Bacillus mobilis BM84 and application thereof in prevention and treatment of tobacco root rot

Abstract

The invention provides bacillus mobilis BM84 and application of the bacillus mobilis BM84. The preservation number of the bacillus mobilis BM84 is CCTCC (China Center For Type Culture Collection) NO: M20241799. The bacillus mobilis 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 present invention relates to the technical field of microbiology, and particularly to a Bacillus mobilis BM84 and its application in preventing and controlling tobacco root rot disease. Background Art

[0002] As one of the important cash crops in China, tobacco has a wide planting area across the country. However, with the expansion of the tobacco planting scale, tobacco root rot disease has occurred extensively in tobacco-growing areas in China, seriously affecting the yield and quality of tobacco. The continuous spread of root rot disease has brought huge economic losses to tobacco farmers and the tobacco industry, becoming one of the important problems restricting the development of the tobacco industry. Therefore, preventing and controlling tobacco root rot disease has become a key task to improve tobacco production efficiency and ensure the sustainable development of the industry.

[0003] Tobacco root rot disease caused by Fusarium spp. is a fungal disease in tobacco production in China, which can occur in both the seedbed stage and the field stage, with an annual incidence rate of 3% - 5%. Due to the increase in continuous cropping years, changes in soil microecology and cultivation systems, tobacco Fusarium root rot disease has gradually risen to a major disease. The commonly used control measures in production are to select disease-resistant varieties and chemical control, but the disease-resistant resources are limited, the breeding cycle is long, and chemical pesticides are prone to residue pollution, threatening human health. Biological control has the characteristics of no residue and environmental friendliness, and can be used for the prevention and control of tobacco Fusarium root rot disease. The utilization of beneficial microorganisms has become a research hotspot in plant disease control. Plant rhizosphere soil bacteria are natural resource bacteria for the biological control of plant diseases and pests, with very extensive theoretical research and development and application value. Many strains in rhizosphere microorganisms have extensive biological functions such as disease prevention, growth promotion, and biological nitrogen fixation for host plants, and can colonize and conduct around the plant body, playing a long-term role.

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

[0005] The object of the present invention is to provide a Bacillus mobilis BM84 and its application. This Bacillus mobilis BM84 has a good disease prevention effect on tobacco root rot disease and a significant growth promotion effect on tobacco plants.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a Bacillus mobilis BM84 is provided, and the preservation number of the Bacillus mobilis BM84 is: CCTCC NO: M20241799.

[0008] In a second aspect of the present invention, a fermentation inoculant is provided, and the fermentation inoculant includes:

[0009] A fermentation broth or bacterial suspension obtained by fermenting and culturing the Bacillus mobilis BM84;

[0010] Or a dry powder inoculant obtained by spray-drying the fermentation broth.

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

[0012] A fermentation broth obtained by inoculating the Bacillus mobilis BM84 into a liquid medium for fermentation and culture.

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

[0014] In a third aspect of the present invention, there is provided the use of the Bacillus mobilis BM84 or the fermentation inoculant in preventing and treating tobacco root rot.

[0015] In a third aspect of the present invention, there is provided the use of the Bacillus mobilis BM84 or the fermentation inoculant in promoting the germination of tobacco seeds.

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

[0017] The Bacillus mobilis provided in the embodiments of the present invention has a good disease prevention effect on tobacco root rot and a significant growth-promoting effect on tobacco plants. Thus, it can be seen that this 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 its mechanism of action, but also for the research and development of soil biological fertilizers and biological fungicides. At the same time, it may also become a good carrier for constructing multi-functional engineering bacteria such as disease prevention and growth promotion, providing a new strain resource for the biological control of tobacco root rot.

[0018] The preservation date of the Bacillus mobilis BM84 of the present invention is August 16, 2024, and the preservation number is CCTCC NO.20241799. Its taxonomic name is Bacillus mobilis BM84, and the name of the preservation unit is China Center for Type Culture Collection, with the address at Wuhan University, Wuhan, Hubei Province, China, and the postal code: 430072. Description of the Drawings

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is the antibacterial effect diagram of Bacillus motilis BM84 against Fusarium oxysporum;

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

[0022] Figure 3 It is the disease prevention effect of the strain on tobacco root rot; among which Figure 3 A is the growth situation of tobacco plant seedlings, Figure 3 B is the comparison diagram of the seedlings and the root (rhizome) part.

[0023] Figure 4 It is the strain phylogenetic tree. Specific Embodiments

[0024] The following will specifically elaborate on the present invention in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0025] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0026] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or by existing methods.

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

[0028] The inventors of the present application collected tobacco-growing soil from disease-free fields in Nanzhang, Zaoyang and other places. Through isolation, purification and screening, a strain of bacteria was obtained, and it was found that this strain has an antagonistic effect on Fusarium oxysporum f. sp. nicotianae. After colony morphology, biochemical and 16S rRNA sequencing analysis of this bacterium, the homology of this strain with multiple strains of Bacillus mobilis is more than 96%. Combining physiological and biochemical characteristics, this strain was initially determined to belong to (Bacillus mobilis), and was named Bacillus mobilis.

[0029] The Bacillus mobilis of the present invention has a good promoting effect on the germination and growth of tobacco seeds and has a good disease prevention effect on tobacco root rot, providing a theoretical basis for the subsequent development of biological bacterial agents.

[0030] Next, a strain of Bacillus mobilis BM84 of the present application and its application in preventing and treating tobacco root rot will be described in detail in combination with examples and experimental data.

[0031] Example 1: Isolation, purification and identification of Bacillus mobilis BM84

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

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

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

[0035] Weigh 10 g of soil and place it in a 250 mL conical flask. Then add 100 mL of sterile water, and place the conical flask on an oscillator at 180 r / min for 20 min. Absorb the original solution and prepare a rhizosphere soil suspension with dilution factors of 10-1 to 10-5; absorb 100 μL of soil suspension with different dilution factors of 10-2 to 10-5 and spread them evenly on the culture medium plates. Set 2 - 3 parallels for each dilution factor, and then place them in an incubator at 28 °C for 48 h. After the colonies grow, perform 2 - 3 times of streak purification according to multiple characteristics such as color, morphology, structure, glossiness, etc. Write down the numbers of the purified strains and store them in a 4 °C refrigerator for later use.

[0036] 2. Isolation and screening of strains

[0037] (1) Primary screening: Using Fusarium oxysporum, the causative agent of tobacco root rot, as the indicator bacterium, the inhibitory activity of the tested strains against Fusarium oxysporum was determined by the plate confrontation method. Fusarium oxysporum was pre-activated on a PAD plate, and a mycelial plug with a diameter of 5 mm was punched from the edge of the colony and inoculated in the center of a new PDA plate. Different bacteria obtained by isolation and purification were streaked parallelly above and below 2.5 cm away from the center of the plate. Only inoculating the pathogenic bacterium was used as the blank control. Each treatment was set with 3 replicates, and the plates were incubated in an incubator at 28 °C. When the control colony grew to 2 / 3 of the petri dish, the presence of obvious antagonistic effects was observed, and the strains that inhibited the growth of Fusarium oxysporum were selected for secondary screening.

[0038] 136 strains of bacteria were isolated from the soil. After primary screening, 20 strains with antagonistic effects against tobacco Fusarium were selected.

[0039] Secondary screening: The 20 strains obtained from primary screening were subjected to secondary screening by the plate confrontation method. The screening method was the same as above, but after inoculating the fungus on each plate, the same strain was inoculated on both sides. The control was not inoculated, and the inhibition zone of the strain was recorded after the plate was fully covered with the fungus. The above-mentioned primary screening plates were taken out, the size of the inhibition zone was measured, and the inhibition rate was calculated. The calculation formula is as follows:

[0040]

[0041] After secondary screening, a strain with good antagonistic effects against tobacco Fusarium was finally selected and named BM84. The antagonistic effect is as Figure 1 shown. The results of the inhibition test of BM84 of the present invention against tobacco root rot are shown in Table 1.

[0042] Table 1 Inhibition test of BM84 against tobacco Fusarium

[0043]

[0044] As can be seen from Table 1: The width of the inhibition zone of strain BM84 against tobacco Fusarium is 18.67 mm, the inhibition rate can reach 76.67%, and the generation of antagonistic effects can be clearly observed on the plate. Therefore, it was selected as the target strain for screening.

[0045] 3. Identification of the strain

[0046] Single colonies of the strain were picked and added to LB liquid medium for overnight culture, followed by precipitation and centrifugation. Genomic DNA of the bacteria was extracted, and the gene DNA of the strain was extracted by the boiling water bath method of the bacterial suspension. Universal bacterial primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1541R (5’-AAGGAGGTGATCCAGCCGCA-3’) were used for PCR amplification. The PCR reaction system contained 1 μL of DNA template, 1 μL of each upstream and downstream primer, 12.5 μL of Taq polymerase, and ddH2O was added to make up to 25 μL. The PCR amplification program was: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min 30 s, for 35 cycles, and finally extension at 72 °C for 5 min. The PCR reaction products were detected by 1% agarose gel electrophoresis. After the PCR amplification products of 16S rRNA were recovered and purified, they were sequenced by Wuhan Qingke Biotechnology Co., Ltd. The sequencing results were submitted to the NCBI database for BLAST homology analysis, and then sequence alignment and phylogenetic tree construction were carried out using MEGA 11.0 software. Data processing was analyzed using Excel and SPSS27 software.

[0047] The 16S rRNA sequence of the test strain obtained by PCR was subjected to BLAST alignment analysis in NCBI. It was found that the sequence homology of this strain with Bacillus mobilis was 93%. Combining the morphological, physiological and biochemical characteristics of this strain, the strain was identified as Bacillus mobilis. This strain has been deposited in the China General Microbiological Culture Collection Center, and the deposit number is CCTCC NO.20241799.

[0048] Example 2: Determination of the antibacterial activity of the sterile fermentation filtrate of the strain

[0049] 1. Preparation of the strain fermentation broth

[0050] The Bacillus mobilis BM84 strain was inoculated into 5 mL of LB liquid medium and cultured at 37 °C and 220 r / min for 12 h to prepare a seed solution. Then, it was inoculated into LB culture medium at an inoculation amount of 1% and cultured under the conditions of 37 °C and 220 r / min for 48 h to obtain the strain fermentation broth. Before the pot experiment, the viable cell concentration in the fermentation broth was adjusted to 1×10 8 cfu / mL with LB culture medium.

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

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

[0053] (2) Effect of the sterile fermentation filtrate on the mycelial growth of pathogenic bacteria: The antibacterial effect of the sterile fermentation filtrate of the antagonistic bacterium was tested by the method of punching holes on a plate. Use a hole punch to evenly punch 3 holes at a distance of 2.5 cm from the center of the plate (a petri dish with a diameter of 9 mm). Inject 40 μL of the fermentation filtrate into each hole. Use sterile LB liquid medium as a control. Inoculate a pathogenic bacteria block with a diameter of 5 mm at the center of the medium. Each treatment is repeated 3 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 determination results of the effect of the sterile fermentation filtrate of the strain on the mycelial growth of the pathogen of tobacco root rot are as Figure 2 shown in Table 2.

[0056] Table 2 Antibacterial activity of the sterile fermentation filtrate of BM84

[0057]

[0058] From Figure 2 the results of Table 2, it can be seen that the fermentation filtrate of BM84 also has an inhibitory effect on the mycelial growth of Fusarium oxysporum f. sp. nicotianae ( Figure 2 ), the size of the inhibition zone is 10.67 mm, and the relative antibacterial rate is 32.64%. The antibacterial effect is lower than that of the live bacteria, with a decrease of 44.03% (Table 2).

[0059] Example 3. Determination of the promoting effect on the germination of tobacco seeds

[0060] Select tobacco seeds with uniform and regular particles. Disinfect the surface with 75% ethanol for 30 s, wash three times with sterile water, and dry with sterile filter paper. Then immerse the seeds in the fermentation broth of the strain for 4 h. Take out the seeds and place them evenly and equidistantly in a petri dish lined with 2 layers of moist filter paper, with 50 seeds in each dish. Use clear water (CK1) and LB (CK2) liquid medium as negative controls. Set 3 replicates for each treatment. Culture in the dark in an illumination incubator for 15 d (humidity 80%, temperature 28 ± 2 °C). Uniformly supplement a certain amount of sterile water every 2 d to keep the filter paper moist. Count the number of germinated seeds and calculate the germination rate.

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

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

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

[0064] Vitality index = Germination index × S. In the above expression, S is the total length of the roots and buds of the seedlings.

[0065] The results are shown in Table 3.

[0066] Table 3 Effects of BM84 on the germination of tobacco seeds

[0067]

[0068] As can be seen from the results in Table 3, 10 days after the seeds were soaked with the strain, the germination rate of the seeds increased significantly, reaching 87.33%, which was 58% higher than that of the control group. This indicates that it has a good promoting effect on the germination of tobacco seeds.

[0069] Example 4: Indoor potted plant control efficacy experiment of the strain against tobacco root rot

[0070] The experimental design included a blank group CK1, a control group T1, and a treatment group T2. First, tobacco seedlings with consistent growth were selected. The ends of the roots of the seedlings were cut off by 1 cm using sterilized scissors, and 5 roots were cut from each plant. The rhizomes were pricked with sterilized needles, and 5 wounds were made on each rhizome. Then, they were transferred to sterilized nutrient pots (containing nutrient substrates). There were 5 plants in each treatment, with 3 replicates, totaling 15 plants. Two days after transplantation, the plants were watered by the root irrigation method. Each plant in the blank group and the control group was watered with 15 mL of sterile water; the treatment group was watered with the biocontrol bacteria fermentation broth prepared in Example 2 at a rate of 15 mL per plant. 48 hours later, the control group and the treatment group were watered with a Fusarium oxysporum suspension at a rate of 15 mL per plant. Among them, the concentration of fungal spores in the suspension was 5×10 6 cells / mL, and the concentration of the biocontrol bacteria fermentation broth was 1×10 5 CFU / mL. The disease incidence was counted 15 days after the control group developed the disease. The grading standard for tobacco root rot referred to the Tobacco Industry Standard of the People's Republic of China (GB / T 23222-2008).

[0071] Disease incidence = (Number of diseased plants / Total number of plants surveyed) × 100%

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

[0073] Relative control efficacy = [(Control disease index - Treatment disease index) / Control disease index] × 100%

[0074] The results are as Figure 3 shown in A. The tobacco seedlings in the control group showed wilting and yellowing. Additionally, from Figure 3As can be seen from B, the roots and stems of the seedlings in the control group inoculated with the pathogenic bacteria rotted, the plants died, and pathological phenotypes similar to those in the field occurred. However, the seedlings in the treatment group (inoculated with BM84) showed no disease symptoms, and the roots and stems grew normally without rot symptoms, indicating that inoculating pathogenic bacteria indoors could cause tobacco to develop and die, but inoculating the potential biocontrol bacterium BM84 could effectively prevent the plants from developing and dying. The specific disease statistics are as follows:

[0075] Table 4 Inhibitory effect of biocontrol bacteria on the incidence of tobacco root rot

[0076]

[0077] As can be seen from Table 4, in the control group, 15 tobacco seedlings wilted, yellowed and died caused by Fusarium oxysporum, the incidence of root rot reached 93.33%, while the incidence of tobacco seedlings inoculated with BM84 was 6.67%. At the same time, the number of diseased plants was alleviated, and the relative control effect was 89.20%.

[0078] In summary, a Bacillus licheniformis BM84 provided by the present invention has a good disease prevention effect on tobacco root rot and a significant growth promotion effect on tobacco plants.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

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

[0081] Although the embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include 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 equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Bacillus mobilis BM84, characterized in that, The preservation number of the Bacillus mobilis BM84 is: CCTCC NO: M20241799.

2. A fermentation inoculant, characterized in that, The fermentation inoculant includes: The fermentation broth or bacterial suspension obtained by fermenting and culturing the Bacillus mobilis BM84 as described in claim 1; Or the dry powder inoculant obtained by spray-drying the fermentation broth.

3. The fermenting inoculant according to claim 2, characterized in that, The preparation method of the bacterial suspension includes: The fermentation broth obtained by inoculating the Bacillus mobilis BM84 as described in claim 1 into a liquid medium for fermentation and culture.

4. The fermenting inoculant according to claim 3, wherein The conditions for the fermentation and culture include: the temperature is 36-38 °C, and the pH is 5-9.

5. Application of the Bacillus mobilis BM84 as described in claim 1 or the fermentation inoculant as described in claims 2-4 in preventing and controlling tobacco root rot.

6. Application of the Bacillus mobilis BM84 as described in claim 1 or the fermentation inoculant as described in claims 2-4 in promoting tobacco seed germination and tobacco plant growth.

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