Detection method and application of tobacco endophytic nitrogen-fixing bacteria

Abstract

The invention discloses a method for detecting tobacco endophytic nitrogen-fixing bacteria and application. The method comprises the following steps: confirming that tobacco plants have nitrogen-fixing bacteria; culturing the surface sterilized tobacco leaf tissues on a solid nutrient agar culture medium; continuously culturing on a multi-carbon-source low-nitrogen culture medium and a nitrogen-free culture medium, and carrying out multiple times of streak purification to obtain endophytic nitrogen-fixing bacteria single colonies; observing and identifying the morphological structure; carrying out nitrogenase activity detection on the separated strain to confirm that the strain has nitrogen fixation capability; identifying the phosphorus solubilization and potassium solubilization of the separated strain by adopting an inorganic phosphorus culture medium and a potassium solubilization culture medium; carrying out enlarged culture on the separated strain, and extracting DNA for detection and analysis; the method comprises the following steps: sterilizing tobacco seeds, soaking the tobacco seeds in sterile water and bacterial liquid respectively, culturing the tobacco seeds in a culture dish, transferring the tobacco seeds to a nitrogen-free MS solid culture medium after germination, continuously culturing the tobacco seeds, and observing the influence of isolated strains on tobacco growth under a nitrogen-free condition. Compared with sterile water treatment, the tobacco root system treated by the bacterial liquid is more developed, the area of young leaves is larger, and the growth promoting effect on the tobacco is shown.

Description

Technical Field

[0001] The present invention relates to the field of detection of plant endophytic nitrogen-fixing bacteria, and particularly to a detection method and application of a tobacco endophytic nitrogen-fixing bacterium. Background Art

[0002] Chemical fertilizers can significantly improve the yield and quality of crops, and about half of the world's food supply is attributed to the use of chemical fertilizers. However, the overuse of chemical fertilizers will lead to a decline in nutrient utilization efficiency, which not only increases agricultural production input and causes waste of resources, but also brings a series of environmental problems, such as water eutrophication, air pollution, soil acidification, etc., seriously restricting the sustainable use of land and the sustainable development of agriculture.

[0003] Nitrogen fertilizer is the most demanded and important nutrient for plants, but at the same time, the loss after application is also very large. Therefore, in order to protect the ecological environment, reducing the application of chemical nitrogen fertilizer is of top priority. In addition to using organic fertilizers to replace chemical fertilizers, biological nitrogen fixation can also achieve good results. In biological nitrogen fixation, in addition to rhizobia that form root nodules in symbiosis with leguminous plants, there is also a class of endophytic nitrogen-fixing bacteria that widely exist inside plant tissues.

[0004] Plant endophytic nitrogen-fixing bacteria refer to a class of microorganisms that carry out associative nitrogen fixation with host plants and colonize inside the plants. Different from rhizobia, they do not need to form nodules with plants, can invade and colonize inside host plants, and can directly or indirectly provide the nitrogen source required for plant growth. In addition to nitrogen fixation, functions such as producing IAA, dissolving phosphorus, producing siderophores, and antagonizing pathogenic bacteria have been confirmed, and can be used in the fields of biological control and promoting plant growth. Therefore, it is expected to reduce the input of nitrogen fertilizer and promote the sustainable use of land through biological nitrogen fixation.

[0005] At present, the research on endophytic nitrogen-fixing bacteria at home and abroad mostly focuses on major crops such as sugarcane, rice, corn, and wheat, and there are few reports on the isolation, identification and growth-promoting effects of tobacco endophytic nitrogen-fixing bacteria. Tobacco occupies a very large area and requires a large amount of nitrogen fertilizer. Isolating and screening tobacco endophytic nitrogen-fixing bacteria is of great significance for reducing the use of chemical nitrogen fertilizer and improving the yield and quality of tobacco leaves. Summary of the Invention

[0006] In view of this, the present invention provides a detection method and application of a tobacco endophytic nitrogen-fixing bacterium.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A detection method of a tobacco endophytic nitrogen-fixing bacterium, comprising the following steps:

[0009] Step 1: By measuring the nitrogenase activity of each organ of tobacco, confirm the existence of nitrogen-fixing bacteria in the tobacco plant;

[0010] Step 2: Immerse the tobacco leaf tissue in 75% ethanol and 5.5% sodium hypochlorite for surface sterilization, and place the surface-sterilized tobacco leaf tissue on a solid nutrient agar medium for culturing to isolate endophytic bacteria from the tobacco leaves;

[0011] Step 3: The isolated endophytic bacteria from the tobacco leaves are continuously cultured on a multi-carbon-source low-nitrogen medium and a nitrogen-free medium in sequence, and single colonies of endophytic nitrogen-fixing bacteria are obtained through multiple streak purifications;

[0012] Step 4: Observe the purified strains and identify their morphological structures;

[0013] Step 5: Detect the nitrogenase activity of the isolated strains to confirm their nitrogen-fixing ability;

[0014] Step 6: Use an inorganic phosphorus medium and a potassium-solubilizing medium to further identify the phosphorus-solubilizing property and potassium-solubilizing property of the isolated strains;

[0015] Step 7: After the isolated strains are enlarged in culture, extract DNA and perform 16S rDNA detection and analysis;

[0016] Step 8: After sterilizing the tobacco seeds, soak them in sterile water and the bacterial solution respectively, place them in a culture dish for culturing, and transfer them to a nitrogen-free MS solid medium for continuous culturing after germination, and observe the effect of the isolated bacterial species on the growth of tobacco under nitrogen-free conditions.

[0017] Preferably, in Step 1, soil and tobacco root, stem, and leaf samples are taken respectively, and the roots are rinsed clean with running water; the above samples are subjected to the acetylene reduction method, and whether acetylene is reduced to form ethylene and the amount of ethylene are detected by a gas chromatograph to indirectly determine whether there is nitrogenase activity and its level.

[0018] Preferably, in Step 2, soak in 75% ethanol for 15 s, soak in sterile water for 30 s, soak in 5.5% sodium hypochlorite for 30 s, and soak in sterile water for 30 s and repeat 3 times.

[0019] Preferably, in Step 2, the formula of the nutrient agar medium is (adjust the pH to 7.0):

[0020]

[0021] Incubate in an inverted position at 27 °C on the solid nutrient agar medium for 3 days.

[0022] Preferably, in Step 3, the formula of the multi-carbon-source low-nitrogen medium is (adjust the pH to 7.0):

[0023]

[0024]

[0025] The improved Ashby nitrogen-free medium formula is (adjust the pH to 7.0):

[0026]

[0027] Culture for 1 day at 28 °C on the multi-carbon source low-nitrogen medium and the nitrogen-free medium.

[0028] Preferably, in step 5, scrape the colonies cultured on the nutrient agar medium and transfer them to the liquid Ashby nitrogen-free medium. After suspension, take a small amount and use the acetylene reduction method to indirectly determine whether there is nitrogenase activity by detecting whether acetylene is reduced to form ethylene and the amount of ethylene through a gas chromatograph.

[0029] Preferably, in step 6, after centrifuging the bacterial solution cultured overnight in the nutrient agar medium, discard the nutrient solution, suspend it with sterile water and adjust the concentration of the bacterial solution to make its OD600 about 0.5; take 3 μL of the bacterial solution and inoculate it into a quadrant culture dish respectively. Among them, area A: nutrient agar medium, area B: improved Ashby nitrogen-free medium, area C: inorganic phosphorus medium, area D: potassium-solubilizing medium;

[0030] The inorganic phosphorus medium formula is (adjust the pH to 7.0):

[0031]

[0032]

[0033] The potassium-solubilizing medium formula is (adjust the pH to 7.0):

[0034]

[0035] Incubate in an inverted position at 27 °C for 2 days.

[0036] Preferably, in step 8, sterilize the tobacco seeds as follows: soak them in 5.5% sodium hypochlorite for 2 min, wash them 5 times with sterile water, soak them in 75% ethanol for 30 s, and wash them 5 times with sterile water.

[0037] Preferably, in step 8, soak the sterilized tobacco seeds in sterile water and the bacterial solution for 10 min respectively, place them in a culture dish and culture them in the dark at 25 °C for 6 days; after taking root and germinating, transfer them to the nitrogen-free MS medium. The nitrogen-free MS medium formula is:

[0038]

[0039]

[0040] Culture at 25 °C / 20 °C under day / night conditions for 22 days.

[0041] Application of a detection method for an endophytic nitrogen-fixing bacterium in tobacco in tobacco production

[0042] The present invention has achieved the following technical effects compared with the prior art:

[0043] Compared with the sterile water treatment in the present invention, after the treatment with the bacterial solution, the tobacco roots are more developed and the young leaf area is larger, showing a growth-promoting effect on tobacco. Description of the Drawings

[0044] Figure 1 It is a graph showing the ranking of nitrogenase activities from large to small of the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0045] Figure 2 It is a graph showing the culture results of a solid nutrient agar medium for the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0046] Among them, left: tobacco leaf tissue; right: rinsing solution;

[0047] Figure 3 It is a graph showing the culture results of a solid nutrient agar medium for the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0048] Among them, left: nutrient agar medium; right: multi-carbon source low-nitrogen medium;

[0049] Figure 4 It is a graph showing the culture results of an Ashby nitrogen-free medium for the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0050] Among them, left: modified Ashby nitrogen-free medium; right: nutrient agar medium;

[0051] Figure 5 It is a microscopic image of an isolated strain for the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0052] Figure 6 It is a scanning electron microscope image of an isolated strain for the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0053] Figure 7 It is a graph showing the detection of nitrogenase activity of an isolated strain for the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0054] Figure 8 It is a graph showing the culture results of a quadrant petri dish for the detection method and application of an endophytic nitrogen-fixing bacterium in tobacco according to the present invention;

[0055] Among them, left: just inoculated; right: after 2 days of culture;

[0056] Figure 9 Sequence alignment diagram in NCBI for the detection method and application of an endophytic nitrogen-fixing bacterium of the present invention in tobacco

[0057] Figure 10 Root effect diagram of the detection method and application of an endophytic nitrogen-fixing bacterium of the present invention in tobacco

[0058] Among them, left: treated with sterile water; right: treated with soaking in bacterial liquid

[0059] Figure 11 Young leaf effect diagram of the detection method and application of an endophytic nitrogen-fixing bacterium of the present invention in tobacco

[0060] Among them, left: treated with sterile water; right: treated with soaking in bacterial liquid Detailed implementation manners

[0061] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention

[0062] Example 1

[0063] The present invention discloses a detection method for an endophytic nitrogen-fixing bacterium in tobacco, comprising the following steps

[0064] Step 1: By measuring the nitrogenase activity of each organ of tobacco, it is confirmed that nitrogen-fixing bacteria exist in the tobacco plant

[0065] In the tobacco (Yunyan 85) field in Luowugou, Puge County, Liangshan Prefecture, soil and tobacco root, stem, and leaf samples are taken respectively, and the roots are washed clean with running water. The above test samples are subjected to the acetylene reduction method, and whether acetylene is reduced to form ethylene and the amount of ethylene are detected by a gas chromatograph to indirectly determine whether there is nitrogenase activity and its level

[0066] The results show that nitrogenase activity is detected in each organ of tobacco and in the soil. The nitrogenase activity is ranked from large to small as soil, root, stem, and leaf. As Figure 1 shown, it indicates that nitrogen-fixing bacteria exist in the tobacco plant

[0067] Step 2: Soak the tobacco leaf tissue in 75% ethanol for 15 s and in 5.5% sodium hypochlorite for 30 s to achieve the surface sterilization effect. The surface-sterilized tobacco leaf tissue is cultured on a nutrient agar medium for 3 days to isolate the endophytic bacteria in the tobacco leaves

[0068] Using tobacco leaves as materials, in order to isolate endophytes, the tobacco leaves were first surface sterilized. Cut about 25 mm 2 sized tobacco leaf tissues. In a laminar flow hood, the tobacco leaf tissues were sequentially placed in the following solutions: soaked in 75% ethanol for 15 s, soaked in sterile water for 30 s, soaked in 5.5% sodium hypochlorite for 30 s, and soaked in sterile water for 30 s and repeated 3 times;

[0069] Finally, rinse the surface-sterilized tissues with sterile water and collect the rinsing solution. Take 400 μL of the collected rinsing solution and spread it on a solid nutrient agar medium. After using sterile absorbent paper to dry the surface moisture of the tobacco leaf tissues, place the surface-sterilized tobacco leaf tissues on the solid nutrient agar medium;

[0070] The above-mentioned medium was cultured upside down at 27 °C for 3 days. The results showed that colonies grew around the tobacco leaf tissues, but no colonies grew on the medium spread with the rinsing solution. As Figure 2 described, it indicates that the surface sterilization of the tobacco leaves was sufficient, and the grown bacteria belong to the endophytes of the tobacco leaves;

[0071] The formula of the nutrient agar medium is (adjust the pH to 7.0):

[0072]

[0073] Step 3: The isolated endophytes of tobacco leaves were continuously cultured on a multi-carbon source and low-nitrogen medium and a nitrogen-free medium in sequence, and single colonies of endophytic nitrogen-fixing bacteria were obtained after multiple streak separations

[0074] Transfer the Figure 2 colonies to the solid nutrient agar medium and the multi-carbon source and low-nitrogen medium respectively, and culture them at 28 °C for 1 day. The results showed that the transferred colonies grew significantly weaker on the multi-carbon source and low-nitrogen medium than on the nutrient agar medium. As Figure 3 shown, transfer the colonies on the multi-carbon source and low-nitrogen medium to the modified Ashby nitrogen-free medium for streak separation to obtain single colonies, and then purify them by multiple streakings on the nutrient agar medium. As Figure 4 shown;

[0075] The formula of the multi-carbon source and low-nitrogen medium is (adjust the pH to 7.0):

[0076]

[0077]

[0078] The formula of the modified Ashby nitrogen-free medium is (adjust the pH to 7.0):

[0079]

[0080] Step 4: The purified strains were observed under an optical microscope and a scanning electron microscope respectively, and their morphological structure was identified as rod-shaped Gram-positive bacteria

[0081] The Gram staining method was used to identify the strain morphology. 3 μL of the bacterial solution was dropped in the center of a sterilized glass slide in a sterile environment, and the bacterial solution was spread evenly with a pipette tip; the glass slide was quickly passed over the flame of an alcohol lamp 2-3 times to volatilize the liquid, and after cooling, 1 drop of ammonium oxalate crystal violet staining solution was added and stained for 1 min; after rinsing the glass slide with a small stream of water until the water was colorless, an appropriate amount of Gram staining solution was added and stained for 1 min. After rinsing the glass slide with a small stream of water, 95% ethanol was added dropwise to the glass slide until the flowing ethanol was not purple, and after about half a minute, it was rinsed with sterile water; 2 drops of safranin staining solution were added and stained for 1 min, rinsed with sterile water and air-dried at room temperature, and observed under an optical microscope;

[0082] The results showed that the isolated strains presented blue-violet, as Figure 5 shown, and they were Gram-positive bacteria;

[0083] The overnight cultured bacterial solution was centrifuged at 10000 g for 5 min to enrich the precipitate, 1 mL of PBS was added and rinsed 2 times, and the supernatant was removed by centrifugation at 5000 g; 0.8 mL of pre-cooled 2.5% glutaraldehyde was added and fixed overnight at 4°C; the supernatant was removed by centrifugation at 5000 g, and it was washed 3 times with PBS, 15 min each time. Then it was dehydrated successively with a series of gradient ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%), dehydrated once with each concentration of ethanol for 15 min each time, and then dehydrated thoroughly with 100% ethanol 2 times; 1 μL of the sample was pipetted onto a 5 mm×5 mm silicon wafer, and after natural drying, it was subjected to conductive treatment by vacuum sputtering, and after completion, it was observed under a scanning electron microscope;

[0084] The results showed that the bacteria presented rod-shaped, with a length of about 4 μm, as Figure 6 shown;

[0085] Step 5: The nitrogenase activity of the isolated strains was detected to confirm their nitrogen fixation ability

[0086] The colonies cultured on the nutrient agar medium were scraped and transferred to the liquid Ashby nitrogen-free medium. After suspension, a small amount was taken and the acetylene reduction method was used. Whether acetylene was reduced to form ethylene and the amount of ethylene were detected by a gas chromatograph to indirectly determine whether there was nitrogenase activity;

[0087] The results showed that the isolated strains had high nitrogenase activity, as Figure 7 shown;

[0088] Step 6: The phosphorus-solubilizing and potassium-solubilizing properties of the isolated strains were further identified using an inorganic phosphorus medium and a potassium-solubilizing medium

[0089] After centrifuging the bacterial solution cultured overnight in nutrient agar medium, discard the nutrient solution, suspend it with sterile water and adjust the concentration of the bacterial solution to make its OD600 about 0.5; Take 3 μL of the bacterial solution and inoculate it into a quadrant petri dish respectively. Among them, Area A: nutrient agar medium; Area B: modified Ashby nitrogen-free medium; Area C: inorganic phosphorus medium; Area D: potassium-solubilizing medium, and incubate it in an inverted position at 27 °C for 2 days;

[0090] The results showed that the strain could grow on both the inorganic phosphorus medium and the potassium-solubilizing medium. As Figure 8 shown, although the growth was weaker than that on the nutrient agar medium, it was better than that on the modified Ashby nitrogen-free medium, indicating that the strain had strong phosphorus-solubilizing and potassium-solubilizing functions;

[0091] The formula of the inorganic phosphorus medium (adjust the pH to 7.0) is:

[0092]

[0093]

[0094] The formula of the potassium-solubilizing medium (adjust the pH to 7.0) is:

[0095]

[0096] Step 7: After expanding the culture of the isolated strain, extract DNA and perform 16S rDNA detection and analysis;

[0097] Use the Ezup column bacterial genomic DNA extraction kit (B518255) to extract the DNA of the isolated strain, perform PCR amplification and then sequence analysis;

[0098] The results of 16S rDNA sequence analysis are as follows:

[0099]

[0100] The above sequence was aligned in NCBI, and the strain was identified as Bacillus, as Figure 9 shown;

[0101] Step 8: After sterilizing tobacco seeds, soak them in sterile water and bacterial liquid for 10 min respectively, place them in a petri dish and culture them in the dark for 6 days. After germination, transfer them to a nitrogen-free MS solid medium and continue to culture for 22 days to observe the effect of the isolated strain on tobacco growth under nitrogen-free conditions.

[0102] Sterilize tobacco (Yunyan 85) seeds as follows: soak them in 5.5% sodium hypochlorite for 2 min, wash them 5 times with sterile water, soak them in 75% ethanol for 30 s, and wash them 5 times with sterile water.

[0103] The sterilized tobacco seeds were soaked in sterile water and bacterial liquid for 10 min respectively, placed in a petri dish and cultured at 25 °C in the dark for 6 days. After taking root and germinating, transfer them to a nitrogen-free MS medium and culture them at 25 °C / 20 °C (day / night) for 22 days.

[0104] The results showed that compared with the sterile water treatment, the tobacco roots were more developed after the bacterial liquid treatment, manifested as longer roots and more branches, as Figure 10 shown, and the area of young leaves was larger, as Figure 11 shown; it indicates that the isolated strain has a promoting effect on the growth of tobacco under nitrogen-deficient conditions.

[0105] The formula of the nitrogen-free MS medium is:

[0106]

[0107]

[0108] The present invention also discloses a detection method of an endophytic nitrogen-fixing bacterium in tobacco and its application in tobacco production.

[0109] The above is only a preferred embodiment of the present invention, and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A detection method for an endophytic nitrogen-fixing bacterium in tobacco, characterized in that It includes the following steps: Step 1: By measuring the nitrogenase activity of each organ of tobacco, it is confirmed that nitrogen-fixing bacteria exist in tobacco plants; Step 2: Immerse the tobacco leaf tissue in 75% ethanol and 5.5% sodium hypochlorite for surface sterilization, and place the surface-sterilized tobacco leaf tissue on a solid nutrient agar medium for culture to isolate endophytic bacteria in tobacco leaves; Step 3: The isolated endophytic bacteria in tobacco leaves are continuously cultured on a multi-carbon-source low-nitrogen medium and a nitrogen-free medium in sequence, and single colonies of endophytic nitrogen-fixing bacteria are obtained after multiple streak purifications; Step 4: Observe the purified strains and identify their morphological structures; Step 5: Detect the nitrogenase activity of the isolated strains to confirm their nitrogen-fixing ability; Step 6: Use an inorganic phosphorus medium and a potassium-solubilizing medium to further identify the phosphorus-solubilizing and potassium-solubilizing properties of the isolated strains; Step 7: After the isolated strains are enlarged in culture, extract DNA and perform 16S rDNA detection and analysis; Step 8: After the tobacco seeds are sterilized, soak them in sterile water and the bacterial solution respectively, place them in a culture dish for culture, and transfer them to a nitrogen-free MS solid medium for continuous culture after germination, and observe the effect of the isolated bacterial species on the growth of tobacco under nitrogen-free conditions.

2. The detection method of an endophytic nitrogen-fixing bacterium of tobacco according to claim 1, characterized in that, In step 1, soil and tobacco root, stem, and leaf samples are taken respectively, and the roots are washed clean with running water; the above samples are subjected to the acetylene reduction method, and whether acetylene is reduced to form ethylene and the amount of ethylene are detected by a gas chromatograph to indirectly determine whether there is nitrogenase activity and its level.

3. The detection method of an endophytic nitrogen-fixing bacterium in tobacco according to claim 1, characterized in that, In step 2, soak in 75% ethanol for 15 s, soak in sterile water for 30 s, soak in 5.5% sodium hypochlorite for 30 s, and soak in sterile water for 30 s and repeat 3 times.

4. The detection method of an endophytic nitrogen-fixing bacterium in tobacco according to claim 1, characterized in that, In step 2, the formula of the nutrient agar medium is (adjust the pH to 7.0): Invert and culture on the solid nutrient agar medium at 27°C for 3 days.

5. The detection method of an endophytic nitrogen-fixing bacterium of tobacco according to claim 1, characterized in that, In step 3, the formula of the multi-carbon-source low-nitrogen medium is (adjust the pH to 7.0): The formula of the modified Ashby nitrogen-free medium is (adjust the pH to 7.0): Culture on the multi-carbon-source low-nitrogen medium and the nitrogen-free medium at 28°C for 1 day.

6. The detection method of an endophytic nitrogen-fixing bacterium of tobacco according to claim 1, characterized in that In step 5, scrape the colonies cultured on the nutrient agar medium and transfer them to a liquid Ashby nitrogen-free medium, take a small amount after suspension, and use the acetylene reduction method to detect whether acetylene is reduced to form ethylene and the amount of ethylene by a gas chromatograph to indirectly determine whether there is nitrogenase activity.

7. The detection method of an endophytic nitrogen-fixing bacterium in tobacco according to claim 1, characterized in that, In step 6, after centrifuging the bacterial solution cultured overnight in the nutrient agar medium, discard the nutrient solution, suspend it with sterile water and adjust the bacterial solution concentration to make its OD600 about 0.5; take 3 μL of the bacterial solution and inoculate it into a quadrant culture dish respectively, where area A: nutrient agar medium, area B: modified Ashby nitrogen-free medium, area C: inorganic phosphorus medium, area D: potassium-solubilizing medium; The formula of the inorganic phosphorus medium is (adjust the pH to 7.0): The formula of the potassium-solubilizing medium is (adjust the pH to 7.0): Invert and culture at 27°C for 2 days.

8. The detection method of an endophytic nitrogen-fixing bacterium of tobacco according to claim 1, characterized in that In step 8, sterilize the tobacco seeds, and the operation is as follows: soak in 5.5% sodium hypochlorite for 2 min, wash with sterile water 5 times, soak in 75% ethanol for 30 s, and wash with sterile water 5 times.

9. The detection method of an endophytic nitrogen-fixing bacterium of tobacco according to claim 1, characterized in that, In step 8, the sterilized tobacco seeds are soaked in sterile water and the bacterial solution for 10 min respectively, placed in a culture dish and cultured in the dark at 25 °C for 6 days; after taking root and germinating, they are transferred to an MS medium without nitrogen, and the formula of the MS medium without nitrogen is as follows: Cultivate for 22 days at 25 °C / 20 °C under day / night conditions.

10. Application of the detection method of an endophytic nitrogen-fixing bacterium in tobacco according to claims 1-9 in tobacco production.

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