Azotobacter coxakella for promoting plant growth and application of azotobacter coxakella

Abstract

The invention belongs to the field of agricultural microorganisms, and particularly relates to coxakella cocori and application thereof. The coxakia cokei is preserved in the China General Microbiological Culture Collection Center, the classification name of the coxakia cokei is HHQGLZ30-1 (Kosakia cokei HHQGLZ30-1), the preservation date of the coxakia cokei is January 29, 2024, and the preservation number of the coxakia cokei is CGMCC No.29798. The coxakia cokei is named as the coxakia cokei HHQGLZ30-1. The coxakella cocori can secrete auxin, has nitrogen fixation capacity and siderophore production capacity, can convert iron elements which are difficult to absorb and utilize by plants in soil into siderophore chelated iron, and can promote plant growth and antagonize phytopathogens. By applying the growth-promoting and disease-preventing microbial agent prepared from the Corisakia cocori HHQGLZ30-1, the stress resistance of crops can be effectively improved, the growth of the crops is promoted, the crops are healthier, and the leaf surfaces are brighter and greener.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural microorganisms, and relates to a Kosakonia cowanii, in particular to a Kosakonia cowanii and its application. Background Art

[0002] According to the statistics of the Food and Agriculture Organization of the world, China's nitrogen fertilizer consumption accounts for 30%, but the average utilization rate of nitrogen fertilizer is only 28%-41%, which is significantly lower than the world level. The loss of nitrogen fertilizer into the environment will cause a series of environmental problems, such as soil compaction, soil pollution, etc., which not only cause waste of resources, but also seriously affect the sustainable development of agriculture and human health. Therefore, reducing the application of chemical fertilizers and improving the utilization rate of nitrogen fertilizer are important tasks in current agricultural production.

[0003] Nitrogen-fixing bacteria refer to those microorganisms that fix nitrogen in association with host plants, mainly bacteria. They live, reproduce, and spread in plant tissues, especially vegetative tissues, without causing obvious damage to the plants. Plant nitrogen-fixing bacteria is an efficient nitrogen-fixing system different from symbiotic nitrogen fixation and associative nitrogen fixation. It mainly lives in non-leguminous plants, avoiding competition with indigenous microorganisms that are inhibitors of combined nitrogen, and has a higher nitrogen-fixing efficiency. Utilizing biological nitrogen fixation is currently the most effective way to reduce the application amount of nitrogen fertilizer.

[0004] Indole-3-acetic acid (IAA) plays an important role in plant morphogenesis, organogenesis, and various physiological processes, especially crucial for plant growth and development. Iron (Fe) is an essential mineral nutrient element for plant growth and plays an important role in plant respiration, photosynthesis, and nitrogen metabolism. Although iron is relatively abundant in the earth's crust, due to the oxygen-rich environment of the earth, iron oxides exist in a form with extremely low solubility and are not easily directly absorbed and utilized by plants. Siderophores are small molecule (1-2 ku) compounds with a strong specific chelating ability for Fe 3 + (the chelating coefficient can reach 1020-1030). Many plant rhizosphere microorganisms can synthesize such substances to uptake iron in the environment and provide the excess iron for plants to use. They can compete with harmful pathogenic bacteria with very low rhizosphere iron carrier production in plants for iron elements, thereby inhibiting the growth and reproduction of harmful microorganisms.

[0005] Therefore, screening microorganisms with biological nitrogen fixation, IAA production, and siderophore production plays a very important role in the development of growth-promoting and disease-preventing microbial agents. Summary of the Invention

[0006] The object of the present invention is to provide a Kosakonia cowanii HHQGLZ30-1 with nitrogen fixation ability and siderophore production ability, which can secrete plant growth hormone, convert iron elements in the soil that are difficult for plants to absorb and utilize into siderophore-chelated iron, promote plant growth and antagonize plant pathogens, and develop a microbial inoculant for promoting growth and preventing diseases.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: A strain of Kosakonia cowanii, classified and named Kosakonia cowanii HHQGLZ30-1, was deposited at the General Microbiological Center of the China Microbial Culture Collection Center on January 29, 2024, with the deposit number: CGMCC No. 29798, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Preferably, the Kosakonia cowanii HHQGLZ30-1 can secrete plant growth hormone (IAA), has nitrogen fixation ability and siderophore production ability.

[0009] Preferably, the Kosakonia cowanii HHQGLZ30-1 is made into a microbial inoculant for promoting growth and preventing diseases, including the following components: Kosakonia cowanii HHQGLZ30-1, Trichoderma harzianum, fucoidan oligosaccharide, 5-aminolevulinic acid.

[0010] Preferably, the specific concentration of the microbial inoculant for promoting growth and preventing diseases containing Kosakonia cowanii HHQGLZ30-1 is 2×10 9 -1×10 10 CFU / mL.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The Kosakonia cowanii HHQGLZ30-1 isolated from the rhizosphere soil sample of plants in Linze Farm, Linze County, Gansu Province has nitrogen fixation ability and siderophore production ability, can secrete plant growth hormone, convert iron elements in the soil that are difficult for plants to absorb and utilize into siderophore-chelated iron, promote plant growth and antagonize plant pathogens. After the crop is applied with the microbial inoculant for promoting growth and preventing diseases prepared from this strain, the stress resistance of the crop is effectively improved, the growth of the crop is promoted, the crop becomes healthier, and the leaf surface is greener and tenderer. Description of the Drawings Figure 1 Colony morphology of Kosakonia cowanii HHQGLZ30-1 on the Ashby nitrogen-free solid medium plate.

[0013] Figure 2 Microscopic examination image of the cells of Kosakonia cowanii HHQGLZ30-1.

[0014] Figure 3Functional identification diagram of Kosakonia cowanii HHQGLZ30-1 for indole-3-acetic acid production

[0015] Figure 4 Chromogenic circle produced by Kosakonia cowanii HHQGLZ30-1 on CAS plate Detailed implementation mode

[0016] The present invention provides a nitrogen-fixing endophyte promoting plant growth Kosakonia cowanii and its application. To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention

[0017] Example 1 Isolation, screening and identification of strains Kosakonia cowanii HHQGLZ30-1 provided by the present invention was isolated from the rhizosphere soil sample of plants in Linze Farm, Linze County, Gansu Province. The steps are as follows: Weigh 10 g of the rhizosphere soil of plants in Linze Farm, Linze County, Gansu Province, add it to a 250 mL conical flask containing 90 mL of sterile water and glass beads, shake for 30 min, then place it in a water bath at 80 °C for 30 min, shake well again, dilute it by 10-fold serial dilution, and coat it on an Ashby nitrogen-free medium (the components are 10 g of sucrose, 0.2 g of NaCl, KH2PO 0.2 g, 0.2 g of MgSO4·7H2O, 0.1 g of CaSO4·2H2O, 5 g of CaCO3, 1000 mL of distilled water, pH 7.0) plate, and culture it at 30 °C for 2-3 d until single colonies grow. After the colonies grow, use the streaking method to purify the strain on the Ashby nitrogen-free medium plate 5 times to obtain a nitrogen-fixing bacterium, numbered: HHQGLZ30-1

[0018] 16S rDNA gene sequencing was used to classify and identify Kosakonia cowanii HHQGLZ30-1: The strain HHQGLZ30-1 was sent to the sequencing and identification department of Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification, and the sequencing results were compared and analyzed on the ribosomal database. The 16S rDNA sequence analysis results showed that the strain of the present invention had a homology of up to 99.8% with the Kosakonia cowanii of the genus Kosakonia, that is, the strain was Kosakonia cowanii, Specifically Kosakonia cowanii HHQGLZ30-11

[0019] This strain has the following properties 1. Colony morphological characteristics Kosakonia cowanii HHQGLZ30-1 belongs to Gram-positive bacilli, arranged singly, non-motile. The colonies on the LB medium plate are light yellow, with a smooth surface, opaque, moist, regular in shape, neat in edge, and without wrinkles. The optimal growth temperature is 35 °C, the pH range is 4.5 - 9.5 (optimum 7.0), and the NaCl tolerance range is 0 - 8% (w / v); the colonies Figure 1 are shown as and the microscopic examination pictures are as Figure 2 shown.

[0020] 2. Physiological characteristics of the strain Kosakonia cowanii HHQGLZ30-1 is positive for VP, catalase, citrate utilization, glucose fermentation, and Gram staining, and negative for phenylalanine deaminase, methyl red, urease, amylase, and nitrate reduction. The colonies grow normally at salt concentrations of 4%, 6%, and 8%, and do not grow at 10%. It can use galactose, sucrose, mannose, urea, inositol, lactose, and glucose as carbon sources, and ammonium nitrate, sodium nitrate, and methionine as nitrogen sources.

[0021] Example 2 Determination of the ability of Kosakonia cowanii HHQGLZ30-1 strain to produce phytohormones The isolated Kosakonia cowanii HHQGLZ30-1 strain was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured on a shaker at 30 °C and 180 r•min-1 for 24 h. Take 100 μL of the bacterial suspension on a white ceramic plate, and then drop 100 μL of Salkowski colorimetric solution (1 mL of 0.5 mol / L FeCl3 + 50 mL of 35% HClO4). Use the uninoculated LB liquid culture as a control and add an equal volume of Salkowski colorimetric solution. Place the white ceramic plate in the dark for 30 min, take it out and observe. A red color indicates the ability to produce indole-3-acetic acid, and the darker the color, the higher the indole-3-acetic acid content. The test results show that the strain HHQGLZ30-1 can produce indole-3-acetic acid, as Figure 3 shown.

[0022] Example 3 Qualitative detection of siderophores of Kosakonia cowanii HHQGLZ30-1 strain The isolated Kosakonia cowanii HHQGLZ30-1 strain was inoculated on a CAS plate and observed for the production and size of orange-red transparent zones on the plate after culturing at 35 °C for 3 - 5 days. HHQGLZ30-1 produced orange-red transparent zones on the CAS plate. The test results show that the strain HHQGLZ30-1 has the function of producing siderophores, as Figure 4 shown.

[0023] Example 4 Promotion of seed germination by Kosakonia cowanii HHQGLZ30-1 strain Select plump pepper seeds of the same size, disinfect them with 75% absolute ethanol for 30 s and 2% NaClO for 3 min, then rinse them thoroughly with sterile water. Soak the seeds in water and the HHQGLZ30-1 bacterial solution for 12 h respectively, and then place the seeds in a petri dish with filter paper, with 30 seeds in each dish. Set up the CK group and the MD10 group in the experiment. There are 5 petri dishes for each treatment, and 5 replicates are set. Place them in a plant culture room at 25 °C with 12 h of daily light, supplement sterile water to the petri dishes every day, and record the number of germinated seeds every day. After two weeks of treatment, calculate the germination rate, germination potential and germination index of pepper seeds.

[0024]

[0025] As can be seen from Table 1, the germination rate of the HHQGLZ30-1 group increased by 7% compared with the CK group. For the effect on the germination potential, the germination rate of the HHQGLZ30-1 group increased by 30% compared with the CK group; in terms of the germination index, the value of the HHQGLZ30-1 group increased by 47% compared with the CK group; the experimental results show that Kosakonia cowanii HHQGLZ30-1 can promote the germination of pepper seeds.

[0026] Example 5 Kosakonia cowanii Growth promotion test of HHQGLZ30-1 on rice Pick single colonies from the LB medium plate and inoculate them into the liquid LB medium, shake and culture at 30 °C for 2 days to obtain the HHQGLZ30-1 fermentation broth. Dilute it 200 - 600 times with water before use. Select rice seedlings with two leaves and one heart for the test samples, irrigate the roots once every 7 days, and collect samples after using it 4 times to measure the fresh weight and dry weight of the above-ground parts of the test rice. Use water as the negative control.

[0027]

[0028]

[0029] As can be seen from Table 2, the fresh weight of the above-ground parts of the fermentation broth treated at 400-fold and 500-fold dilutions increased by 25.96% and 28.22% respectively compared with the control, and the differences both reached the extremely significant level; the fresh weight of the above-ground parts of the 200-fold dilution treatment increased by 19.47% compared with the control, and the difference reached the significant level; the fresh weight of the above-ground parts of the 200-fold and 600-fold dilutions increased by 10.52% and 17.14% respectively compared with the control, and the differences were not significant.

[0030] As can be seen from Table 3, the dry weights of the above-ground parts treated with the fermentation broth diluted 400 times and 500 times increased by 38.26% and 38.75% respectively compared with the control, and the differences reached an extremely significant level; the dry weight of the above-ground part treated with the dilution of 300 times increased by 28.97% compared with the control, and the difference reached a significant level; the dry weights of the above-ground parts diluted 200 times and 600 times increased by 15.82% and 23.23% respectively compared with the control, and the differences were not significant.

[0031] In summary, the use of Kosakonia cowanii The fresh weight and dry weight of the above-ground parts of rice seedlings treated with the HHQGLZ30-1 fermentation broth diluted 500-100 times were both higher than those of the control group, indicating that the HHNXJCJ4G-3 fermentation broth has a growth-promoting effect on rice seedlings.

[0032] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A Kosakonia cowanii, characterized in that, The described Kosakonia cowanii is deposited in the China General Microbiological Culture Collection Center, and its taxonomic name is Kosakonia cowanii HHQGLZ30-1 ( Kosakonia cowanii HHQGLZ30-1), and the deposit number is CGMCC No. 29798.

2. The Kosakonia cowanii HHQGLZ30-1 according to claim 1, characterized in that, It can secrete auxin (IAA), has the ability of nitrogen fixation and siderophore production, and can convert iron elements in the soil that are difficult for plants to absorb and utilize into siderophore-chelated iron.

3. The Kosakonia cowanii HHQGLZ30-1 according to claim 1, characterized in that, It can promote plant growth and antagonize plant pathogens.

4. A growth-promoting and disease-preventing microbial inoculant prepared from Kosakonia cowanii HHQGLZ30-1 according to claim 1, characterized in that, The microbial inoculant described above comprises the following components: Kosakonia cowanii HHQGLZ30-1, Trichoderma harzianum, fucoidan oligosaccharide, 5-aminolevulinic acid.

5. The specific concentration of the growth-promoting and disease-preventing microbial inoculant containing Kosakonia cowanii HHQGLZ30-1 according to claim 4 is 2×10 9 -1×10 10 CFU / mL.

6. The growth-promoting and disease-preventing microbial inoculant according to claim 5 can effectively improve the stress resistance of crops, promote the growth of crops, make the crops healthier, and the leaves are more tender and green.

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