A strain of Priesterol NM17 and its application in controlling tobacco black shank disease

Abstract

The present invention provides a strain of Priesteria gigantea NM17 and its application in preventing and controlling tobacco black shank disease, and its deposit number is CGMCC NO.31273. The strain of Priesteria gigantea NM17 has a good disease prevention effect on tobacco black shank disease and has the activity of producing elongin and siderophore. The strain provides a new strain resource for biological control of tobacco black shank disease and provides a theoretical basis for developing environmentally friendly, biosafe, and long-lasting composite biocontrol agents.

Description

Technical Field

[0001] The invention relates to the technical field of microbiology, and in particular to a Priesteria gigantea NM17 strain and application thereof in preventing and treating tobacco black shank disease. Background Art

[0002] Black shank disease caused by Phytophthora nicotianae is one of the most destructive soil-borne plant diseases. Due to its wide host range and diverse transmission pathways, the pathogen forms dormant oospores and survives in the soil for a long time. So far, it is still very difficult to control soil-borne diseases caused by Phytophthora such as tobacco and potato. At present, chemical pesticides and breeding of resistant varieties are commonly used to control tobacco black shank disease in production. However, the long-term use of large amounts of chemical drugs not only pollutes the environment, but also makes the pathogen resistant to drugs; due to the strong environmental adaptability, fast spore formation, short incubation period and rapid onset of Phytophthora, it is more difficult to breed disease-resistant varieties. At the same time, disease resistance is difficult to balance with yield, quality and other traits. Therefore, the use of disease-resistant varieties to control the damage of Phytophthora has entered a bottleneck period in production practice. With the increase in demand for ecological agriculture, the disadvantages 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. Microbial preparations are environmentally friendly and free of pesticide residues, which brings positive effects to the green development of agriculture. They are an effective way to increase plant yields and prevent and control plant diseases.

[0003] 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 Priesteria gigantea NM17 and application thereof. The strain of Priesteria gigantea has good disease prevention effect on tobacco black shank disease and has the activity of producing elongin and siderophore.

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

[0006] In a first aspect of the present invention, a strain of Priesteria gigantea ( Priestia megaterium )NM17, the Priococcus megaterium ( Priestia megaterium )The deposit number of NM17 is: CGMCC NO.31273.

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

[0008] Fermentation culture of the Priesteria megaterium NM17 to obtain a fermentation broth or bacterial suspension;

[0009] Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

[0010] Furthermore, the preparation method of the bacterial suspension comprises:

[0011] The Prioccludinium megaterium NM17 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid.

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

[0013] In the third aspect of the present invention, provided is the use of the Priococcus megaterium NM17 or the fermentation agent in preventing and treating tobacco black shank disease.

[0014] In the third aspect of the present invention, provided is the use of the Priococcus megaterium NM17 or the fermentation agent in producing elongin or siderophore.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0016] The Priesteria gigantea provided in the embodiment of the present invention has a good disease prevention effect on tobacco black shank, and has the activity of producing elongin and producing siderophore. It can be seen that the strain has good theoretical research value and application prospects. It can not only be used as a good experimental material for studying the interaction between soil bacteria and plants and their mechanism of action, but also can be used for the research and development of soil biofertilizers and biofungicides. At the same time, it may also become a good carrier for multifunctional engineering bacteria such as producing elongin and producing siderophore, providing a new strain resource for the biological control of tobacco black shank.

[0017] The deposit date of the Prioccidioides giganteus NM17 of the present invention is July 11, 2024, and the deposit number is CGMCCNO.31273. Its classification name is Prioccidioides giganteus Priestia megaterium 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

[0018] 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.

[0019] Figure 1This is the colony morphology of Priesterol NM17 on NA medium;

[0020] Figure 2 is the strain phylogenetic tree;

[0021] Figure 3 For the determination of IAA production of strains;

[0022] Figure 4 To determine the siderophore production of the strain;

[0023] Figure 5 The inhibitory effect of bacterial strain NM17 on tobacco phytophthora JM01;

[0024] Figure 6 The disease incidence and disease index 15 days after inoculation with Phytophthora nicotianae. ** indicates P < 0.01, where a is the picture 15 days after inoculation with Phytophthora nicotianae, and b is the statistical result of the disease index. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0029] The inventors of the present application obtained a bacterial strain from tobacco-growing soil collected from disease-free fields in Nanzhang, Zaoyang and other places through separation, purification and screening, and found that the strain has a preventive and therapeutic effect on tobacco black shank disease. The bacterial strain was analyzed by colony morphology, biochemistry and 16SrRNA sequencing, and it was found that the strain was different from Priesteria gigantea ( Priestia megaterium ) has the highest similarity of 99.30%. Combined with physiological and biochemical characteristics, it is preliminarily determined that the strain belongs to ( Priestia megaterium ), and named Priesteria gigantea ( Priestia megaterium )NM17.

[0030] The Priesteria gigantea of ​​the present invention has the activity of producing elongin and siderophore, has a good disease prevention effect on tobacco black shank disease, and provides a theoretical basis for the subsequent development of biological bacterial agents.

[0031] The following will describe in detail a strain of Priococcus giganteus NM17 of the present application and its application in preventing and controlling tobacco black shank disease in combination with examples and experimental data.

[0032] Example 1: Isolation, purification and identification of Priococcus megaterium NM17

[0033] 1. Sample collection

[0034] 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.

[0035] 2. Isolation and purification of soil microorganisms

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 3. Identification of strains

[0040] (1) Colony morphology of Priococcus megaterium NM17

[0041] After 48 h of cultivation on nutrient agar medium, the colony diameter reached 1-4 mm; the colony was light yellow, smooth and regular round ( Figure 1 ).

[0042] (2) 16S rRNA gene sequence determination

[0043] The bacterial 16S rRNA gene sequence was amplified using colony PCR technology using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGA CTT-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:

[0044] TCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCCTGGGGAGTACGGTCGCAAGACTGAACTCAAAGGGAATTGACGGGGCCCGCACAGGCGG

[0045] After the alignment is completed, download the sequences of the model strains with the highest homology respectively, and perform CLUSTALX multiple alignment using MEGA5.0, with the bootstrap value set to 1000 ( Figure 2 ).

[0046] The 16S rRNA sequence of the strain to be tested obtained by PCR was compared and analyzed by BLAST in NCBI. It was found that the 16S rRNA gene sequence of strain NM17 had the highest similarity with Priestia megaterium, which was 99.30%. Combined with the morphological, physiological and biochemical characteristics of the strain and the phylogenetic tree analysis, strain NM17 was determined to be Priestia megaterium. The strain has been deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.31273.

[0047] Example 2: Determination of Growth-Promoting Indexes of Priococcus megaterium NM17

[0048] 1. Use a pipette to draw 2 mL of bacterial solution in a clean bench and add it to a 2 mL centrifuge tube for centrifugation (4°C, 8000r / min for 10 min). Take 1 mL of supernatant and add 1 mL of colorimetric solution (Salkowski reagent) to a 10 mL centrifuge tube. After standing in the dark for 30 min, quickly use a spectrophotometer in an ELISA reader to colorimetrically measure the absorbance at a wavelength of 530 nm, and calculate the IAA content based on the absorbance.

[0049] The results showed that strain NM17 had the activity of producing cytochrome P60, with an IAA concentration of 64.86 μg / mL. Compared with other strains in the same batch, the content of cytochrome P60 was higher ( Figure 3 ).

[0050] 2. Evaluation of vector production capacity:

[0051] Take 1 ml of the supernatant of the strain after centrifugation (4 ℃, 8000 r / min centrifugation for 10 min) and add it to a sterilized 10 ml centrifuge tube. Add 1 mL of CAS detection solution to each centrifuge tube, mix thoroughly and let it stand in the dark for 30 min. Use a pipette to draw 200 μL of each tube of bacterial solution and add it to a 96-well ELISA plate. Quickly use an ELISA reader to measure the absorbance value (As) of the sample at 630 nm. Take an equal volume of blank culture medium and CAS solution and mix them, and use its absorbance value as the reference value (Ar). According to the formula [(Ar-As) / Ar]×100%, the relative content of iron carriers is obtained. The As / Ar ratio represents the iron carrier production capacity of the strain. As / Ar is between 0-1, and a "+" is added for every 0.2 decrease.

[0052] The results showed that strain NM17 had the activity of producing siderophores, and its siderophore production capacity was "++". Compared with other strains from the same batch (other strains screened from the same batch using the method of Example 1), its siderophore production capacity was higher ( Figure 4 ).

[0053] Example 3, Determination of antibacterial activity of Priesterol NM17

[0054] 1. Preparation of strain fermentation broth

[0055] The tobacco fungus JM01 was inoculated into oatmeal medium and cultured for 3 days. The strain NM17 was inoculated into NB medium and cultured in a shaking incubator at 28°C and 180 r / min for 24 h.

[0056] 2. Determination of antibacterial activity of strain fermentation broth

[0057] 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.

[0058] 3. Results

[0059] The results of the test on the effect of the fermentation liquid of the strain on the mycelial growth of tobacco phytophthora are as follows Figure 5 And as shown in Table 1.

[0060] Table 1 Antibacterial activity of NM17 sterile fermentation filtrate

[0061] Indicator bacteria Diameter of inhibition zone (mm) Antibacterial rate (%) Phytophthora nicotianae 15.50±2.89 43.64 %±0.08

[0062] Depend on Figure 5 From the results in Table 1, it can be seen that the fermentation broth of NM17 has an inhibitory effect on the mycelial growth of Phytophthora nicotianae, the size of the inhibition zone is 15.50 mm, and the inhibition rate of strain NM17 on Phytophthora nicotianae reaches 43.64% (Table 1).

[0063] Example 4: Disease prevention effect of Priococcus megaterium NM17

[0064] 1. Preparation of tobacco phytophthora

[0065] 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 Phytophthora nicotianae cakes, 3-5 cakes (6 mm in diameter) per bottle. Incubate at room temperature for 14 days until the mycelium of black leg pathogen is fully grown, and then it can be used for inoculation.

[0066] 2. Biocontrol potted plant test of strain NM17

[0067] NM17 was inoculated into NB medium, shaken at 28°C, 180 rpm / min for 24 h, centrifuged at 4°C, 6000 r / min for 5 min, and resuspended in sterile water to OD 600=0.5; sow tobacco seeds (small gold) into seedling trays, and transplant after emergence. After 30 days of cultivation, select tobacco seedlings with consistent growth; weigh 200 g of substrate soil, add 2 g of fungus grain to each pot, stir evenly and place in a 9×9×8 cm plastic pot; take out the tobacco seedlings, carefully shake off the soil at the roots and transplant; water 20 mL of NM17 bacterial solution along the roots of the tobacco seedlings, set up 3 replicates, and set 6 pots in each replicate. The control group was not watered with NM17 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.

[0068] 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.

[0069] Incidence rate = (number of diseased plants / total number of plants investigated) × 100%

[0070] 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

[0071] Relative control effect = [(control disease index - treatment disease index) / control disease index] × 100%

[0072] 3. Results

[0073] like Figure 6 As shown in the figure, the use of NM17 bacterial solution can significantly reduce the infection of black shank pathogen on tobacco, and the control effect reaches 80.77%, ** indicates P < 0.01.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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 Priesteria gigantea NM17, characterized in that: The taxonomic name of the P. megaterium NM17 is Priestia megaterium, The deposit number is: CGMCC NO.31273.

2. A fermentation agent, characterized in that: The fermentation agent comprises: A fermentation broth or bacterial suspension obtained by fermenting the Priestera gigantea NM17 as claimed in claim 1; Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

3. The fermentation agent according to claim 2, characterized in that: The preparation method of the bacterial suspension comprises: The fermentation liquid is obtained by inoculating the Prioccludinium giganteum NM17 described in claim 1 into a liquid culture medium for fermentation culture.

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 Priococcus megaterium NM17 according to claim 1 or the fermentation agent according to any one of claims 2 to 4 in preventing and treating tobacco black shank disease.

6. Use of the Priococcus megaterium NM17 according to claim 1 or the fermentation agent according to any one of claims 2 to 4 in producing elongin or siderophore.

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