A kind of Burkholderia SN11 bacterial agent and its application

Abstract

The invention discloses a Burkholderia contaminans SN11, a bacterial agent and an application thereof, and belongs to the technical field of biological bacterial agents; the Burkholderia contaminans SN11 has a preservation number of CCTCCM20242491, and the preservation unit is the China Center for Type Culture Collection (CCTCC); the Burkholderia contaminans SN11 and the bacterial agent thereof can effectively promote the growth of plants under acidic soil stress, thereby achieving the improvement of the ability of plants to resist acid stress, and can effectively improve the yield of plants, and provide technical support for the development of a functional microbial preparation dedicated to acidic soil areas for enhancing the acid stress resistance of crops and improving crop yield, and is of great significance.

Description

Technical Field

[0001] The present invention relates to the technical field of biological microbial agents, and in particular to a Burkholderia SN11, a microbial agent and applications thereof. Background Art

[0002] Farmland soil acidification destroys soil structure and causes soil compaction, leading to soil nutrient loss, reduced nutrient effectiveness, and decreased fertility, which is not conducive to crop nutrient absorption, resulting in a decrease in crop yield and quality. It has become one of the global problems that affect crop yield and endanger soil productivity. Natural soil acidification is a slow process, but farmland soil is accelerated by frequent human activities, thus bringing a series of negative impacts to the farmland soil-microorganism-crop system.

[0003] Compared with breeding methods, improving the acid resistance of crops through rhizosphere microorganisms is faster and more effective. Microbial agents can effectively improve the physical and chemical properties of soil, thereby increasing the quality and yield of crops, and at the same time can enhance the stress resistance of crops. The development of functional microbial preparations that enhance crop acid stress resistance and increase crop yields in acidic soils using plant root growth-promoting strains has great application prospects. Alkali-producing microorganisms can affect the form of heavy metals in the soil by increasing the pH value of the soil, thereby reducing the degree of heavy metal pollution. It can also loosen soil compaction, alleviate acidification, and reduce the occurrence of pests and diseases, thereby improving crop yield and quality.

[0004] Under acid stress, basal growth-promoting bacteria can increase nutrient absorption rate. Different strains have significantly different strategies for coping with acid stress. Microorganisms may preferentially regulate SOD and CAT activities to cope with acid stress, or regulate glutathione transferase activity to change the content and ratio of oxidized and reduced glutathione to alleviate the effects of acid stress. Common basal growth-promoting bacteria that enhance plant tolerance to acid stress include nitrogen-fixing bacteria, acetobacillus, and Agrobacterium, but Burkholderia tumefaciens that enhances plant tolerance to acid stress has not yet been found. Summary of the invention

[0005] The object of the present invention is to provide a Burkholderia SN11, a bacterial agent and its application. The Burkholderia SN11 can effectively improve the ability of plants to resist acid stress and increase the yield of plants to solve the deficiencies in the prior art. The specific contents are as follows.

[0006] The present invention provides a Burkholderia contaminans SN11, with a preservation number of CCTCCM20242491, a preservation address of the School of Life Sciences of Wuhan University, a preservation unit of the China Center for Type Culture Collection (CCTCC), and a preservation time of November 8, 2024.

[0007] The present invention also provides a bacterial agent containing the Burkholderia SN11.

[0008] Preferably, the bacterial agent is a fermentation broth of Burkholderia SN11 or a bacterial suspension containing Burkholderia SN11.

[0009] Preferably, the bacterial suspension containing Burkholderia SN11 includes bacterial bodies of Burkholderia SN11 and bacterial powder of Burkholderia SN11.

[0010] Preferably, the OD600 value of the bacterial agent is 0.9-1.1, more preferably 1.0.

[0011] The present invention also provides a method for preparing the fermentation broth of Burkholderia SN11:

[0012] The Burkholderia SN11 was inoculated into a TSB liquid culture medium for fermentation until the OD600 value was 0.9 to 1.1, thereby obtaining the fermentation liquid of the Burkholderia SN11.

[0013] Preferably, the fermentation culture temperature is 28-32°C, more preferably 30°C.

[0014] The present invention also provides a method for preparing a bacterial suspension containing Burkholderia SN11:

[0015] The fermentation liquid of Burkholderia SN11 is centrifuged, and the precipitate is washed to obtain washed Burkholderia SN11 bacterial cells, which are then centrifuged, freeze-dried, and ground to obtain bacterial powder.

[0016] The present invention also provides the use of the bacterial agent in improving the acid stress resistance of plants and / or promoting plant growth.

[0017] Preferably, the application in promoting plant growth includes increasing the germination rate of plant seeds and / or promoting the growth and development of plant plants.

[0018] Preferably, the promoting the growth and development of the plant comprises at least one of increasing the plant height, increasing the soil pH, increasing the fresh weight of the aboveground part, increasing the fresh weight of the underground part, increasing the dry weight of the aboveground part and increasing the dry weight of the underground part.

[0019] Preferably, the plant comprises tomato.

[0020] Preferably, the application includes any one of the following:

[0021] 1) 7 days after transplanting the plants, watering the bacterial agent on the roots of the plant seedlings;

[0022] 2) adding the bacterial agent to a plant seedling substrate, and then transplanting the plant seedlings to a field for planting;

[0023] 3) soaking the plant seedlings with the bacterial agent, and then transplanting the soaked seedlings into the field for planting;

[0024] 4) applying the bacterial agent to the field when the plants are transplanted;

[0025] 5) Spraying the bacterial agent on the leaves of seedlings during the seedling stage.

[0026] Preferably, the bacterial agent in 1) is used in a pot plant experiment.

[0027] Preferably, after watering in 1), the amount of inoculation on the roots of the plant seedlings is (0.75-1.25)×10 8 CFU / g root, more preferably 108 CFU / g root.

[0028] Preferably, the dosage of the bacterial agent in 2) is (0.75-1.25)×10 8 CFU / g root.

[0029] Preferably, the root soaking time in 3) is 12 to 24 hours, and the concentration of the bacterial agent is 10 8 CFU / mL.

[0030] Preferably, the application of the microbial agent in 4) during plant transplanting is:

[0031] Apply 2 kg of microbial agent mixed with 140-200 catties of water per mu of land, and soak the seedlings for 3-5 hours before transplanting.

[0032] Preferably, the method of foliar spraying in 5) is:

[0033] Mix the fungal agent and water evenly, add 100 to 140 kilograms of water per kilogram and spray three times a week.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention provides a Burkholderia SN11, and has completed biological preservation. The Burkholderia SN11 of the present invention is isolated from acidic soil in Dehong, Yunnan (23°50′N, 97°31′E), and is confirmed to be Burkholderia through colony characteristics and 16SrDNA sequence identification. The colony of the Burkholderia of the present invention on LB solid culture medium is small, convex and smooth, and milky white ( Figure 1), the Burkholderia SN11 described in the present invention has 100.0% similarity with the Burkholderia contaminans strain; on this basis, the present invention also provides a bacterial agent containing the Burkholderia SN11, and the Burkholderia SN11 and its bacterial agent can effectively improve the germination rate of plant seeds and promote plant growth under acid stress, thereby improving the ability of plants to resist acid stress, and at the same time can effectively increase plant yields, providing technical support for the development of functional microbial preparations dedicated to acid land to enhance crop acid stress resistance and increase crop yields, and is of great significance.

[0036] (2) The strain of the present invention has excellent alkali production ability. Compared with the blank LB test tube without the strain, the pH value of the LB test tube added with the strain of the present invention increased by 0.62 after 16 hours of culture, an increase of 13.71%; increased by 1.42 after 24 hours, an increase of 31.19%; increased by 1.58 after 48 hours, an increase of 34.60%; and increased by 2.35 after 24 hours, an increase of 51.38%.

[0037] (3) The Burkholderia SN11 bacterial agent of the present invention has a significant effect on promoting plant growth. Figures 5-6 It can be concluded that by the end of potted planting, compared with the control group, the soil pH of the experimental group treated with bacterial agents significantly increased by 0.31 units, an increase of 6.69%; the plant height significantly increased by 2.44cm, an increase of 16.26%; the dry weight of the aboveground and underground parts significantly increased by 0.53g and 0.09g, respectively, an increase of 14.19% and 36.62%.

[0038] Biological deposit information

[0039] Burkholderia contaminans SN11 was deposited in the China Center for Type Culture Collection (CCTCC) on November 8, 2024. The deposit address is the School of Life Sciences, Wuhan University, and the deposit number is CCTCCM20242491. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a purification diagram of the Burkholderia contaminans SN11 strain of the present invention;

[0041] Figure 2 It is a phylogenetic tree diagram of the Burkholderia contaminans SN11 strain of the present invention;

[0042] Figure 3This is a graph showing the effect of Burkholderia contaminans SN11 on environmental pH;

[0043] Figure 4 This is a colorimetric test result of the alkali production ability of Burkholderia contaminans SN11;

[0044] Figure 5 This is a diagram showing the effect of the bacterial agent of the present invention on acidic soil;

[0045] Figure 6 This is a diagram showing the effects of the bacterial agent of the present invention on tomato plant height, above-ground and underground stems. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0048] Example 1

[0049] Isolation and identification of bacterial species

[0050] The Burkholderia SN11 described in the present invention was isolated from acidic soil in Dehong, Yunnan (23°50′N, 97°31′E), and was confirmed to be Burkholderia by colony characteristics and 16S rDNA sequence identification. The colony of the Burkholderia SN11 described in the present invention on LB solid culture medium is small, convex and smooth in surface, and milky white ( Figure 1 ), the Burkholderia SN11 described in the present invention has 100.0% similarity with the Burkholderiacontaminans strain.

[0051] The 16S rDNA sequence of Burkholderia SN11 of the present invention is as follows, specifically:

[0052] CAGCGAAGCTTACCATGCAAGTCGACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTTGGCTCTAATACAGTCGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGCTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATCCTGCTGAGAGTGGGAGTGCTCGAAAGAGACCGGCGCACA。

[0053] Example 2

[0054] This embodiment provides a method for preparing a bacterial liquid and bacterial powder containing Burkholderia SN11:

[0055] The activated Burkholderia SN11 was inoculated into TSB liquid culture medium for fermentation.

[0056] Each liter of TSB liquid culture medium contains the following components: 15 g of tryptone, 5 g of soy peptone, and 5 g of sodium chloride. After the culture medium is prepared, the pH value is adjusted to 7.2 and sterilized at 121°C for 20 minutes.

[0057] During the fermentation culture, the fermentation temperature is 30° C., the rotation speed is 170 rpm, and the fermentation is performed until OD600=1.0. The fermentation product is the bacterial liquid.

[0058] The bacterial liquid is centrifuged, freeze-dried and crushed to obtain bacterial powder.

[0059] Example 3

[0060] This example verifies the alkali production capacity of Burkholderia SN11

[0061] 1. Pick the purified strains and place them in a fresh LB test tube (the pH of the culture medium in the test tube was adjusted to 4.5 first), shake on a shaker at 30°C and 200 rpm, and culture for 16h, 24h, 48h, and 72h. Use a pH meter to measure the pH value of the LB test tube in these four time periods;

[0062] The purification process involves picking colonies with a sterile inoculation loop and streaking them on a new LB solid culture medium with a pH of 4.5. After growing for 48 hours, the single colony that grows is the purified strain.

[0063] 2. At the same time, perform a color development experiment and prepare bromocresol blue culture medium for staining. The culture medium will appear purple. Drop the bacterial liquid on the culture medium and culture it for 48h to 72h. Observe the color change of the culture medium.

[0064] Figure 3 This is a graph showing the effect of the strain on the environmental pH. It can be seen that compared with the blank-treated LB test tube without the strain, the pH value increased by 0.62 after 16 hours of culture, an increase of 13.71%; increased by 1.42 after 24 hours, an increase of 31.19%; increased by 1.58 after 48 hours, an increase of 34.60%; and increased by 2.35 after 24 hours, an increase of 51.38%, proving that the strain has excellent alkali production ability.

[0065] Figure 4This is the result of the color development experiment of the alkali production ability of the strain. It can be seen that the color around the bacterial liquid is purple, indicating that Burkholderia SN11 has a certain alkali production ability.

[0066] Example 4

[0067] This embodiment provides an application of Burkholderia SN11 in promoting the growth of tomato potted plants in soil

[0068] 1. After surface disinfection and germination of tomato seeds, seedlings with consistent germination status were selected and transplanted into a designated soil with a soil background pH of 4.68 for an acid stress growth test.

[0069] 2. After 7 days, water the bacterial solution prepared in Example 2 around the roots of the seedlings, maintaining the inoculation concentration of each bacteria at 10 8 CFU / g, and the same volume of sterile water was irrigated as the control group. Under the conditions of 30℃, 16h / 8h photoperiod, and light intensity of 8000 lux, various physiological and biochemical indicators of tomato plants were measured after 14 days of growth.

[0070] 3. The test indicators include plant height, fresh weight of aboveground and underground parts, and dry weight of aboveground and underground parts. The plant height is measured using a tape measure, and the fresh weight of aboveground and underground parts, as well as the dry weight of aboveground and underground parts (dried to constant weight) are measured using an electronic balance. Each test indicator is measured with 4 biological replicates and the average value is taken.

[0071] 4. Set up two treatment groups, add bacterial agent S experimental group and control group CK, set up 6 biological replicates for each treatment group, 1 kg of soil for each pot, and keep the nutrients equal during the treatment. 0.4 g of nitrogen fertilizer (0.86 g of urea) was applied to each pot, 0.14 g of phosphorus fertilizer (2.123 g of superphosphate) was applied to each pot, and 0.3 g of potassium fertilizer (0.67 g of potassium sulfate) was applied to each pot. The background pH of the soil was 4.68.

[0072] Figure 5 This is the effect of bacterial agents on acidic soil. Figure 6 The effect of the bacterial agent on tomato plant height, aboveground and underground dry matter content is shown in Figure 2. Figures 5-6It can be concluded that by the end of potted planting, compared with the control group, the soil pH of the experimental group treated with bacterial agents significantly increased by 0.31 units, an increase of 6.69%; the plant height significantly increased by 2.44cm, an increase of 16.26%; the dry weight of the aboveground and underground parts significantly increased by 0.53g and 0.09g, respectively, an increase of 14.19% and 36.62%. Comparison shows that the Burkholderia SN11 inoculant provided by the present invention can significantly improve the growth of tomato seedlings in soil culture under acid stress. The soil pH of the roots irrigated with the Burkholderia SN11 inoculant is significantly higher than that of the acid soil treatment group without inoculation; the plant height of the tomato seedlings irrigated with the Burkholderia SN11 inoculant is significantly higher than that of the acid soil treatment group without inoculation; the underground dry weight of the tomato seedlings irrigated with the Burkholderia SN11 inoculant is significantly higher than that of the acid soil treatment group without inoculation, and the aboveground dry weight of the tomato seedlings irrigated with the Burkholderia SN11 inoculant is significantly higher than that of the acid soil treatment group without inoculation. Therefore, the Burkholderia SN11 inoculant of the present invention has a significant effect on promoting plant growth.

[0073] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that all equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A Burkholderia contaminans SN11, characterized in that The deposit number is CCTCCM20242491, the deposit address is the School of Life Sciences, Wuhan University, the deposit unit is China Center for Type Culture Collection (CCTCC), and the deposit time is November 8, 2024.

2. A bacterial agent containing the Burkholderia SN11 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is one of a fermentation broth of Burkholderia SN11 and a bacterial suspension containing Burkholderia SN11.

4. The bacterial agent according to claim 3, characterized in that The bacterial suspension containing Burkholderia SN11 includes bacterial bodies of Burkholderia SN11 and bacterial powder of Burkholderia SN11.

5. The bacterial agent according to claim 3, characterized in that The OD600 value of the bacterial agent is 0.9-1.

1.

6. Use of the bacterial agent according to claims 2 to 5 for improving the acid stress resistance of plants and / or promoting plant growth.

7. The use according to claim 6, characterized in that: The plants include tomatoes.

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