ and 200 r / min for 24 h, and the fermentation broth was collected by centrifugation and filtered for sterilization. The inhibition rate of each treatment was checked according to the mycelial growth rate method, and the optimal culture medium formula was selected.

[0060] Table 3 Orthogonal primers and levels

[0061]

[0062] As shown in Table 4, the order of influence of various factors on the inhibition rate is fermentation time > carbon source > nitrogen source > inorganic salts. The optimal inhibition conditions are: D3 > B2 > C3 > A1. However, this result is inconsistent with the results of the third experimental group, A1B3C3D3, which showed the highest inhibition rate in the experiment. Therefore, the inhibition rates of the A1B3C3D3 and A1B2C3D3 experimental groups need to be re-examined. The verification experiment results show that the fermentation filtrate of strain R12 in both the A1B3C3D3 and A1B2C3D3 experimental groups effectively inhibited the growth of *Fusarium foetens* L1 strain. Figure 7 As shown, the inhibition rates of experimental groups A1B3C3D3 and A1B2C3D3 were 81.7% and 84.8%, respectively, indicating that experimental group A1B2C3D3 was superior to experimental group A1B3C3D3. Therefore, experimental group A1B2C3D3 was selected as the optimal fermentation condition, namely 4 g / L CaCl2, 12 g / L corn flour, and 15 g / L soybean meal, fermented for 36 h. The fermentation filtrate of this combination showed inhibition rates of 78.5% and 83.6% against Fusarium oxysporum and Penicillium janthinellum F7 strains, respectively.

[0063] Table 4. Results of the Orthogonal Experiment

[0064]

[0065]

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

[0067] (1) The inhibitory effect of strain R12 on the fungus causing lily bulb rot in soil

[0068] Preparation of R12 strain seed culture: The activated R12 strain was inoculated into beef extract peptone medium and cultured with shaking at 37℃ and 200r / min for 24h to obtain fermentation broth. The OD600 was diluted with sterile physiological saline to 0.8 and then set aside.

[0069] After soil sterilization, spores of *Fusarium foetens* L1, *Fusarium oxysporum*, and *Penicillium janthinellum* F7 strains were mixed in, adjusting the pathogen concentration to 1×10⁻⁶. 7 CFU / g soil. The bacterial suspension of strain R12 was applied to soil containing lily bulb rot pathogen at a weight ratio of 0.5-1.5%. Soil without bacterial suspension was used as a control group. The number of the three pathogens was measured and the inhibition rate of R12 bacteria against the three pathogens was calculated.

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

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

[0072] Preparation of R12 strain fermentation filtrate: Following the optimized fermentation medium formula in Example 2 (4 g / L CaCl2, 12 g / L corn flour, and 15 g / L soybean meal), fermentation was carried out for 36 hours. The bacterial cells were removed by centrifugation, and the mixture was then filtered for sterilization. The addition of *Lilium bulb rot* pathogen was prepared according to (1) in Example 3. The R12 strain fermentation filtrate was applied at a weight ratio of 0.3–1% to soil containing *Lilium bulb rot* pathogen. Soil without the R12 strain fermentation filtrate served as a control group. The number of the three pathogens was measured, and the inhibition rate of the R12 strain fermentation filtrate against the three pathogens was calculated.

[0073] The R12 strain fermentation filtrate reduces the spore number of Fusarium foetens L1 strain, Fusarium oxysporum strain and Penicillium janthinellum F7 strain. As shown in the table, the inhibition rates of R12 on the three pathogenic bacteria are 87.5%, 74.5% and 77.3% respectively, indicating that the R12 strain fermentation filtrate has the effect of inhibiting and killing pathogenic bacteria in soil environment. Figure 9

[0074] In summary, the R12 strain and its fermentation liquid have strong inhibitory effect on the three lily bulb rot disease pathogenic bacteria, and the optimal conditions of the R12 strain fermentation filtrate for antagonizing pathogenic bacteria are screened through single factor experiment and orthogonal optimization experiment, the strain culture method is simple, the antagonistic effect is good, the growth of common pathogenic bacteria of lily can be effectively inhibited, especially the R12 strain fermentation filtrate has the potential to be developed into a high-efficiency lily bulb rot disease pathogenic bacteria preparation.

[0075] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. A biocontrol bacterium antagonizing Plasmodiophora paeoniae, characterized in that, The biocontrol bacteria is Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) R12, and the strain preservation number is CCTCC NO: M 20241785.

2. The application of the antagonistic bacteria against Lilium squamae-rotum in preventing and treating the Lilium squamae-rotum.

3. Use according to claim 2, characterized in that, The method for preventing and treating the Lilium squamae-rotum by using the Bacillus velezensis R12 strain or the fermentation liquor of the Bacillus velezensis R12 strain to prepare a prevention and treatment preparation is as follows:

4. Use according to claim 3, characterized in that, The method for preventing and treating the Lilium squamae-rotum by using the Bacillus velezensis R12 strain to prepare a prevention and treatment preparation is as follows: S1: inoculating the activated Bacillus velezensis R12 strain into a beef extract peptone culture medium, and culturing at 37°C and 200 r / min for 24 hours to obtain a fermentation liquor; S2: Diluted the bacterial suspension to a concentration OD 600 = 0.8 with sterile physiological saline, and applied to the soil containing the pathogen of lily bulb rot at a rate of 0.5-1.5% by weight.

5. Use according to claim 3, characterized in that, The method for preventing and treating the Lilium squamae-rotum by using the Bacillus velezensis R12 strain to prepare a prevention and treatment preparation is as follows: inoculating the Bacillus velezensis R12 strain into a culture medium containing 4 g / L CaCl2, 12 g / L corn powder and 15 g / L soybean meal, and fermenting for 36 hours; removing the bacterial bodies by centrifugation, removing bacteria by filtration, and applying the filtrate to the soil containing the Lilium squamae-rotum pathogen according to a weight ratio of 0.3-1%.

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