Bradyrhizobium sp. arachidis ZB15, culture method and application thereof

Abstract

The application belongs to the field of microorganisms and relates to rhizobium, in particular to a peanut slow-growing rhizobium ZB15 and a culture method and application thereof. The peanut slow-growing rhizobium ZB15 is classified as Bradyrhizobium sp. WYCCWR13023 = ZB15, which has been preserved in the China Center for Type Culture Collection on October 14, 2021, and the preservation number is CCTCC NO: M 20211276. In addition to the specific nitrogen fixation ability of rhizobium, the rhizobium also has super strong IAA production capacity, up to 85.116 mg / L. After the peanut culture group treated by the slow-growing rhizobium of the application, the number of nodule formation of the peanut plant is 91 per plant, the plant height is increased by 45.23%, the root length is increased by 57.14%, the chlorophyll content is increased by 105.03%, and the dry weight is increased by 134.41%. The effect is remarkable.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and relates to rhizobia, specifically a slow-growing peanut rhizobium ZB15 and its culture method and application. Background Technology

[0002] Peanuts, belonging to the legume family, are one of my country's important economic crops. Sustainable agricultural development in my country still faces significant challenges, and reducing fertilizer use while increasing efficiency has become an inevitable choice for my country to pursue green agriculture and sustainable agricultural development. In recent years, microbial inoculants have become a research hotspot for improving crop yields and resisting pests and diseases. Microbial inoculants improve the soil microecological environment through the life activities and metabolic products of a large number of beneficial microorganisms, ultimately promoting crop growth, reducing crop diseases, and improving crop yield and quality. In the context of sustainable agricultural development, microbial inoculants can increase crop yields at low cost, helping to mitigate the environmental damage caused by chemical fertilizers. Numerous studies have shown that microbial inoculants have been applied in the cultivation of crops such as soybeans, milkvetch, potatoes, and corn, significantly improving crop yields and other growth indicators. In recent years, rhizobia and Bacillus have been commonly used strains for producing microbial inoculants. Peanut slow-growing rhizobia can increase plant biomass and achieve yield increases by forming nodules with peanut roots. Bacillus can release potassium from the soil, improving soil fertility, and secrete hormones, amino acids, and other substances to promote crop growth. Currently, rhizobium inoculants are developing into multifunctional compound inoculants, which combine rhizobium with beneficial strains for symbiotic nodulation and growth in legumes.

[0003] Liu Ye et al. published a paper titled "Screening, Identification and Effect Study of Multifunctional and Highly Efficient Growth-Promoting Bacteria in Peanut Rhizosphere," *Biotechnology Bulletin*, 2017, 33(10):125-34. This paper disclosed the screening of an IAA-producing Bacillus strain from soil in North China, which secreted up to 49.06 mg / mL of IAA, significantly promoting peanut growth. Patent 201610141897.9 discloses a slow-growing rhizobium in peanuts and its application; this strain primarily promotes peanut growth by enhancing nitrogen fixation. Our research group discovered a rhizobium strain capable of high IAA production and conducted in-depth research on it. Summary of the Invention

[0004] This invention provides a strain of slow-growing rhizobium ZB15 in peanuts, its culture method and application. This strain, in addition to the nitrogen-fixing performance of nitrogen-fixing bacteria, also has the characteristic of high IAA production, with an IAA production of up to 82.585 mg / L.

[0005] The technical solution of this invention is implemented as follows: A peanut slow-growing rhizobium strain ZB15, classified as Bradyrhizobium sp. WYCCWR13023=ZB15, was deposited at the China Center for Type Culture Collection on October 14, 2021, with accession number CCTCC NO: M 20211276.

[0006] The above-mentioned method for culturing peanut slow-growing rhizobium ZB15 involves the following steps: Inoculating peanut slow-growing rhizobium ZB15 onto YMA solid medium and incubating at 28℃ for 7 days; picking single colonies and transferring them to YMA liquid medium; and culturing in a shaker at 180 rpm and 28℃ until the logarithmic growth phase; adjusting the OD of the rhizobium culture solution... 600 =0.8.

[0007] The YMA solid culture medium consists of 10 g mannitol, 0.25 g KH2PO4, 0.25 g K2HPO4, 0.1 g anhydrous MgSO4, 0.1 g NaCl, 3 g yeast extract, and deionized water to a final volume of 1000 mL. The pH is 6.8-7.2, and 18 g agar powder is added to the solid culture medium.

[0008] The above-mentioned application of peanut slow-growing rhizobium ZB15 in high-yield IAA.

[0009] The above-mentioned application of peanut slow-growing rhizobium ZB15 in promoting peanut plant growth.

[0010] Microbial inoculants containing the aforementioned peanut slow-growing rhizobium ZB15.

[0011] The present invention has the following beneficial effects: 1. This invention discloses a slow-growing rhizobium strain of peanut, Bradyrhizobium sp. WYCCWR13023=ZB15. In addition to the nitrogen-fixing ability unique to rhizobia, this rhizobium strain also has a super strong IAA production capacity, up to 85.116 mg / L.

[0012] 2. The strain described in this application can significantly improve the symbiotic efficiency of peanuts and promote their growth during the sterile potted seedling cultivation process. In peanut culture groups treated with the slow-growing rhizobium of this application, the number of nodules per plant was 91, plant height increased by 45.23%, root length increased by 57.14%, chlorophyll content increased by 105.03%, and dry weight increased by 134.41%. The effect is significant, and it can be used as a highly efficient growth-promoting microbial agent for peanuts. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 The standard curve for IAA production by peanut rhizobium.

[0015] Figure 2 Images of potted peanut plants inoculated with rhizobium ZB15 and control peanut plants.

[0016] Figure 3 Images of peanut plants inoculated with rhizobium ZB15 and control peanut plants.

[0017] Figure 4 Phylogenetic tree of the 16S rRNA sequence of Rhizobium ZB15. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] A peanut slow-growing rhizobium strain ZB15, classified as Bradyrhizobium sp. WYCCWR13023=ZB15, with accession number CCTCC NO: M 20211276, deposited on October 14, 2021, at the China Center for Type Culture Collection.

[0020] The publicly available formula for YMA solid culture medium containing slow-growing rhizobium: Mannitol 10 g, KH2PO4 0.25 g, K2HPO4 0.25 g, anhydrous MgSO4 0.1 g, NaCl 0.1 g, yeast powder 3 g, agar powder 18 g, distilled water 1 L, pH 6.8-7.2.

[0021] Example 1: Isolation and screening of Bradyrhizobium sp. WYCCWR13023=ZB15, a peanut rhizobium Soil samples were collected from the peanut planting area in Zhouwan Village, Dougou Town, Zhengyang County, Henan Province. A five-point sampling method was used, with soil samples collected at a depth of 10-20 cm at each sampling point. The samples were stored in sterile sealed bags, recorded, and transported back to the laboratory under low temperature conditions. Peanut seeds were treated with 95% ethanol (v / v) for 30 seconds under sterile conditions, followed by disinfection with 0.2% mercuric chloride (w / w) for 5 minutes, and washed 7-8 times with sterile water. They were then cultured in the dark at 28°C until germination. After intermittent sterilization twice with vermiculite mixed with low-nitrogen plant nutrient solution, the seeds were planted using a vermiculite-soil-vermiculite planting method. A double-layer pot system was used for planting peanuts. The upper pot contained sterile vermiculite and germinated seeds, while the lower pot held water. The two layers were connected by gauze strips to provide moisture for plant growth. After planting, the plants were placed in an artificial climate incubator and allowed to grow for 45 days. The peanut roots were then rinsed clean, and the root nodules were removed with sterile tweezers. The nodules were then disinfected with 0.2% mercuric chloride for 30 seconds. The nodules were crushed, and the liquid was streaked in a "Z" pattern on YMA medium. The medium was then incubated in the dark at 28°C for 3–5 days. The nodules were purified by streaking three times until no other colonies appeared on the plates. A total of 35 strains were obtained and labeled as ZB1 to ZB35.

[0022] Example 2: Experiment on high IAA production by peanut slow-growing rhizobium strain ZB15 The method for producing IAA using the above-mentioned slow-growing rhizobia: (1) Preparation of the standard curve: Prepare an IAA solution with a concentration of 10 mg / mL. Add 0, 100, 200, 300, 400, 500, and 600 μL of the solution to each test tube, and replenish with deionized water to a final volume of 5 mL to prepare IAA standard solutions of 0, 10, 20, 30, 40, 50, and 60 μg / mL. Mix the standard solution with the standard Salkowski colorimetric solution at a volume ratio of 1:1 and let stand at room temperature in the dark for 30 min. Measure the OD530 of the standard solution using a UV spectrophotometer. Use the 1:1 mixture of deionized water and Salkowski colorimetric solution as a blank control and zero the UV spectrophotometer. Plot a scatter plot with the concentration of the IAA standard solution on the x-axis and OD530 on the y-axis to obtain the IAA standard curve.

[0023] (3) Quantitative determination of IAA production capacity of peanut rhizobium: Single colonies were picked and inoculated into 5 mL YMA liquid test tubes and cultured at 28℃ and 180 rpm in a shaker until the logarithmic growth phase. The OD600 of the bacterial solution was adjusted to 1.0, and 0.5 mL of the bacterial solution was taken at a ratio of 1:100 and inoculated into 50 mL YMA liquid medium. The culture was carried out at 28℃ and 180 rpm in a shaker for 6-8 days (the OD600 of the bacterial solution was between 1.0 and 1.2). The bacterial solution was collected under aseptic conditions and centrifuged at 6000 r / min for 15 min. The supernatant was collected. The supernatant was mixed with an equal volume of Salkowski colorimetric solution and incubated at room temperature in the dark for 30 min. The OD530 was measured. The blank YMA liquid medium without inoculated strains was used as a control. The corresponding IAA concentrations of the 35 strains were calculated by using the IAA standard curve. The IAA production range of strains ZB1-ZB35 was 2.123 - 82.585 mg / L, with strain ZB15 producing the highest IAA level at 82.585 mg / L. The IAA values ​​of the other strains were all less than 16.476 mg / L. The IAA standard curve for strain ZB15 is shown below. Figure 1 The quantitative detection results of IAA are shown in Table 1: Table 1. Quantitative Detection of IAA Production by ZB15 Peanut Rhizobium

[0024] As can be seen from the above, the slow-growing rhizobium Bradyrhizobium sp. WYCCWR13023=ZB15 of peanut in this application has a strong IAA production capacity of 82.585±2.531 mg / L.

[0025] Quantitative detection of IAA production was performed on 67 bacterial strains isolated from soil. A high-IAA-producing strain, ZB15, was screened out. After 16S rRNA sequencing and phylogenetic analysis, it was identified as belonging to the genus *Bradyrhizobium* and named *Bradyrhizobium* sp. WYCCWR13023=ZB15. The 16S rRNA sequence of ZB15 is shown in SEQ ID No:1, and the phylogenetic tree is shown below. Figure 4 As shown.

[0026] Example 3: Method for detecting the symbiotic effect of peanut slow-growing rhizobium ZB15 on peanuts. (1) Inoculate the peanut slow-growing rhizobium strain into 5 mL of TY liquid medium and culture it in a constant temperature shaker at 28 ℃ at 180 rpm (OD600 = 0.8 ~ 1.0). Before planting peanut seedlings, adjust the OD600 of the bacterial solution to 0.8.

[0027] (2) Select peanut seeds with intact surfaces and uniform size, add an appropriate amount of 95% ethanol and stir for 30 s. Add an appropriate amount of 0.2% mercuric chloride solution for disinfection for 5 min. Finally, treat with sterile water 8 times. Place the seeds evenly on the surface of water agar medium and incubate in the dark at 28 ℃ for 3-4 days until the seed root length reaches 2-3 cm.

[0028] (3) Peanuts were planted using a double-layer pot system (the upper pot was sterilized and filled with sterile vermiculite, while the lower pot contained water, connected by gauze strips to provide necessary moisture for plant growth). Using sterile tweezers, a normally germinating peanut seed was planted in the soil layer of the upper pot, with the root facing down. After planting, the seedlings were placed in an artificial climate incubator with the following cultivation parameters: 25 ℃ light for 16 h, 20 ℃ darkness for 8 h. When the seedlings grew to the point where the bud tip touched the sealing film, the sealing film was cut in a cross shape, and water was replenished intermittently during this period.

[0029] (4) After 45 days of growth, carefully remove each plant and wash away the vermiculite attached to the roots with clean water. During the operation, be careful to avoid damaging the root nodules and the integrity of the plant. Then measure the root length and use a portable chlorophyll detector to detect the chlorophyll content of the plant leaves. Finally, dry the plant to constant weight and measure its dry weight.

[0030] The conventional culture method without adding rhizobium culture solution was used as a control.

[0031] Peanut plants treated with the slow-growing rhizobium Bradyrhizobium sp. WYCCWR13023=ZB15 and the control group showed the following results after 45 days of cultivation: Figure 2 As shown in Table 2, the statistics of symbiosis indicators are as follows: Table 2: Statistical analysis of root data between the Bacillus subtilis culture group and the control group

[0032] Depend on Figure 2 , Figure 3 As shown in Table 2, after using the slow-growing rhizobium ZB15 of this application, the number of nodules per plant was 91, the plant height increased by 45.23%, the root length increased by 57.14%, the chlorophyll content increased by 105.03%, and the dry weight increased by 134.41%. The effect is significant.

[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. <110> Zhengzhou University of Light Industry <120>A strain of Bradyrhizobium from peanut ZB15, its culture method and application <141> 2021-12-24 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 929 <212> DNA <213> Bradyrhizobium sp <400> 1 ataccggata agcccttacg gggaaagatt tatcgccgaa agatcggccc gcgtctgatt 60 agctagttgg tagggtaatg gcctaccaag gcgacgatca gtagctggtc tgagaggatg 120 atcagccaca ttgggactga gacacggccc aaactcctac gggaggcagc agtggggaat 180 attggacaat gggggcaacc ctgatccagc catgccgcgt gagtgatgaa ggccctaggg 240 ttgtaaagct cttttgtgcg ggaagataat gacggtaccg caagaataag ccccggctaa 300 cttcgtgcca gcagccgcgg taatacgaag ggggctagcg ttgctcggaa tcactgggcg 360 taaagggtgc gtaggcgggt ttttaagtca ggggtgaaat cctggagctc aactccagaa 420 ctgcctttga tactgaagat cttgagtccg ggagaggtga gtggaactgc gagtgtagag 480 gtgaaattcg tagatattcg caagaacacc agtggcgaag gcggctcact ggcccggtac 540 tgacgctgag gcacgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 600 cgtaaacgat gaatgccagc cgttagtggg tttactcact agtggcgcag ctaacgcttt 660 aagcattccg cctggggagt acggtcgcaa gattaaaact caaaggaatt gacgggggcc 720 cgcacaagcg gtggagcatg tggtttaatt cgacgcaacg cgcagaacct taccagccct 780 tgacatgtcc aggaccggtc gcagagatgt gaccctctct tcggagcctg gaacacaggt 840 gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 900 aacccccgtc cttagttgct accatttag 929

Claims

1. A slow-growing rhizobium strain ZB15 of peanut, characterized by: The classification name of the peanut slow-growing rhizobium ZB15 is: Bradyrhizobium sp .WYCCWR13023=ZB15 was deposited at the China Center for Type Culture Collection on October 14, 2021, with accession number CCTCC NO: M 20211276.

2. The method for culturing peanut slow-growing rhizobium ZB15 according to claim 1, characterized in that, The steps are as follows: Inoculate peanut slow-growing rhizobium ZB15 onto YMA solid medium and incubate at 28℃ for 7 days. Pick single colonies and transfer them to YMA liquid medium, then incubate in a shaker at 180 rpm and 28℃ until the logarithmic growth phase. Adjust the OD of the rhizobium culture solution. 600 =0.

8.

3. The cultivation method according to claim 2, characterized in that: The YMA solid culture medium consists of 10 g mannitol, 0.25 g KH2PO4, 0.25 g K2HPO4, 0.1 g anhydrous MgSO4, 0.1 g NaCl, 3 g yeast extract, and deionized water to a final volume of 1000 mL. The pH is 6.8-7.2, and 18 g agar powder is added to the solid culture medium.

4. The application of the peanut slow-growing rhizobium ZB15 as described in claim 1 in high-yield IAA.

5. The application of the peanut slow-growing rhizobium ZB15 as described in claim 1 in promoting peanut plant growth.

6. A microbial inoculum containing the peanut slow-growing rhizobium ZB15 as described in claim 1.

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