A strain of Bacillus altitudinis NM62 and its application in promoting growth and disease resistance

Abstract

The present invention provides a strain of Bacillus altitudinis NM62 and its application in promoting growth and disease resistance, with the preservation number of CGMCC NO.31272. This strain of Bacillus altitudinis has a good disease prevention effect on tobacco black shank disease and can promote the growth of tobacco plants. This strain provides a new strain resource for the biological control of tobacco black shank disease and provides a theoretical basis for the development of compound biocontrol agents that are environmentally friendly, biosafe and have a long-lasting control effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly relates to a strain of Bacillus altitudinis NM62 and its application in promoting growth and disease resistance. Background Art

[0002] During the growth process, tobacco is constantly attacked by pathogens such as fungi, bacteria, and viruses. These pathogens can cause various diseases, threatening the safety of tobacco leaf production and causing serious economic losses. Black shank caused by Phytophthora nicotianae is one of the most destructive plant soil-borne diseases. With the increasing demand for ecological agriculture, the disadvantages of completely relying on the use of chemical fertilizers and pesticides have gradually emerged. Currently, a large number of studies have shown that plant rhizosphere microorganisms can be used as biocontrol agents and growth-promoting bacteria to control plant diseases and promote plant growth. Microbial agents prepared from them have the characteristics of environmental friendliness and no pesticide residues, bringing a positive effect to the green development of agriculture and being an effective way to increase plant yield and control plant diseases.

[0003] In recent years, due to its environmentally friendly characteristics, biological control has become one of the important measures for plant disease control. Microbial agents have been widely used in the control of tobacco black shank and have achieved certain results. However, the microbial agents currently used still have unstable control effects. Therefore, there is an urgent need to find new biocontrol bacteria with stable effects and develop microbial biocontrol agents. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Bacillus altitudinis NM62 and its application in promoting growth and disease resistance. This strain of Bacillus altitudinis has a good disease prevention effect on tobacco black shank and can promote the growth of tobacco plants.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a strain of Bacillus altitudinis NM62 is provided. The taxonomic naming of the Bacillus altitudinis NM62 is Bacillus altitudinis , and the preservation number is: CGMCC NO.31272.

[0007] In the second aspect of the present invention, a fermentation agent is provided. The fermentation agent includes:

[0008] A fermentation broth or bacterial suspension obtained by fermenting and culturing the Bacillus altitudinis NM62;

[0009] Or a dry powder bacterial agent obtained by spray-drying the fermentation broth.

[0010] Further, the preparation method of the fermentation broth includes:

[0011] Inoculate the Bacillus altitudinis NM62 in a liquid medium for fermentation culture to obtain a fermentation broth.

[0012] Furthermore, the conditions for the fermentation culture include: the temperature is 26 - 30 °C, and the pH is 5 - 9.

[0013] In the third aspect of the present invention, there is provided the use of the Bacillus altitudinis NM62 in controlling tobacco black shank.

[0014] In the fourth aspect of the present invention, there is provided the use of the fermentation bacterium agent in controlling tobacco black shank.

[0015] In the fifth aspect of the present invention, there is provided the use of the Bacillus altitudinis NM62 in promoting the growth of tobacco.

[0016] In the sixth aspect of the present invention, there is provided the use of the fermentation bacterium agent in promoting the growth of tobacco.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0018] The Bacillus altitudinis provided in the embodiments of the present invention has a good disease prevention effect on tobacco black shank and can promote the germination of tobacco seeds and the growth of 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 can 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 multi-functional engineering bacteria such as promoting the growth of tobacco, providing a new strain resource for the biological control of tobacco black shank.

[0019] The preservation date of the Bacillus altitudinis NM62 of the present invention is July 11, 2024, and the preservation number is CGMCC NO. 31272. Its taxonomic name is Bacillus altitudinis Bacillus altitudinis , and the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1Colony morphology of Bacillus altitudinis NM62 on NA medium;

[0022] Figure 2 Phylogenetic tree of the strain;

[0023] Figure 3 Inhibitory effect of strain NM62 on Phytophthora nicotianae JM01;

[0024] Figure 4 Disease incidence and disease index 15 days after inoculation with Phytophthora nicotianae with or without inoculation of NM62. ** indicates P < 0.01. Among them, a is the picture 15 days after inoculation with or without Phytophthora nicotianae, and b is the statistical result of the disease index;

[0025] Figure 5 Comparison of disease resistance effects of different strains screened in the same period;

[0026] Figure 6 Pictures of the growth-promoting effect of strain NM62. Detailed implementation manners

[0027] The following will specifically describe the present invention in combination with the detailed 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 detailed implementation manners and examples are used to illustrate the present invention, rather than limiting the present invention.

[0028] 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 belongs. In case of contradiction, this specification shall prevail.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be obtained by existing methods.

[0030] The technical solution of the embodiment of the present application is to solve the above technical problems, and the general idea is as follows:

[0031] The inventor of the present application collected tobacco-growing soil from a tobacco experimental base in Jimo, Qingdao. Through isolation, purification, and screening, a strain of bacteria was obtained. It was found that this strain has a control effect on tobacco black shank. After colony morphology, biochemical, and 16S rRNA sequencing analysis of this bacterium, this strain has the highest similarity with Bacillus altitudinis ( Bacillus altitudinis ), which is 100%. Combining physiological and biochemical characteristics and phylogenetic tree analysis, it was initially determined that this strain belongs to ( Bacillus altitudinis ), and it was named Bacillus altitudinis ( Bacillus altitudinis ) NM62.

[0032] The Bacillus altitudinis of the present invention can promote the growth of tobacco plants and has a good disease prevention effect on tobacco black shank disease, providing a theoretical basis for the subsequent development of biological bacterial agents.

[0033] Next, a strain of Bacillus altitudinis NM62 of the present application and its application in promoting growth and disease resistance will be described in detail in combination with examples and experimental data.

[0034] Example 1: Isolation, purification and identification of Bacillus altitudinis NM62

[0035] 1. Sample collection

[0036] In July 2023, an experiment was carried out in a diseased tobacco field at the tobacco experimental base in Jimo, Qingdao. Methyl ferulate was externally applied to the diseased tobacco plants, and then healthy tobacco rhizosphere soil was collected. The whole tobacco plant with roots was dug out, the non-rhizosphere soil was shaken off, and the roots and the attached rhizosphere soil were put into a sterile self-sealing bag and transported back to the laboratory at low temperature for strain isolation and culture.

[0037] 2. Isolation and purification of soil microorganisms

[0038] Isolation and culture of bacteria: Weigh 1.0 g of the soil samples treated as above and dissolve it in 10 mL of sterile water. After shaking and mixing evenly, let it stand. Take the supernatant without soil particles on the upper layer for gradient dilution, and use sterile water to dilute the supernatant to 10 -2 、10 -3 、10 -4 respectively for later use. Take 100 μL of soil suspensions with different concentrations and spread them evenly on NA and 1 / 10 TSA solid media respectively, and then incubate them overnight at 28°C. Each treatment is repeated 2 times.

[0039] Select the concentration treatment with uniform and clearly visible colony distribution. Pick about 100 single colonies with different morphologies and sizes from the culture dish of this concentration and streak them on new 1 / 10 TSA and NA media, and then incubate them overnight at 28°C. After that, streak and purify all the bacteria according to the same steps.

[0040] Pick the single colonies of the purified bacteria and inoculate them into 6 mL of 1 / 10 TSA and NA liquid media, and then shake and culture them overnight at 28°C and 180 rpm / min. Take 800 μL of the bacterial liquid and an equal amount of 50% glycerol and add them to a 2 mL cryopreservation tube, and store them in a -80°C refrigerator.

[0041] 3. Identification of strains

[0042] (1)Colony morphology of Bacillus altitudinis NM62

[0043] Cultured on nutrient agar medium for 48 h, the colony diameter reached 1-2 mm; the morphology was a light yellow, smooth and regular round colony ( Figure 1 ).

[0044] (2)16S rRNA gene sequence determination

[0045] Using the colony PCR technique, the bacterial 16S rRNA gene sequence was amplified with the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). The PCR product was sent to Qingdao Ruibo Biotechnology Co., Ltd. for sequencing. The obtained bacterial sequence was uploaded to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for alignment after verification. The sequence is as shown in SEQ ID NO.1:

[0046] CCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGAC GATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAATGAAGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTT GACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCC ACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTC

[0047] After the alignment was completed, the model strain sequences with the highest homology were downloaded and CLUSTALX multiple alignment was performed using MEGA5.0, with the bootstrap value set to 1000 ( Figure 2 ).

[0048] The 16S rRNA sequence of the strain to be tested obtained by PCR was compared with that of Bacillus subtilis (Bacillus altitudinis The similarity is the highest, reaching 100%. Combining the morphological, physiological and biochemical characteristics of the strain and sequence analysis, the strain was identified as Bacillus altitudinis. This strain has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC NO. 31272.

[0049] Example 2: Determination of the antibacterial activity of Bacillus altitudinis NM62

[0050] 1. Preparation of the strain fermentation broth

[0051] Phytophthora nicotianae JM1 was inoculated on an oatmeal medium and cultured for 3 days. The strain NM62 was inoculated into an NB medium and cultured on a shaker at 28 °C and 180 r / min for 24 h.

[0052] 2. Determination of the antibacterial activity of the strain fermentation broth

[0053] A 6-mm mycelial disc was punched from the edge of the Phytophthora nicotianae colony and inoculated in the center of an MKB plate. Four 6-mm holes were punched at a distance of 2.5 cm from the edge of the plate, and 50 μL of the bacterial solution was added. Using the addition of sterile water as a control, after culturing for 3 days, the mycelial diameter was measured and the antibacterial rate was calculated.

[0054] 3. Results

[0055] The results of the determination of the effect of the strain fermentation broth on the mycelial growth of Phytophthora nicotianae are as Figure 3 shown in Table 1.

[0056] Table 1 Antibacterial activity of the sterile fermentation filtrate of NM62

[0057]

[0058] From Figure 3 the results in and Table 1, it can be seen that the fermentation broth of NM62 has an inhibitory effect on the mycelial growth of Phytophthora nicotianae ( Figure 3 ), the size of the antibacterial zone is 12.67 mm, and the antibacterial rate of the strain NM62 against Phytophthora nicotianae reaches 82.48% (Table 1).

[0059] Example 3: Disease prevention effect of Bacillus altitudinis NM62

[0060] 1) Preparation of Phytophthora nicotianae valley

[0061] Activate the mycelial blocks of Phytophthora nicotianae JM01 on oat agar (OA) medium in advance for 4 - 5 days. Boil millet until it is 2 / 3 in bloom, then take it out and air it until it can be formed into a ball when held by hand and dispersed when loosened. Divide it into 500 mL Erlenmeyer flasks and sterilize at high temperature. After cooling, inoculate the activated Phytophthora nicotianae cake, with 3 - 5 cakes (diameter 6 mm) inoculated into each flask. Incubate statically at room temperature for 14 d until the mycelia of black shank pathogen cover the surface, and then it can be used for inoculation.

[0062] 2) Biocontrol pot experiment of strain NM62

[0063] Inoculate NM62 into NB medium, shake culture at 28℃ and 180 rpm / min for 24 h, centrifuge at 4℃ and 6000 r / min for 5 min, and resuspend with sterile water to OD600 = 0.5; Sow tobacco seeds (Xiaohuangjin) into the seedling tray, perform pickling after emergence, culture for 30 d, and select tobacco seedlings with consistent growth; Weigh 200 g of substrate soil, add 2 g of fungus-infected millet to each pot, stir evenly and place it in a 9×9×8 cm plastic pot; Take out the tobacco seedlings, carefully shake off the soil on the roots and transplant; Pour 20 mL of NM62 bacterial solution along the roots of the tobacco seedlings, set 3 replicates, with 6 pots in each replicate. Use the non-irrigation of NM62 bacterial solution as the control group; Place the transplanted tobacco seedlings in an environment of 28℃ and 70% humidity for cultivation, and count the disease grade and incidence after the disease occurs, calculate the disease index and disease control effect.

[0064] Disease grading standard for tobacco black shank: Grade 0, the whole plant is disease-free; Grade 1, the lesion on the stem does not exceed 1 / 3 of the stem circumference, or the leaves below 1 / 3 wilt; Grade 3, the lesion on the stem surrounds 1 / 2 - 2 / 3 of the stem circumference, or 1 / 3 - 1 / 2 of the leaves wilt slightly; Grade 5: The lesion on the stem exceeds 1 / 2 of the stem circumference but does not completely surround the stem circumference, or 1 / 2 - 2 / 3 of the leaves wilt; Grade 7, the lesion on the stem completely surrounds the stem circumference, or more than 2 / 3 of the leaves wilt; Grade 9: The diseased plant is basically dead.

[0065] Incidence = (Number of diseased plants / Total number of plants investigated) × 100%

[0066] Disease index = [∑(Number of diseased plants or leaves at each level × Value of that disease level) / (Total number of plants or leaves investigated × Highest disease level value)] × 100

[0067] Relative control effect = [(Control disease index - Treatment disease index) / Control disease index] × 100%

[0068] 3. Results

[0069] As Figure 4 shown, after using NM62 bacterial solution, it can significantly reduce the infection of black shank pathogen on tobacco ( P(< 0.001), and the control effect reached 83.97%. Compared with other strains screened in the same batch (other strains screened in the same batch by the method of Example 1), its disease prevention effect is better ( Figure 5 ).

[0070] Example 4. Growth promotion effect of strain NM62

[0071] Sow the tobacco variety Xiaohuangjin 1025 susceptible to black shank disease with sterilized substrate. When 3 true leaves appear, transplant them into a seedling tray. After 6 true leaves grow, remove the plants with uneven growth, and keep the tobacco seedlings with consistent growth for use. Select the tobacco seedlings of Xiaohuangjin 1025 with consistent growth, and transfer the seedlings to a large pot with a diameter of 9 cm. The treatment group irrigated 20 mL of NM62 bacterial liquid with OD600 = 0.5 after resuspension along the roots of the tobacco seedlings, and the normal growth group added an equal amount of sterile water. There were 6 pots for each treatment, and the treatment was repeated 3 times. After 10 days, investigate the agronomic traits such as plant height, leaf length and leaf width.

[0072] The results are shown in Table 2.

[0073] Table 2 Effects of strain NM62 on the agronomic traits of tobacco

[0074]

[0075] As can be seen from Table 2 and Figure 6 it can be known that NM62 can significantly increase the plant height, maximum leaf length, maximum leaf area and fresh weight of tobacco seedlings, and has an obvious growth promotion function on the growth of tobacco seedlings.

[0076] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by 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.

[0077] 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.

[0078] 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.

[0079] Obviously, those skilled in the art can make various modifications and variations 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,

[0080] the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Bacillus subtilis ( Bacillus altitudinis )NM62, its deposit number is: CGMCC NO.31272.

2. A fermentation agent, characterized in that: The fermentation bacterial agent comprises: a fermentation liquid or a bacterial suspension obtained by fermenting and culturing the Bacillus subtilis NM62 as claimed in claim 1; or a dry powder bacterial agent obtained by spray-drying the fermentation liquid.

3. The fermentation agent according to claim 2, characterized in that: The Bacillus subtilis NM62 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 26-30° C. and pH of 5-9.

5. Use of the Bacillus subtilis NM62 according to claim 1 or the fermentation agent according to any one of claims 2 to 4 in preventing and controlling tobacco black shank disease.

6. Use of the Bacillus subtilis NM62 according to claim 1 or the fermentation agent according to any one of claims 2 to 4 in promoting the growth of tobacco plants.

Images