Bacillus pseudomycoides BP88 and application thereof in prevention and treatment of tobacco root rot

Abstract

The invention provides bacillus pseudomycoides BP88 and application of the bacillus pseudomycoides BP88. The preservation number of the bacillus pseudomycoides BP88 is CCTCC (China Center For Type Culture Collection) NO.20241800. The invention further provides a preparation method of the bacillus pseudomycoides BP88. The bacillus pseudomycoides has a good disease prevention effect on tobacco root rot and has a remarkable growth promoting effect on tobacco plants. The strain has good theoretical research value and application prospect, can be used as a good test material for researching interaction and action mechanism of soil bacteria and plants, can also be used for research and development of soil bio-fertilizers and biological bactericides, can also become a good carrier of multifunctional engineering bacteria for disease prevention, growth promotion and the like, and has broad application prospects. And a new strain resource is provided for biological prevention and control of tobacco root rot.

Description

Technical Field

[0001] The invention relates to the technical field of microbiology, and in particular to a strain of Bacillus mycoides BP88 and application thereof in preventing and controlling tobacco root rot. Background Art

[0002] China's tobacco industry occupies a pivotal position in the global market. Its planting area and cigarette production are among the highest in the world, becoming an important economic pillar of the country. Tobacco planting provides economic support and employment opportunities for many regions, and plays an irreplaceable role in local economic development. However, the frequent outbreaks of tobacco root rot have seriously affected the growth and yield of tobacco, becoming one of the main problems troubling tobacco farmers. The spread of root rot not only causes huge economic losses to the tobacco industry, but also threatens the stability of the tobacco supply chain. Prevention and control work needs to be strengthened urgently.

[0003] Tobacco root rot is caused by Fusarium spp., which is a common fungal disease affecting tobacco production in my country. It can break out in both the seedbed stage and the field stage, and the incidence rate is stable at 3% to 5%. Due to the extension of continuous cropping time and changes in soil ecological environment and cultivation patterns, Fusarium root rot has gradually become the main disease in tobacco production. The current means of preventing and controlling root rot are mainly disease-resistant varieties and chemical control, but the resources of disease-resistant varieties are limited and the cultivation cycle is long, and chemical control is prone to cause environmental pollution. In contrast, biological control has become a research hotspot because of its non-residue and environmentally friendly characteristics. In particular, actinomycetes in plant rhizosphere soil, as a natural disease prevention resource, can show great potential in pest and disease control, promoting plant growth and enhancing plant disease resistance.

[0004] Therefore, it is necessary to screen and identify a strain that can prevent and treat root rot from soil or plant materials. Summary of the invention

[0005] The invention aims to provide a strain of Bacillus mycoides BP88 and an application thereof. The strain of Bacillus mycoides has a good disease prevention effect on tobacco root rot and has a significant growth-promoting effect on tobacco plants.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a strain of Bacillus paramycoides BP88 is provided, and the deposit number of the strain of Bacillus paramycoides BP88 is: CCTCCNO.20241800.

[0008] In a second aspect of the present invention, a fermentation agent is provided, the fermentation agent comprising:

[0009] Fermenting the Bacillus mycoides BP88 to obtain a fermentation liquid or a bacterial suspension;

[0010] Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

[0011] Furthermore, the preparation method of the bacterial suspension comprises:

[0012] The Bacillus mycoides BP88 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid.

[0013] Furthermore, the fermentation culture conditions include: temperature of 36-38°C and pH of 5-9.

[0014] In the third aspect of the present invention, there is provided the use of the Bacillus mycoides BP88 or the fermentation agent in preventing and controlling tobacco root rot.

[0015] In the third aspect of the present invention, there is provided the use of the Bacillus mycoides BP88 or the fermentation agent in promoting tobacco seed germination.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0017] The pseudomycoides provided by the embodiment of the present invention has a good disease prevention effect on tobacco root rot and has a significant growth-promoting effect on tobacco plants. It can be seen that the strain has good theoretical research value and application prospects. It can not only be used as a good experimental material for studying the interaction between soil bacteria and plants and their mechanism of action, but also can be used for the research and development of soil biofertilizers and biofungicides. At the same time, it may also become a good carrier for multifunctional engineering bacteria such as disease prevention and growth promotion, providing a new bacterial species resource for the biological control of tobacco root rot.

[0018] The deposit date of the pseudomycoides Bacillus BP88 of the present invention is August 16, 2024, and the deposit number is CCTCCNO.20241800. Its classification name is Bacillus paramycoides BP88, and the depository name is China Center for Type Culture Collection, with an address of Wuhan University, Wuhan City, Hubei Province, China, Postal Code: 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 This is a graph showing the antibacterial effect of Bacillus mycoides BP88 on Fusarium oxysporum;

[0021] Figure 2 This is the antibacterial effect diagram of sterile fermentation broth;

[0022] Figure 3 is the disease prevention effect of the strain on tobacco root rot; Figure 3 A is the growth of tobacco seedlings. Figure 3 B is a comparison chart of the seedlings and roots (rhizomes).

[0023] Figure 4 Phylogenetic tree of strains. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0025] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0027] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0028] The inventors of the present application obtained a strain of bacteria from tobacco-growing soil collected from non-disease, continuous cropping / non-continuous cropping fields in Nanzhang, Zaoyang and other places. Through separation, purification and screening, it was found that the strain had an antagonistic effect on tobacco oxysporum Fusarium. The bacteria were analyzed by colony morphology, biochemistry and 16S rRNA sequencing. The homology of the strain with multiple strains of Bacillus paramycoides was more than 96%. Combined with physiological and biochemical characteristics, the strain was preliminarily determined to belong to (Bacillus paramycoides) and named Bacillus paramycoides.

[0029] The pseudomycoides bacillus of the present invention has a good promoting effect on tobacco seed germination and tobacco plant growth, has a good disease prevention effect on tobacco root rot, and provides a theoretical basis for the subsequent development of biological microbial agents.

[0030] The following will describe in detail a strain of Bacillus mycoides BP88 of the present application and its application in preventing and controlling tobacco root rot in combination with examples and experimental data.

[0031] Example 1: Isolation, purification and identification of Bacillus mycoides BP88

[0032] 1. Isolation and purification of tobacco field soil microorganisms

[0033] Test culture medium: NA medium: 3 g beef extract, 1 g yeast extract, 5 g peptone, 10 g glucose, 15 g agar, make up to 1000 mL with distilled water, pH 7.2, sterilize.

[0034] LB solid medium: 10 g sodium chloride, 10 g peptone, 5 g yeast powder, 1000 mL water, 15 g agar, sterilized. Potato dextrose medium (PDA): 200 g potato, 20 g glucose, 15-20 g agar, 1000 mL distilled water, pH 7.0-7.4, sterilized.

[0035] Weigh 10g of soil and place it in a 250mL conical flask, then add 100mL of sterile water, and place the conical flask on an oscillator at 180r / min for 20min. Pipette the stock solution to prepare a rhizosphere soil suspension with a dilution of 10-1 to 10-5; take 100μL of soil suspensions with different dilutions of 10-2 to 10-5, evenly spread them on the culture medium plate, set 2 to 3 parallels for each dilution, and then place them in a 28℃ constant temperature incubator for 48h. After the colonies grow, perform 2 to 3 times of streaking purification according to various characteristics such as color, morphology, structure, and glossiness. The purified strains are numbered and stored in a 4℃ refrigerator for standby use.

[0036] 2. Isolation and screening of strains

[0037] (1) Primary screening: Using Fusarium oxysporum, a tobacco root rot pathogen, as an indicator bacterium, the plate confrontation method was used to determine the inhibitory activity of the test strain against Fusarium oxysporum. Pre-activate Fusarium oxysporum on a PAD plate, use a sterile punch with a diameter of 5 mm to punch a bacterial cake at the edge of the colony, inoculate it in the center of a new PDA plate, and inoculate different bacteria isolated and purified 2.5 cm from the center of the plate in parallel lines up and down. Use only the inoculated pathogen as a blank control. Set up 3 replicates for each treatment, and place the plate in a 28°C incubator for culture. When the control colony grows to 2 / 3 of the culture dish, observe whether there is an obvious antagonistic effect, and select the strain that has an inhibitory effect on the growth of Fusarium oxysporum for re-screening.

[0038] 136 bacterial strains were isolated from the soil, and after initial screening, 20 strains were found to have antagonistic effects against tobacco Fusarium.

[0039] (2) Rescreening: The 20 strains obtained from the initial screening were rescreened using the plate confrontation method. The screening method is the same as above, but after each plate is inoculated with fungi, the same strain is inoculated on both sides. The control is not inoculated with fungi and the inhibition zone of the strain is recorded after the plate is full of growth. The above initial screening plate is taken out, the size of the inhibition zone is measured and the inhibition rate is calculated. The calculation formula is as follows:

[0040]

[0041] After repeated screening, a strain with good antagonistic effect on tobacco Fusarium was finally screened out and named BP88. Figure 1 The results of the antibacterial test of BP88 of the present invention on tobacco root rot are shown in Table 1.

[0042] Table 1 Antibacterial assay of BP88 against tobacco Fusarium

[0043]

[0044] It can be seen from Table 1 that the width of the inhibition zone of strain BP88 against tobacco Fusarium is 19.00 mm, the inhibition rate can reach 76.25%, and the antagonistic effect can be clearly observed on the plate, so it was selected as the target strain for screening.

[0045] 3. Identification of strains

[0046] The strain was picked up and added to LB liquid medium for overnight culture, and then the sediment was centrifuged. The genomic DNA of the bacteria was extracted, and the strain gene DNA was extracted by the bacterial suspension boiling water bath method. The bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCCGCA-3') were used for PCR amplification. The PCR reaction system included 1 μL of DNA template, 1 μL of upstream and downstream primers, 12.5 μL of Taq polymerase, and ddH2O was added to 25 μL. The PCR amplification program was: 98℃ pre-denaturation for 5min, 98℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 1min30s, 35 cycles, and finally 72℃ extension for 5min. The PCR reaction products were detected by 1% agarose gel electrophoresis, and the PCR amplification products of 16S rRNA were recovered and purified and sequenced by Wuhan Qingke Biotechnology Co., Ltd. The sequencing results (sequence shown in SEQ ID NO.1) were submitted to the NCBI database for BLAST homology analysis, and then MEGA 11.0 software was used for sequence alignment and phylogenetic tree construction. Data processing was performed using Excel and SPSS27 software.

[0047] The 16S rRNA sequence of the strain to be tested obtained by PCR was compared with BLAST in NCBI, and it was found that the sequence homology between the strain and Bacillus paramycoides was 87%. Combined with the morphology and physiological and biochemical characteristics of the strain, the strain was determined to be Bacillus paramycoides. The strain has been deposited in the China Center for the Collection of Common Microbiological Cultures, with the deposit number of CCTCC NO.20241800.

[0048] Example 2, Determination of antibacterial activity of aseptic fermentation filtrate of strains

[0049] 1. Preparation of strain fermentation broth

[0050] The Bacillus mycoides BP88 strain was inoculated into 5 mL of LB liquid medium and cultured at 37°C, 220 r / min for 12 h to prepare seed solution. Then, it was inoculated into LB culture medium at a 1% inoculum amount and cultured at 37°C, 220 r / min for 48 h to obtain the strain fermentation liquid. Before the pot test, the concentration of live bacteria in the fermentation liquid was adjusted to 1×10 8 cfu / mL.

[0051] 2. Determination of antibacterial activity of sterile fermentation filtrate of strains

[0052] (1) Preparation of sterile fermentation filtrate of biocontrol strains: The cultured bacterial broth was centrifuged at 12000 r / min for 10 min, and the supernatant was sterilized and filtered twice with a bacterial filter (0.22 μm) to obtain a sterile fermentation filtrate.

[0053] (2) Effect of sterile fermentation filtrate on the growth of pathogenic mycelium: The plate punching method was used to test the antibacterial effect of the sterile fermentation filtrate of antagonistic bacteria. Use a hole puncher to evenly punch three holes 2.5 cm away from the center of the plate (a petri dish with a diameter of 9 mm). Inject 40 μL of fermentation filtrate into each hole. Use sterile LB liquid culture medium as the control. Inoculate a pathogenic bacterial block with a diameter of 5 mm in the center of the culture medium. Repeat each treatment three times. After culturing in the dark at 28°C for 5 days, measure the colony radius of the control group and the treatment group, and calculate the relative antibacterial rate.

[0054] 3. Results

[0055] The results of the test on the effect of the aseptic fermentation filtrate of the strain on the mycelial growth of tobacco root rot pathogens are as follows Figure 2 And as shown in Table 2.

[0056] Table 2 Antibacterial activity of BP88 sterile fermentation filtrate

[0057]

[0058] Depend on Figure 2As shown in Table 2, the fermentation filtrate of BP88 also has an inhibitory effect on the mycelial growth of Fusarium oxysporum ( Figure 2 ), the size of the inhibition zone was 11.00 mm, the relative inhibition rate was 36.24%, and the inhibition effect was lower than that of living bacteria, which was reduced by 40.01% (Table 2).

[0059] Example 3: Determination of the effect of promoting tobacco seed germination

[0060] Select tobacco seeds with uniform particles, disinfect them with 75% ethanol for 30 seconds, wash them with sterile water three times, and soak them in the fermentation liquid of the strain for 4 hours after drying with sterile filter paper. Take out the seeds and place them evenly and equidistantly in a culture dish covered with 2 layers of moist filter paper, 50 seeds per dish, and use clear water (CK1) and LB (CK2) liquid culture medium as negative controls, with 3 replicates for each treatment. Cultivate in the dark in a light incubator for 15 days (humidity 80%, temperature 28±2℃), add a certain amount of sterile water every 2 days to keep the filter paper moist, count the number of seeds that germinate, and calculate the germination rate.

[0061] Seed germination rate = (number of germinated seeds ÷ number of test seeds) × 100%

[0062] Germination potential (%) = number of seeds germinated in the first 5 days / total number of test seeds × 100%;

[0063] Germination index (GI) = ∑(Gt / Dt). In the above expression, Gt is the total number of seeds that germinated on day t, and Dt is the corresponding germination date;

[0064] Vitality index = germination index × S. In the above expression, S is the total length of the seedling root and bud.

[0065] The results are shown in Table 3.

[0066] Table 3 Effect of BP88 on tobacco seed germination

[0067]

[0068] From the results in Table 3, it can be seen that 10 days after the strain soaking treatment, the seed germination rate increased significantly, reaching 80.00%, which was 50.67% higher than that of the control group, indicating that it has a good promoting effect on tobacco seed germination.

[0069] Example 4: Experiment on the effectiveness of strains in preventing tobacco root rot in indoor potted plants

[0070] The experiment was designed as blank group CK1, control group T1, and treatment group T2. Pre-select tobacco seedlings with consistent growth, use sterilized scissors to cut off 1 cm of the root end of the seedling, cut 5 roots for each plant, and pierce the rhizome with a sterilized needle. Each rhizome was pierced with 5 wounds, and then moved to a sterilized nutrient pot (with nutrient matrix). Each treatment had 5 plants, 3 replicates, and a total of 15 plants. The plants were watered by root irrigation 2 days after transplanting. Each plant in the blank group and the control group was irrigated with 15 mL of sterile water; the treatment group was irrigated with the biocontrol bacteria fermentation liquid prepared in Example 2 at a rate of 15 mL per plant, and the control group and the treatment group were irrigated with a suspension of Fusarium oxysporum at a rate of 15 mL per plant after 48 hours. Among them, the concentration of fungal spores in the bacterial suspension is 5×10 6 / mL, the concentration of the biocontrol bacteria fermentation liquid was 1×10 5 CFU / mL. When the control group became ill after 15 days of growth, the incidence was counted. The grading standard of tobacco root rot was based on the tobacco industry standard of the People's Republic of China (GB / T23222-2008).

[0071] Incidence rate = (number of diseased plants / total number of plants surveyed) × 100%

[0072] Disease index = [∑ (number of diseased plants or leaves at each level × value of the disease level) / (total number of plants or leaves surveyed × highest level value)] × 100

[0073] Relative control effect = [(control disease index - treatment disease index) / control disease index] × 100%

[0074] The results are as follows Figure 3 As shown in A, the tobacco seedlings in the control group wilted and turned yellow. Figure 3 B shows that the root and stem of the seedlings in the control group inoculated with pathogens rotted and the plants died, producing a pathological phenotype similar to that in the field. The seedlings in the treatment group (inoculated with BP88) showed no symptoms of disease, and the root and stem grew normally without rot symptoms, proving that indoor inoculation with pathogens can cause tobacco to become sick and die, but inoculation with potential biocontrol bacteria BP88 can effectively prevent the plant from becoming sick and dying. The specific disease statistics are as follows:

[0075] Table 4 Inhibition of tobacco root rot by biocontrol bacteria

[0076]

[0077] As shown in Table 4, in the control group, 15 tobacco seedlings wilted, turned yellow and died due to Fusarium oxysporum, and the incidence of root rot reached 100.00%, while the incidence of seedlings inoculated with BP88 was 6.67%. At the same time, the disease index of the diseased plants was reduced, and the relative control effect was 89.20%.

[0078] In summary, the Bacillus mycoides BP88 provided by the present invention has a good disease prevention effect on tobacco root rot and has a significant growth-promoting effect on tobacco plants.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0080] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0081] Although the embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the embodiments and all changes and modifications falling within the scope of the present invention.

[0082] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Bacillus paramycoides BP88, characterized in that: The deposit number of the Bacillus paramycoides BP88 is: CCTCC NO.20241800.

2. A fermentation agent, characterized in that: The fermentation agent comprises: Fermenting the Bacillus mycoides BP88 as claimed in claim 1 to obtain a fermentation liquid or a bacterial suspension; Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

3. The fermentation agent according to claim 2, characterized in that: The preparation method of the bacterial suspension comprises: The Bacillus mycoides BP88 of claim 1 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid.

4. The fermentation agent according to claim 3, characterized in that: The fermentation culture conditions include: temperature of 36-38° C. and pH of 5-9.

5. Use of the Bacillus mycoides BP88 according to claim 1 or the fermentation agent according to claims 2-4 in preventing and controlling tobacco root rot.

6. Use of the Bacillus mycoides BP88 according to claim 1 or the fermentation agent according to claims 2 to 4 in promoting tobacco seed germination and tobacco plant growth.

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