A slow-growing rhizobium of soybean resistant to seed coating agent of precise azoles · fludioxonil

Abstract

The present application relates to the technical field of microorganism, and in particular to a soybean Bradyrhizobium elkanii Y63-1 K3500 resistant to prochloraz seed coating agent and application thereof.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a slow-growing soybean rhizobium *Bradyrhizobium elkanii* Y63-1 K3500 resistant to styraxone and fludioxonil seed coating agent, and its applications. Background Technology

[0002] Soybeans are an important source of plant protein and edible vegetable oil, and are also an excellent crop for crop rotation and intercropping in agricultural production. However, they are also a crop with high nitrogen requirements; nitrogen deficiency can severely impact yield, while excessive application of nitrogen fertilizer increases production costs and causes environmental pollution. The symbiotic nitrogen fixation between soybeans and rhizobia plays a crucial role in reducing the application of chemical nitrogen fertilizers, improving soil fertility, and promoting the sustainable development of green agriculture.

[0003] Soybean production in my country generally relies on the application of chemical nitrogen fertilizers, and the soil contains a large number of native rhizobia with weak nitrogen-fixing ability but strong nodulation competition. These factors lead to poor application of exogenous rhizobium inoculants and low utilization rate of symbiotic nitrogen fixation. Therefore, identifying and utilizing highly efficient, broad-spectrum and adaptable soybean rhizobia is crucial for promoting the development of green agriculture.

[0004] Bradyrhizobium elkanii Y63-1 is a slow-growing rhizobium strain in soybean, which has the technical effect of improving the nodulation and nitrogen fixation ability of soybean (CN117535180A). Currently, seed coating is crucial for pest and disease control in soybean production in my country. However, commonly used seed coating agents (such as cyprodinil·fludioxonil) usually inhibit the proliferation and nitrogen fixation function of rhizobium, which seriously limits the integrated application of highly efficient rhizobium inoculants and seed coating technology.

[0005] This invention, through drug resistance screening, obtained a slow-growing rhizobium of soybean resistant to cyprodinil-fludioxonil seed coating agent. It not only retains the original nitrogen-fixing ability but also tolerates cyprodinil-fludioxonil seed coating agent, thus providing a comprehensive solution for soybean pest and disease control and improving nitrogen-fixing ability. Summary of the Invention

[0006] The purpose of this invention is to provide a slow-growing rhizobium of soybean resistant to cyprodinil and fludioxonil seed coating agent.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a *Syntrophus elutipes* strain, characterized in that the *Syntrophus elutipes* strain with accession number CGMCC No. 28151 has undergone the following mutation:

[0009] The sequence shown in SEQ ID NO. 1 has a mutation at position 483 where A is changed to G; and

[0010] The sequence shown in SEQ ID NO. 2 has a G mutation at position 498, which is changed to T; and

[0011] In the sequence shown in SEQ ID NO. 3, position 396 C mutates to T; position 399 C mutates to T; position 435 T mutates to C; and...

[0012] The sequence shown in SEQ ID NO. 4 has a mutation at position 3220, where T is changed to G; and

[0013] The sequence shown in SEQ ID NO. 5 has a mutation at position 805 where G is replaced by A; and

[0014] The sequence shown in SEQ ID NO. 6 has a mutation at position 1036 (G) to A; a mutation at position 1038 (T) to A; and

[0015] The sequence shown in SEQ ID NO. 7 has a T mutation at position 440 that changes to G; a T mutation at position 443 that changes to G; and

[0016] The sequence shown in SEQ ID NO. 8 has a mutation at position 734 (A) to C, position 739 (A) to C, and position 740 (A) to C.

[0017] The aforementioned Bradyrhizobium elkanii strain is a strain deposited at the China Center for Type Culture Collection (CCTCC NO: M 2026795) on April 23, 2026, and is classified as Bradyrhizobium elkanii Y63-1K.

[0018] The present invention also provides a microbial agent, characterized in that the microbial agent contains the above-mentioned *Syntrophus esculenta*.

[0019] The present invention also provides a mixture, characterized in that the mixture is composed of the above-mentioned *Syntrophus esculenta* or the above-mentioned inoculum and a seed coating agent of methyl fludioxonil.

[0020] In some embodiments, the concentration of methyl fludioxonil in the above mixture is 2000 mg / L to 3500 mg / L.

[0021] The present invention also provides the application of the above-mentioned *Syntrophus esculenta*, or the above-mentioned inoculant, or the above-mentioned mixture in soybean cultivation.

[0022] The present invention also provides a method for simultaneously improving the nitrogen-fixing capacity, biomass, yield, plant height, chlorophyll content, root length, and disease resistance of soybeans, characterized in that the method involves applying the above mixture to soybean seeds.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention obtains a slow-growing rhizobium of soybean resistant to cyprodinil-fludioxonil seed coating agent through drug resistance screening. It not only retains the original nitrogen fixation ability, but also can tolerate cyprodinil-fludioxonil seed coating agent. Attached Figure Description

[0024] Figure 1 Colony growth of strain Y63-1 after culturing on YEM solid medium containing different concentrations of coating agent active ingredient for 5 days. The concentration of coating agent active ingredient is marked in the upper left corner of the petri dish, in mg / L.

[0025] Figure 2 Figures showing the growth of different Y63-1 strains on YEM solid medium with different concentrations of the active ingredient in the coating agent. The concentration of the active ingredient in the coating agent is marked in the upper left corner of the petri dish. The location of the strain type streaked on each petri dish is shown in the diagram.

[0026] Figure 3 Growth of the Y63-1 antibiotic-resistant strain on four-antibiotic YEM solid medium. "CK" represents YEM solid medium without antibiotics; "Resistant" represents YEM solid medium with four antibiotics. The location of the strain type streaked on each petri dish is shown in the diagram.

[0027] Figure 4 Four different coating treatments were used for Zhongdou 63. S63: ultrapure water + original strain Y63-1; B63: coating agent + original strain Y63-1; S63M: ultrapure water + strain Y63-1K3500; B63M: coating agent + strain Y63-1K3500.

[0028] Figure 5 Growth of Zhongdou 63 plants after inoculation with different coating agents.

[0029] Figure 6 Root nodulation of Zhongdou 63 after inoculation with different coating agents. Detailed Implementation

[0030] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent literature, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s *Molecular Cloning: A Laboratory Manual* (Sambrook J & Russell DW, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available and conventional methods well known to those skilled in the art.

[0032] To facilitate understanding of the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0033] Example 1: Targeted screening of the coating agent resistance characteristics of soybean slow-growing rhizobia

[0034] By sequentially increasing the concentration of a seed coating agent, metalaxyl-fludioxonil (a fungicide composed of metalaxyl and fludioxonil), in a solid culture medium for rhizobia, and screening using the streak plate method, highly resistant mutant strains to metalaxyl-fludioxonil (or metalaxyl-fludioxonil) seed coating were created. Further resistance experiments were then conducted on the selected mutant strains to ultimately identify highly resistant seed coating mutant strains. The specific experimental procedure is as follows:

[0035] I. Experimental Materials

[0036] (1) Rhizobium strains

[0037] The original strain Y63-1 was isolated by the applicant's team and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28151.

[0038] (2) YEM medium

[0039] Yeast extract 0.4 g (Sigma-Aldrich), CaCl2·6H2O 0.1 g, NaCl 0.1 g, K2HPO4 0.5 g, MgSO4 0.2 g, mannitol 10 g, agarose 25 g, and 4 mL of Rh trace element solution (5 g / L H3BO3, 5 g / L Na2MnO4) were added to a final volume of 1000 mL with ddH2O. The solid culture medium was then supplemented with 1.5% agar and sterilized at 115℃ for 20 min. Except for the yeast extract, all other reagents were purchased from Shanghai Guoyao Reagent Company.

[0040] (3) Bright Shield Coating Agent

[0041] Shield seed coating agent, the main component of which is metalaxyl-mancozeb (active ingredient concentration 62.5 g / L), product registration certificate number: PD20150641, manufactured by Syngenta.

[0042] II. Experimental Methods

[0043] Add Bright Shield coating agent to YEM solid medium until the initial concentration of the active ingredient, metalaxyl-methyl·fludioxonil, is 50 mg / L. Activate Y63-1 colonies using the streak plating method and incubate at 28℃ for 3–5 days. Select vigorous colonies and transfer them to YEM solid medium containing 100 mg / L of the coating agent. Repeat this process, gradually increasing the concentration. From each screening concentration plate, select vigorous colonies and transfer them to plates with higher concentrations. This targeted screening process aims to ultimately obtain a mutant strain resistant to concentrations up to 3500 mg / L.

[0044] III. Experimental Results

[0045] The growth of strain Y63-1 on YEM solid medium with different concentrations of coating agent active ingredients is as follows: Figure 1 As shown, the obtained drug-resistant strains were named "K+ screening concentration" (concentration unit is mg / L).

[0046] To verify the effectiveness of the technology, the applicant again selected the control group (Y63-1 colony), Y63-1K1000, Y63-1K2000, and Y63-1K3500 strains and streaked them on YEM solid medium containing 1000 mg / L, 2000 mg / L, and 3500 mg / L. The results are as follows. Figure 2 As shown. On these three YEM solid media containing high concentrations of coating agent, strain K3500 grew faster than the other three strains. In particular, in K3500 medium, colonies of the control group, K1000, and K2000 were almost invisible, while K3500 showed clusters of colonies, and its colony reproduction was significantly better than that of the other strains.

[0047] The applicant has deposited the selected Y63-1K3500 strain at the China Center for Type Culture Collection on April 23, 2026, with the deposit name Y63-1K and accession number CCTCC NO: M 2026795.

[0048] Example 2: Identification of antibiotic resistance to Y63-1K

[0049] This invention further identified the antibiotic resistance of the screened resistant strains. The antibiotics used were purchased from Solarbio and included: ampicillin (Amp), rifampin (Rif), cephalosporin (Cef), and carbenicillin (Car). Amp, Cef, and Car were dissolved in sterile water, and Rif was dissolved in DMSO. The stock solutions of the four antibiotics were prepared at a concentration of 10 mg / L, filtered, sterilized, and stored at -20 °C. During the experiment, 100 μg / mL of Amp, Rif, Cef, and Car were added to YEM solid medium to prepare a four-antibiotic medium. The original Y63-1 strain, K1000, K2000, and K3500 strains were then streaked onto this medium. The growth of the strains was observed after approximately 5-7 days.

[0050] The results are as follows Figure 3 As shown, the original Y63-1 strain, K1000, K2000, and K3500 can all grow on the four-antibiotic solid medium. The results indicate that these screened resistant strains did not lose their antibiotic resistance and even showed a slight increase in resistance.

[0051] Example 3: Identification of the nodulation-symbiotic effect of Y63-1K3500

[0052] This invention further identified the nodulation symbiotic effect of the Y63-1K3500 resistant strain screened above, using the soybean variety Zhongdou 63 (a high-yield, high-oil summer soybean variety bred by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, which can be purchased from public sources) for identification.

[0053] The preserved strains of soybean rhizobium Y63-1 and Y63-1K3500 were activated on YEM plates, and then four antibiotics were inoculated according to the method in Example 2 to prepare a four-antibiotic YEM liquid medium. The colonies were inoculated into the four-antibiotic YEM liquid medium and cultured at 28°C with shaking for 5-7 days.

[0054] Four coating agents were used for treatment: coating agent + Y63-1 original strain (B63), coating agent + K3500 strain (B63M), ultrapure water + Y63-1 original strain (S63), and ultrapure water + K3500 strain (S63M). Zhongdou 63 soybean seeds were pre-selected and sterilized with chlorine before coating treatment. Specifically, the cultured Y63-1 original strain and K3500 strain bacterial suspensions (OD value 1.0) were each divided into two portions. After centrifugation, a portion of the supernatant was discarded, and the suspension was resuspended to obtain bacterial cells. One portion was mixed with the coating agent stock solution, and the other portion was mixed with an equal volume of water. These mixtures were injected into a plastic bag, and soybean seeds were added and mixed thoroughly for coating. The proportions of each component added are as follows: according to the seed weight, four mixed liquids of coating agent / water and Y63-1 / K3500 bacterial solution are added respectively to balance the effects of coating agent and bacterial solution, and the final concentration of coating agent is controlled between 2000 mg / L and 3500 mg / L. The addition amounts in this embodiment are as follows: B63: 37 g of Zhongdou 63 seeds + 0.296 mL of coating agent stock solution + 5.92 mL of Y63-1 bacterial cells (obtained by centrifugation of 300 mL bacterial solution); B63M: 37 g of Zhongdou 63 seeds + 0.296 mL of coating agent stock solution + 5.92 mL of K3500 bacterial cells (obtained by centrifugation of 300 mL bacterial solution); S63: 37 g of Zhongdou 63 seeds + 0.296 mL of sterile water + 5.92 mL of Y63-1 bacterial cells (obtained by centrifugation of 300 mL bacterial solution); S63M: 37 g of Zhongdou 63 seeds + 0.296 mL of sterile water + 5.92 mL of K3500 bacterial cells (obtained by centrifugation of 300 mL bacterial solution). After drying in a fume hood until the surface is dry, sow immediately. See the example images after different coating treatments. Figure 4 .

[0055] The coated seeds were sown in pots containing sterilized vermiculite (4-5 seeds per pot). After germination in about 5-7 days, two healthy and uniform seedlings were kept and the others were removed. The seedlings were then watered with a 1 / 2 nitrogen nutrient solution 1-2 times (depending on the condition of the cotyledons), followed by a 1 / 10 nitrogen nutrient solution. Samples were taken 28 days after sowing to measure nodulation-related indicators such as aboveground fresh weight, plant height, chlorophyll content, number of root nodules, root nodule dry weight, and root dry weight.

[0056] The 1 / 2 nitrogen nutrient solution was prepared as follows: 110.98 mg CaCl2, 43.56 mg K2SO4, 68.045 mg KH2PO4, 0.023 mg CoCl2·6H2O, 0.169 mg MnSO4·H2O, 0.024 mg Na2MoO4·2H2O, 150 mg N&N, 30.09 mg MgSO4, 0.12 mg H3BO3, 18.55 mg ferric citrate, 0.14 mg ZnSO4·7H2O, and 0.049 mg CuSO4·5H2O. The solution was then brought to a final volume of 1000 mL with ddH2O.

[0057] The 1 / 10 nitrogen nutrient solution is prepared as follows: 30.015 mg N&N, 43.56 mg K2SO4, 30.09 mg MgSO4, 0.12 mg H3BO3, 0.049 mg CuSO4·5H2O, 0.223 mg MnSO4·4H2O, 0.14 mg ZnSO4·7H2O, 110.98 mg CaCl2, 68.045 mg KH2PO4, 0.023 mg CoCl2·6H2O, 18.55 mg ferric citrate, and 0.024 mg Na2MoO4·2H2O. Finally, bring the volume to 1000 mL with ddH2O.

[0058] The results of the aboveground growth, chlorophyll content, plant height, and aboveground fresh weight of Zhongdou 63 under four coating treatments are as follows: Figure 5 As shown in the figure. The results showed that the chlorophyll content of the aboveground parts of soybean plants treated with S63 was significantly lower than that of other treatments; there were no significant differences in the aboveground fresh weight and plant height of soybeans under the four treatments. The root nodulation of Zhongdou 63 under the four coating treatments is shown in the figure. Figure 6 As shown, regardless of whether it was mixed with water or a coating agent, the mutant strain Y63-1K3500 showed no significant difference in the number of nodules or the dry weight of the original strain Y63-1, indicating that the Y63-1K3500 mutation does not affect the nodulation and symbiotic ability of the strain. However, regardless of whether it was mixed with water or a coating agent, the root dry weight of the mutant strain Y63-1K3500 was higher than that of the original strain Y63-1.

[0059] The above results indicate that, in addition to being highly resistant to metalaxyl-fludioxonil seed coating agent, the mutant strain K3500 did not exhibit weakened nodulation and growth-promoting abilities, and even showed a slight enhancing effect.

[0060] Example 4: Identification of mutation sites in Y63-1K3500

[0061] To identify the mutation site of strain Y63-1K3500, the inventors performed whole-genome sequencing on strain Y63-1K3500 and compared the sequence with that of Y63-1 to identify the mutation site.

[0062] The information related to the mutation sites of the Y63-1K3500 strain that were finally identified is shown in Table 1. The results showed that a total of 8 genes were mutated, including the gene encoding Fe-S cluster assembly protein SufB, chaperonin GroEL, membrane fusion protein, and multidrug efflux system protein.

[0063]

[0064] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A type of slow-growing rhizobium, characterized in that, The *Syntrophus esculenta* strain with accession number CGMCCNo28151 underwent the following mutation: The sequence shown in SEQ ID NO. 1 has a mutation at position 483 where A is changed to G; and The sequence shown in SEQ ID NO. 2 has a G mutation at position 498, which is changed to T; and In the sequence shown in SEQ ID NO. 3, position 396 C mutates to T; position 399 C mutates to T; position 435 T mutates to C; and... The sequence shown in SEQ ID NO. 4 has a mutation at position 3220, where T is changed to G; and The sequence shown in SEQ ID NO. 5 has a mutation at position 805 where G is replaced by A; and The sequence shown in SEQ ID NO. 6 has a mutation at position 1036 (G) to A; a mutation at position 1038 (T) to A; and The sequence shown in SEQ ID NO. 7 has a T mutation at position 440 that changes to G; a T mutation at position 443 that changes to G; and The sequence shown in SEQ ID NO. 8 has a mutation at position 734 (A) to C, position 739 (A) to C, and position 740 (A) to C.

2. The *Syntrophus esculenta* according to claim 1, characterized in that, The *Syntrophus esculenta* strain is a strain deposited at the China Center for Type Culture Collection with accession number CCTCC NO: M 2026795.

3. A microbial agent, characterized in that, The inoculum contains *Staphylococcus esculenta* as described in any one of claims 1-2.

4. A mixture, characterized in that, The mixture consists of *Staphylococcus esculenta* as described in any one of claims 1-2, or the inoculum agent as described in claim 3, and a seed coating agent of methyl thiophanate-methyl.

5. The mixture according to claim 4, characterized in that, The concentration of methyl fludioxonil in the mixture is 2000 mg / L to 3500 mg / L.

6. The application of the *Syntrophus esculenta* according to any one of claims 1-2, or the inoculant according to claim 3, or the mixture according to any one of claims 4-5 in soybean cultivation.

7. A method for simultaneously improving soybean nodulation and nitrogen fixation capacity / biomass / yield / plant height / chlorophyll content / root length and disease resistance, characterized in that, The method involves applying the mixture described in any one of claims 4-5 to soybean seeds.

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