Bacillus altitudinis NM62 and application thereof in growth promotion and disease resistance

Abstract

The invention provides bacillus altitudinis NM62 and application of the bacillus altitudinis NM62 in growth promotion and disease resistance. The preservation number of the bacillus altitudinis NM62 is CGMCC NO.31272. The bacillus altitudinis strain has a good disease prevention effect on tobacco black shank and can promote the growth of tobacco plants. The strain provides a new strain resource for biological prevention and control of tobacco black shank, and provides a theoretical basis for developing a composite biocontrol agent which is environment-friendly, biosafe and lasting in prevention and control effect.

Description

Technical Field

[0001] The invention relates to the technical field of microbiology, and in particular to a strain of Bacillus subtilis NM62 and application thereof in growth promotion and disease resistance. Background Art

[0002] During its growth, tobacco is constantly attacked by pathogens such as fungi, bacteria and viruses. These pathogens can cause a variety of diseases, threatening tobacco production safety and causing serious economic losses. Black shank disease caused by Phytophthoranicotianae is one of the most destructive soil-borne plant diseases. With the increasing demand for ecological agriculture, the drawbacks of relying entirely on the use of chemical fertilizers and pesticides have gradually been exposed. At present, a large number of studies have shown that plant rhizosphere microorganisms can be used as biocontrol bacteria and growth-promoting bacteria to prevent and control plant diseases and promote plant growth. The microbial preparations prepared with them as raw materials are environmentally friendly and free of pesticide residues, which has a positive effect on the green development of agriculture and is an effective way to increase plant yields and prevent and control plant diseases.

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

[0004] The invention aims to provide a strain of Bacillus subtilis NM62 and its application in growth promotion and disease resistance. The strain of Bacillus subtilis NM62 has a good disease prevention effect on tobacco black shank disease and can promote the growth of tobacco plants.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: In a first aspect of the present invention, a strain of Bacillus subtilis NM62 is provided. The taxonomic name of the strain is Bacillus altitudinis , the deposit number is: CGMCC NO.31272.

[0006] In a second aspect of the present invention, a fermentation agent is provided, the fermentation agent comprising: Fermenting and culturing the Bacillus subtilis NM62 to obtain a fermentation liquid or bacterial suspension; Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

[0007] Furthermore, the method for preparing the fermentation broth comprises: The Bacillus subtilis NM62 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid.

[0008] Furthermore, the fermentation culture conditions include: temperature of 26-30°C and pH of 5-9.

[0009] In the third aspect of the present invention, the use of the Bacillus subtilis NM62 in preventing and treating tobacco black shank disease is provided.

[0010] In the fourth aspect of the present invention, the use of the fermentation bacteria agent in preventing and treating tobacco black shank disease is provided.

[0011] In the fifth aspect of the present invention, the application of the Bacillus subtilis NM62 in promoting tobacco growth is provided.

[0012] In the sixth aspect of the present invention, there is provided the use of the fermentation bacteria agent in promoting tobacco growth.

[0013] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The highland Bacillus provided in the embodiment of the present invention has a good disease prevention effect on tobacco black shank, and can promote tobacco seed germination and tobacco plant growth. 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 biological fertilizers and biological fungicides. At the same time, it may also become a good carrier of multifunctional engineering bacteria such as promoting tobacco growth, providing a new strain resource for the biological control of tobacco black shank.

[0014] The deposit date of the Bacillus subtilis NM62 of the present invention is July 11, 2024, and the deposit number is CGMCCNO.31272. Its classification name is Bacillus subtilis Bacillus altitudinis The name of the depository is the General Microbiology Center of China Microbiological Culture Collection Administration, and its address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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.

[0016] Figure 1 This is the colony morphology of Bacillus subtilis NM62 on NA medium; Figure 2 is the strain phylogenetic tree; Figure 3The inhibitory effect of strain NM62 on tobacco phytophthora JM01; Figure 4 The disease incidence and disease index of the patients inoculated with / without NM62 and then inoculated with Phytophthora nicotianae 15 days later. ** indicates P < 0.01, where a is the picture of the patients inoculated with / without Phytophthora nicotianae 15 days later, and b is the statistical result of the disease index. Figure 5 To compare the disease resistance of different strains screened at the same time; Figure 6 The picture shows the growth-promoting effect of strain NM62. DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows: The inventor of the present application obtained a bacterial strain from tobacco-growing soil collected from the Qingdao Jimo Tobacco Experimental Base. Through separation, purification and screening, it was found that the strain had a preventive and therapeutic effect on tobacco black shank disease. The bacterial strain was analyzed by colony morphology, biochemistry and 16SrRNA sequencing, and the strain was different from Bacillus subtilis ( Bacillus altitudinis ) has the highest similarity of 100%. Combined with physiological and biochemical characteristics and phylogenetic tree analysis, it is preliminarily determined that the strain belongs to ( Bacillus altitudinis ), and named it Bacillus subtilis ( Bacillus altitudinis )NM62.

[0021] The highland bacillus of the invention can promote the growth of tobacco plants, has a good disease prevention effect on tobacco black shank disease, and provides a theoretical basis for the subsequent development of biological bacterial agents.

[0022] The following will describe in detail a strain of Bacillus subtilis NM62 of the present application and its application in growth promotion and disease resistance in combination with examples and experimental data.

[0023] Example 1: Isolation, purification and identification of Bacillus subtilis NM62 1. Sample collection In July 2023, an experiment was carried out in the diseased tobacco fields of Qingdao Jimo Tobacco Experimental Base. Methyl ferulate was applied exogenously to the diseased tobacco plants, and then the rhizosphere soil of healthy tobacco was collected. The whole tobacco plants with roots were dug out, the non-rhizosphere soil was shaken off, and the roots and attached rhizosphere soil were placed in sterile self-sealing bags and transported back to the laboratory at low temperature for strain isolation and culture.

[0024] 2. Isolation and purification of soil microorganisms Isolation and culture of bacteria: Weigh 1.0 g of the soil samples treated with different methods above and dissolve them in 10 mL of sterile water. Mix by shaking and let stand. Take the supernatant without soil particles from the upper layer and dilute it to 10 mL with sterile water. -2 , 10 -3 , 10 -4 100 μL of soil suspensions of different concentrations were evenly plated on NA and 1 / 10 TSA solid culture media, and then cultured at 28°C overnight. Each treatment was repeated twice.

[0025] Select a concentration treatment with uniform and clearly visible colonies, pick about 100 single colonies of different morphology and size from the culture dish of this concentration, streak them on new 1 / 10 TSA and NA culture media, and then culture them at 28°C overnight. After that, streak all bacteria according to the same steps.

[0026] Pick a single colony of purified bacteria and inoculate it into 6 mL of 1 / 10 TSA and NA liquid culture medium, then shake and culture overnight at 28°C and 180 rpm / min. Take 800 μL of bacterial solution and an equal amount of 50% glycerol and add them to 2 mL cryovials and store them in a -80°C refrigerator.

[0027] 3. Identification of strains (1) Colony morphology of Bacillus subtilis NM62 After 48 h of cultivation on nutrient agar medium, the colony diameter reached 1-2 mm; the colony was light yellow, smooth and regular round ( Figure 1 ).

[0028] (2) 16S rRNA gene sequence determination The bacterial 16S rRNA gene sequence was amplified using colony PCR technology using 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 proofread and uploaded to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for comparison. The sequence is shown in SEQ ID NO.1: CCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGAC GATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAATGAAGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTT GACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCC ACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTC 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 ).

[0029] The 16S rRNA sequence of the strain to be tested obtained by PCR was compared with that of Bacillus subtilis ( Bacillus altitudinis) has the highest similarity of 100%. Combined with the strain's morphology, physiological and biochemical characteristics and sequence analysis, the strain was determined to be Bacillus subtilis. The strain has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.31272.

[0030] Example 2, Determination of antibacterial activity of Bacillus subtilis NM62 1. Preparation of strain fermentation broth The tobacco fungus JM1 was inoculated into oatmeal medium and cultured for 3 days. The strain NM62 was inoculated into NB medium and cultured in a shaking incubator at 28°C and 180 r / min for 24 h.

[0031] 2. Determination of antibacterial activity of strain fermentation broth A 6 mm bacterial cake was taken from the edge of the tobacco phytophthora colony and inoculated into the center of the MKB plate. Four 6 mm holes were punched 2.5 cm from the edge of the plate and 50 μL of bacterial solution was added. Sterile water was used as a control. After culturing for 3 days, the mycelium diameter was measured and the inhibition rate was calculated.

[0032] 3. Results The results of the test on the effect of the fermentation liquid of the strain on the mycelial growth of tobacco phytophthora nicotianae are as follows Figure 3 And as shown in Table 1.

[0033] Table 1 Antibacterial activity of NM62 sterile fermentation filtrate Indicator bacteria Diameter of inhibition zone (mm) Antibacterial rate (%) Phytophthora nicotianae 12.67±2.89 82.48 %±0.08 Depend on Figure 3 As shown in Table 1, the fermentation broth of NM62 has an inhibitory effect on the mycelial growth of Phytophthora nicotianae ( Figure 3 ), the size of the inhibition zone was 12.67 mm, and the inhibition rate of strain NM62 against tobacco phytopathogenes reached 82.48% (Table 1).

[0034] Example 3: Disease prevention effect of Bacillus subtilis NM62 1) Preparation of tobacco Phytophthora inoculum In advance, culture and activate the mycelium of Phytophthora nicotianae JM01 on oatmeal agar (OA) medium for 4-5 days. Take out the millet when 2 / 3 of it blooms, dry it until it can be held in a ball and loosen, and divide it into 500 mL triangular bottles and sterilize it at high temperature. After cooling, inoculate the activated tobacco phytophthora cakes, 3-5 cakes (6 mm in diameter) per bottle. Incubate at room temperature for 14 days until the black leg fungus mycelium is fully grown, and then it can be used for inoculation.

[0035] 2) Biocontrol potted plant test of strain NM62 NM62 was inoculated into NB medium, shaken at 28℃, 180 rpm / min for 24 h, centrifuged at 4℃, 6000 r / min for 5 min, and resuspended with sterile water to OD600=0.5; tobacco seeds (small gold) were sown into seedling trays, and transplanted after emergence. After 30 days of cultivation, tobacco seedlings with consistent growth were selected; 200 g of substrate soil was weighed, 2 g of fungus grain was added to each pot, and the mixture was placed in a 9×9×8 cm plastic pot after stirring evenly; the tobacco seedlings were taken out, the soil at the roots was carefully shaken off and transplanted; 20 mL of NM62 bacterial solution was poured along the roots of the tobacco seedlings, and 3 replicates were set, with 6 pots in each replicate. The control group was not watered with NM62 bacterial solution; the transplanted tobacco seedlings were cultured at 28℃ and 70% humidity. After the disease occurred, the disease grade and incidence rate were counted, and the disease index and disease prevention effect were calculated.

[0036] Grading standards for tobacco black shank disease: Grade 0, the whole plant is disease-free; Grade 1, stem lesions do not exceed 1 / 3 of the stem circumference, or less than 1 / 3 of the leaves are withered; Grade 3, stem lesions surround 1 / 2~2 / 3 of the stem circumference, or 1 / 3~1 / 2 of the leaves are slightly withered; Grade 5: stem lesions exceed 1 / 2 of the stem circumference, but do not completely surround the stem circumference, or 1 / 2~2 / 3 of the leaves are withered; Grade 7, stem lesions completely surround the stem circumference, or more than 2 / 3 of the leaves are withered; Grade 9: the diseased plant is basically dead.

[0037] Incidence rate = (number of diseased plants / total number of plants investigated) × 100% 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 Relative control effect = [(control disease index - treatment disease index) / control disease index] × 100% 3. Results like Figure 4 As shown in the figure, the use of NM62 bacterial solution can significantly reduce the infection of black leg pathogen on tobacco ( P <0.001), the control effect reached 83.97%. Compared with other strains screened in the same batch (other strains screened in the same batch using the method of Example 1), its disease prevention effect is better ( Figure 5 ).

[0038] Example 4: Growth-promoting effect of strain NM62 The tobacco variety Xiaohuangjin 1025, which is susceptible to black shank, was sown in a sterilized substrate. When three true leaves appeared, the plants were transplanted to the seedling tray. After six true leaves grew, the plants with uneven growth were removed, and the tobacco seedlings with uniform growth were kept for use. The tobacco seedlings of Xiaohuangjin 1025 with uniform growth were selected and transferred to a large pot with a diameter of 9 cm. The treatment group was watered along the roots of the tobacco seedlings with 20 mL of NM62 bacterial solution with OD600=0.5 after resuspension, and the normal growth group was added with an equal amount of sterile water. Six pots were treated each time and the treatment was repeated three times. After 10 days, agronomic traits such as plant height, leaf length and leaf width were investigated.

[0039] The results are shown in Table 2.

[0040] Table 2 Effects of strain NM62 on agronomic traits of tobacco Plant height Maximum leaf length Leaf Width Leaf area Fresh weight Dry weight CK 5.08±0.28 15.25±0.28 9.10±0.21 138.88±4.80 8.52±0.38 0.48±0.03 NM62 6.23±0.24* 16.35±0.29* 9.55±0.18 156.29±5.17* 9.64±0.28* 0.52±0.04 From Table 2 and Figure 6 It can be seen 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-promoting function on the growth of tobacco seedlings.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 subtilis NM62, characterized in that: The taxonomic name of the Bacillus subtilis NM62 is Bacillus altitudinis , the deposit number is: CGMCC NO.31272.

2. A fermentation agent, characterized in that: The fermentation agent comprises: Fermentation culture of the Bacillus subtilis NM62 as claimed in claim 1 to obtain a fermentation broth or 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 method for preparing the fermentation broth comprises: The Bacillus subtilis NM62 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 in preventing and treating tobacco black shank disease.

6. Use of the fermentation agent according to any one of claims 2 to 4 in preventing and treating tobacco black shank disease.

7. Use of the Bacillus subtilis NM62 according to claim 1 in promoting the growth of tobacco plants.

8. Use of the fermentation agent according to any one of claims 2 to 4 in promoting the growth of tobacco plants.

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