A salt-tolerant Bacillus sp. BH74 and its application in controlling tobacco root rot

Abstract

The present invention provides a halotolerant Bacillus strain (Bacillus halotolerans) BH74 and its applications, and its preservation number is CCTCC NO: M 20241798. This halotolerant Bacillus strain has a good disease prevention effect on tobacco root rot and a significant growth promotion effect on tobacco plants. 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 be used 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 multifunctional engineering bacteria such as disease prevention and growth promotion, providing a new strain resource for the biological control of tobacco root rot.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly to a halotolerant Bacillus sp. BH74 and its application in controlling tobacco root rot disease. Background Art

[0002] As one of the main economic and tax crops in China, tobacco ranks first in the world in terms of planting area and cigarette output, and has an important economic status. Tobacco root rot disease occurs widely in tobacco-growing areas of China and causes serious damage. In recent years, with the improvement of land intensification, continuous cropping of tobacco fields has become increasingly common, further exacerbating the occurrence and spread trend of root rot disease.

[0003] Tobacco root rot disease is caused by Fusarium spp. and is a common fungal disease in tobacco production in China. It can occur in both the seedling bed stage and the field stage, and the annual incidence rate is 3% - 5%. With the extension of continuous cropping years and the changes in soil microecology and cultivation systems, Fusarium root rot disease has gradually become the main disease. Currently, the commonly used control measures include the selection of disease-resistant varieties and chemical pesticide control. However, the resources of disease-resistant varieties are limited and the breeding cycle is long, while chemical pesticides may cause residual pollution and threaten human health. In contrast, biological control has the advantages of no residue and environmental friendliness, and has become an effective means for controlling tobacco root rot disease. In particular, actinomycetes in the rhizosphere soil of plants, as natural biological control resource bacteria, can not only control diseases, but also play a long-term protective role through growth promotion and biological nitrogen fixation.

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

[0005] The object of the present invention is to provide a halotolerant Bacillus sp. BH74 and its application. This halotolerant Bacillus sp. BH74 has a good disease control effect on tobacco root rot disease and has a significant growth promotion effect on tobacco plants.

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

[0007] In the first aspect of the present invention, a halotolerant Bacillus sp. BH74 is provided, and the preservation number of the halotolerant Bacillus sp. BH74 is: CCTCC NO: M20241798.

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

[0009] The fermentation broth or bacterial suspension obtained by fermenting and culturing the halotolerant Bacillus sp. BH74;

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

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

[0012] The fermentation broth obtained by inoculating the salt-tolerant Bacillus sp. BH74 into a liquid medium for fermentation culture.

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

[0014] In the third aspect of the present invention, there is provided the use of the salt-tolerant Bacillus sp. BH74 or the fermented bacterial agent in preventing and treating tobacco root rot.

[0015] In the third aspect of the present invention, there is provided the use of the salt-tolerant Bacillus sp. BH74 or the fermented bacterial agent 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 salt-tolerant Bacillus sp. 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, 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 be used 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 salt-tolerant Bacillus sp. BH74 of the present invention is August 16, 2024, and the preservation number is CCTCC NO: M 20241798. Its taxonomic name is Bacillus halotolerans BH74, and the name of the preservation unit is China Center for Type Culture Collection, address: Wuhan University, Wuhan, Hubei Province, China, zip code: 430072. Description of the Drawings

[0019] In order 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 following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is the antibacterial effect diagram of salt-tolerant Bacillus sp. BH74 against Fusarium oxysporum;

[0021] Figure 2 It is the bacteriostatic 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 condition 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 implementation manners

[0024] The following will specifically elaborate on the present invention in combination with specific implementation manners 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 implementation manners and examples are used to illustrate the present invention rather than limit 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 meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0026] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be obtained 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 inventor of the present application collected tobacco-growing soils from disease-free fields in Nanzhang, Zaoyang and other places. Through isolation, purification and screening, a strain of bacteria was obtained. 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 halotolerans is above 96%. Combining physiological and biochemical characteristics, this strain was initially determined to belong to (Bacillus halotolerans), and was named Bacillus halotolerans.

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

[0030] The following will specifically describe a strain of Bacillus halotolerans BH74 of the present application and its application in preventing and treating tobacco root rot in combination with examples and experimental data.

[0031] Example 1: Isolation, Purification and Identification of Halotolerant Bacillus sp. BH74

[0032] 1. Isolation and Purification of Tobacco Field Soil Microorganisms

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

[0034] LB solid medium: Sodium chloride 10 g, peptone 10 g, yeast powder 5 g, 1000 mL water, agar 15 g, sterilized. Potato dextrose agar medium (PDA): Potato 200 g, glucose 20 g, agar 15 - 20 g, distilled water 1000 mL, 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. Pipette the stock solution to prepare a rhizosphere soil suspension with a dilution of 10 -1 ~10 -5 ; Pipette 100 μL of soil suspension with different dilutions of 10 -2 ~10 -5 onto the culture medium plates evenly. Set 2 - 3 parallels for each dilution, and then place them in an incubator at 28℃ for 48 h. After the colonies grow, perform 2 - 3 streak purifications according to multiple characteristics such as color, morphology, structure, glossiness, etc. Write down the numbers of the purified strains and store them in a refrigerator at 4℃ for future use.

[0036] 2. Isolation and Screening of Strains

[0037] (1) Primary screening: Using Fusarium oxysporum causing tobacco root rot as the indicator bacterium, the inhibitory activity of the tested strains against Fusarium oxysporum was determined by the plate confrontation method. Activate Fusarium oxysporum in advance on a PAD plate. Use a sterile punch with a diameter of 5 mm to punch out a mycelial disc at the edge of the colony and inoculate it in the center of a new PDA plate. Inoculate different bacteria obtained by isolation and purification by parallel streaking 2.5 cm above and below the center of the plate. Use only inoculating the pathogen as the blank control. Set 3 replicates for each treatment and place the plates in an incubator at 28℃. When the control colony grows to 2 / 3 of the culture dish, observe whether there is an obvious antagonistic effect, and screen out the strains with inhibitory effects on the growth of Fusarium oxysporum for the secondary screening.

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

[0039] (2) Re-screening: The 20 strains obtained from the primary screening were re-screened 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 grown. Take out the above-mentioned primary screening plates, measure the size of the inhibition zone and calculate the inhibition rate. The calculation formula is as follows:

[0040]

[0041] After re-screening, a strain with good antagonistic effect against Fusarium oxysporum f. sp. nicotianae was finally screened out and named BH74. The antagonistic effect is as Figure 1 shown. The results of the inhibition test of BH74 of the present invention against tobacco root rot are shown in Table 1.

[0042] Table 1 Inhibition test of BH74 against Fusarium oxysporum f. sp. nicotianae

[0043]

[0044] It can be seen from Table 1 that the width of the inhibition zone of strain BH74 against Fusarium oxysporum f. sp. nicotianae is 17 mm, the inhibition rate can reach 78.75%, and the antagonistic effect can be clearly observed on the plate. Therefore, it was selected as the target strain for screening.

[0045] 3. Identification of the strain

[0046] Pick single colonies of the strain and add them to LB liquid medium for overnight culture, then precipitate and centrifuge. Extract the genomic DNA of the bacteria. Extract the gene DNA of the strain by the boiling water bath method of the bacterial suspension. Use the universal bacterial primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1541R (5’-AAGGAGGTGATCCAGCCGCA-3’) for PCR amplification. The PCR reaction system is 1 μL of DNA template, 1 μL of each upstream and downstream primer, 12.5 μL of Taq polymerase, and ddH 2 O is supplemented to 25 μL. The PCR amplification program is: 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, 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 (the sequence is shown in SEQ ID NO.1) were submitted to the NCBI database for BLAST homology analysis, and then the sequence alignment and construction of the phylogenetic tree were carried out using MEGA 11.0 software. Data processing was analyzed using Excel and SPSS27 software.

[0047] The 16S rRNA sequence of the strain to be tested obtained by PCR was subjected to BLAST alignment analysis in NCBI. Combining the morphological, physiological and biochemical characteristics of the strain, the strain was identified as Bacillus halotolerans. This strain has been deposited in the China General Microbiological Culture Collection Center, and the deposit number is CCTCC NO: M 20241798.

[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 halotolerans BH74 strain was inoculated into 5 mL of LB liquid medium and cultured with shaking 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 sterilized and filtered twice with 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 the pathogen: The antibacterial effect of the sterile fermentation filtrate of the antagonistic bacterium was tested by the plate punching method. Three holes were evenly punched at 2.5 cm from the center of the plate (a petri dish with a diameter of 9 mm) with a puncher, and 40 μL of the fermentation filtrate was injected into each hole. The sterile LB liquid medium was used as a control, and a pathogen bacterial block with a diameter of 5 mm was inoculated in the center of the medium. Each treatment was repeated 3 times. After culturing in the dark at 28 °C for 5 d, the colony radius of the control group and the treatment group was measured, and the relative antibacterial rate was calculated.

[0054] 3. Results

[0055] The determination results of the effect of the sterile fermentation filtrate of the strain on the mycelial growth of the tobacco root rot pathogen are as Figure 2 shown in Table 2.

[0056] Table 2 Antibacterial activity of the sterile fermentation filtrate of BH74

[0057]

[0058] From Figure 2As can be seen from the results in Table 2, the fermentation filtrate of BH74 also has an inhibitory effect on the mycelial growth of Fusarium oxysporum f. sp. nicotianae( Figure 2 ), the size of the inhibition zone is 10.00 mm, the relative inhibition rate is 31.25%, and the antibacterial effect is lower than that of the living bacteria, with a decrease of 47.5% (Table 2).

[0059] Example 3. Determination of the growth promotion effect on tobacco seed germination

[0060] Select tobacco seeds with uniform and regular particles. Surface disinfect them with 75% ethanol for 30 s, wash them three times with sterile water, and dry them 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 two layers of moist filter paper, 50 seeds per dish. Use sterile water (CK1) and LB (CK2) liquid media as negative controls, and set 3 replicates for each treatment. Incubate them in the dark in a light 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 formula, 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 formula, S is the total length of the roots and shoots of the seedlings.

[0065] The results are shown in Table 3.

[0066] Table 3 Growth promotion effect of BH74 on tobacco seeds

[0067]

[0068] As can be seen from the results in Table 3, 10 days after the seeds were treated by soaking with the strain, the seed germination rate increased significantly, reaching 88.33%, an overall increase of 59% compared with the control group, indicating that it has a good promoting effect on the germination and growth of tobacco seeds.

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

[0070] Experimental design: blank group CK1, control group T1, and treatment group T2. Select tobacco seedlings with consistent growth vigor in advance, use sterilized scissors to cut off 1 cm of the root tips of the seedlings, cut 5 roots per plant, and prick the rhizomes with sterilized needles, with 5 wounds pricked on each rhizome. Then transfer them to sterilized nutrient pots (containing nutrient matrix), with 5 plants in each treatment, set 3 replicates, a total of 15 plants. Two days after transplantation, irrigate the plants by the method of drenching the roots. 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 broth prepared in Example 2 at a rate of 15 mL per plant. After 48 h, the control group and the treatment group were irrigated with Fusarium oxysporum spore suspension at a rate of 15 mL per plant. Among them, the concentration of fungal spores in the spore suspension was 5×10 6 per mL, and the concentration of the biocontrol bacteria fermentation broth was 1×10 5 CFU / mL. The disease incidence was statistically analyzed after the control group became diseased at 15 days of growth. The grading standard for tobacco root rot refers 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 × the value of that disease level) / (total number of plants or leaves surveyed × the highest disease level value)] × 100

[0073] Relative control effect = [(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, as can be seen from Figure 3 B, the rhizomes of the seedlings in the control group inoculated with the pathogen showed rot, and the plants died, presenting a pathological phenotype similar to that in the field. However, the seedlings in the treatment group (inoculated with BH74) showed no disease symptoms, and the rhizomes grew normally without rot symptoms, indicating that inoculating the pathogen indoors could cause tobacco to become diseased and die, but inoculating the potential biocontrol bacterium BH74 could effectively prevent the plants from getting diseased 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 due to Fusarium oxysporum, and the incidence of root rot reached 100.00%. While the incidence of tobacco seedlings inoculated with BH74 was 26.67%, the incidence was reduced by 73.33%. At the same time, the disease index of the diseased plants was also reduced, and the relative control effect was 66.57%.

[0078] In summary, a salt-tolerant Bacillus sp. BH74 provided by the present invention has a good disease prevention effect on tobacco root rot and a significant growth-promoting 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 "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[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 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 equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A strain of Bacillus halotolerans BH74, characterized in that: The deposit number of the Bacillus halotolerans BH74 is: CCTCC NO:M 20241798.

2. A fermentation agent, characterized in that: The fermentation agent comprises: The bacterial suspension is obtained by fermenting the halotolerant Bacillus sp. BH74 as claimed in claim 1.

3. The fermentation agent according to claim 2, characterized in that: The preparation method of the bacterial suspension comprises: The salt-tolerant Bacillus sp. BH74 according to claim 1 is inoculated into a liquid culture medium for fermentation culture to obtain the product.

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 halotolerant Bacillus sp. BH74 according to claim 1 or the fermentation agent according to claims 2 to 4 in preventing and treating tobacco root rot caused by Fusarium oxysporum.

6. Use of the halotolerant Bacillus sp. BH74 according to claim 1 or the fermentation agent according to claims 2 to 4 in promoting tobacco seed germination.

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