Rhizobium sesbanianum E1 and application thereof

Abstract

The invention provides rhizobium sesbanianum E1 and application thereof, the preservation number of the rhizobium sesbanianum E1 is CCTCC M 20241925, the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the preservation date is September 5, 2024. The strain has strong exopolysaccharide synthesis capability and stress resistance, can be used for planting production of peas, lotus corniculatus and sesbania, can inhibit infection of soil-borne disease ralstonia solanacearum on non-leguminous plants, and has excellent application prospects in soil barren and disease producing areas.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a sesbania rhizobium E1 and an application thereof. Background Art

[0002] Rhizobia are a class of Gram-negative bacteria widely distributed in the soil that can establish a symbiotic relationship with legumes, for which the plants form specialized nitrogen-fixing sites - nodules. Inside the nodules, the rhizobia invade plant cells and transform into bacteroids, converting atmospheric nitrogen into nitrogen. 2 Fixing ammonia allows legumes to grow in nitrogen-poor soils, thereby increasing crop yields.

[0003] The establishment of a symbiotic relationship is the result of the synergistic effects of bacteria, plants and the environment. Rhizobium nitrogen-fixing symbiosis has extremely strict host specificity. As a result of strict signal molecule dialogue between hosts, a strain of rhizobia can only establish symbiosis with a few legumes, and only adapted rhizobia can effectively fix nitrogen. In addition, rhizobia nitrogen-fixing symbiosis is also affected by regional environmental differences and has diversity. Soil pH, particle type and other physical and chemical properties will directly affect the symbiotic effect. Indigenous rhizobia strains often dominate due to their high adaptability to the regional environment, making artificial bacterial inoculation inefficient. Therefore, screening adapted rhizobia based on the regional environment and the main crop type can maximize the role of biological nitrogen fixation and provide greater value for modern agriculture.

[0004] Green manure refers to cultivated or wild green plants that are applied directly or after composting to the soil as fertilizer. The leguminous green manure Sesbania sesbania is a pioneer crop for reclaiming wasteland. It has the function of biological nitrogen fixation, which can not only improve the physical and chemical properties of the soil, but also increase soil fertility and the yield of the next crop.

[0005] In the prior art, for example, a patent application with publication number CN110063161A discloses a method for promoting the growth of Andrographis paniculata and increasing the content of its effective ingredients by using nitrogen-fixing rhizobium of Sesbania nodules; a patent application with publication number CN113179882A discloses a method for promoting nodulation and nitrogen fixation of the root system of nodular mustard by using nitrogen-fixing rhizobium of Sesbania nodules. And a patent application with publication number CN115261245A discloses a rhizobium strain (TJ22) for inducing salt tolerance in plants (soybeans) and its application. Summary of the invention

[0006] The invention aims to provide a sesbania rhizobium E1 and application thereof.

[0007] The sesbania rhizobium E1 (Ensifer sesbaniae) provided by the present invention is a fast-growing rhizobium that is separated from the nodule tissue of sesbania planted in Zhejiang Province and can form nitrogen-fixing nodules with pea and lotus roots. The rhizobium E1 has rich viscous extracellular polysaccharides and can be used as a soil physical and chemical property improver to inhibit the occurrence of soil-borne diseases. The rhizobium E1 can secrete plant growth hormones, increase plant root length, root surface area and the number of lateral root hairs, increase rhizobium infection efficiency, improve the ability of plants to absorb nutrients, have stress resistance, can adapt to adverse habitats, and has good application prospects.

[0008] A Sesifer sesbaniae E1, with a deposit number of CCTCC NO: M20241925. The depository is China Center for Type Culture Collection, the deposit address is Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit date is September 5, 2024.

[0009] The invention also provides the use of the sesbania rhizobium E1 in promoting plant growth.

[0010] The present invention also provides the use of the sesbania rhizobium E1 in preparing a bacterial agent for promoting plant growth.

[0011] Specifically, the plant growth promotion performance includes at least any one of the following: symbiotic nitrogen fixation, production of nitrogenase, production of indoleacetic acid, salt resistance, alkali resistance, acid resistance, and kanamycin resistance.

[0012] Preferably, the plant is pea, Lotus japonica or Sesbania sesbania.

[0013] The present invention also provides the use of the sesbania rhizobium E1 in preparing medicine for preventing and treating bacterial wilt.

[0014] Preferably, the bacterial wilt disease is caused by Ralstonia solanacearum.

[0015] The present invention also provides a bacterial agent for promoting plant growth, wherein the active ingredient of the bacterial agent includes the sesbania rhizobium E1.

[0016] The present invention also provides a medicine for preventing and treating bacterial wilt, wherein the active ingredient of the bacterial agent includes the Sesbania rhizobium E1.

[0017] The present invention also provides the use of the sesbania rhizobium E1 or the bacterial agent for promoting plant growth in the production of biological organic fertilizer.

[0018] The present invention also provides a biological organic fertilizer, which contains the sesbania rhizobium E1 or the bacterial agent for promoting plant growth.

[0019] The strain of the present invention has strong extracellular polysaccharide synthesis ability and stress resistance, can be used for the planting and production of pea, lotus root and sesbania, and can inhibit the infection of soil-borne disease Ralstonia solanacearum on non-leguminous plants, and has excellent application prospects in soil-poor and disease-infected production areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the evolutionary tree diagram of Rhizobium E1;

[0021] Figure 2 This is a schematic diagram of the rhizobium E1 genome;

[0022] Figure 3 is the distribution of structural variations within the rhizobium E1 genome;

[0023] Figure 4 Morphological characteristics of Rhizobium E1;

[0024] Figure 5 It is the exopolysaccharide and biofilm synthesis performance of Rhizobium E1;

[0025] Figure 6 The nodulation characteristics of Rhizobium E1 in different plants, where A represents the nodulation effect on pea, B represents the nodulation effect on Sesbania odorifera, and C represents the nodulation effect on Lotus japonicus;

[0026] Figure 7 The growth curve and motility of Rhizobium E1, where A represents the growth curve, B represents the motility and swimming motility, 0.5% Agar detects flagellar motility, and 0.3% Agar detects ciliary swimming motility;

[0027] Figure 8 is the IAA synthesis ability of rhizobium E1;

[0028] Fig. 9 The effect of Rhizobium E1 in inhibiting bacterial wilt. DETAILED DESCRIPTION

[0029] Example 1: Isolation and identification of rhizobium E. sesbaniae (E1)

[0030] 1. Isolation and preservation of rhizobia

[0031] The roots of Sesbania sesbania plants in the soybean-Sesbania intercropping field in Zhejiang Province were collected, and fresh nodule tissues with a small amount of root segments were cut and washed with running water to remove the surface soil particles. The roots were transferred to a sterile beaker in a clean bench, soaked in 70% alcohol for 5 minutes, washed with sterile water for 3-5 times, soaked in 10% NaClO for 15 minutes, washed with sterile water for 3-5 times, and the nodule tissue was cut into pieces with a sterile blade, placed on yeast mannitol agar (YMA) medium, and inverted at 28°C for 3-5 days. The colonies were picked and streaked on fresh YMA medium for multiple times until a purified single colony was obtained. The pure culture was placed in yeast mannitol broth (YMB) medium at 28°C and 220rpm for 3 days and frozen at -80°C with 25% glycerol.

[0032] 2. Gene sequence alignment and phylogenetic analysis of rhizobium strains

[0033] The rhizobium DNA was extracted and the housekeeping gene 16S rDNA was amplified. The PCR product was detected by 1% agarose gel and sent to Qingke Biotechnology Company for sequencing. The sequencing result sequence is shown in SEQ ID NO.1.

[0034] The primer sequences are as follows:

[0035] 16S rDNA-P 1: 5'-AGAGTTTGATCCTGGCTCAGAACGAACGCT-3', 16S rDNA-P2: 5'-TACGGCTACCTTGTTACGACTTCACCCC-3'.

[0036] The sequence information was submitted to the GenBank database, the sequences of closely related species were downloaded, and the sequences of isolated strains and reference strains were subjected to phylogenetic analysis using MEGA-X ( Figure 1 ).

[0037] The genomic DNA was extracted using the STE method, the purity and integrity of the DNA was detected by agarose gel electrophoresis, and quantified using Qubit. The library was constructed and sequenced using the PacBio and Illumina platforms. The genome diagram is shown in Figure 2 shown.

[0038] DDH (DNA-DNA hybridization) is the gold standard for defining prokaryotic species at the genome level, providing a taxonomic method for digital and relatively stable species definition. The value obtained by ANI (average nucleotide identity) is higher than 94%, and the value of DDH is higher than 70%, which can be considered to be the same species. According to the statistics of the DDH value and ANI value of E1 based on the genome data, the DDH value is 92.8 and the ANI value is 98.57 as shown in Table 1. Therefore, the housekeeping gene sequence of the rhizobium strain provided by the present invention has a high similarity with the typical species CCBAU65729 of E.sesbaniae at the housekeeping gene and genome levels, and it can be determined that the rhizobium belongs to the sesbania rhizobium E.sesbaniae, and the strain number is E1, which was deposited in the China Type Culture Collection Center at Wuhan University, China on September 5, 2024, with the deposit number of CCTCC NO: M 20241925.

[0039] Table 1. Comparison results of ANI, dDDH values ​​and genetic distance

[0040]

[0041] Abbreviation: ANI, average nucleotide identity; dDDH; digital DNA-DNAhybridization.

[0042] Comparative genomic analysis was performed on the genome of the target strain E1 and the reference strain CCBAU65729. Single-nucleotide polymorphisms (SNPs) were counted, which are DNA sequence polymorphisms caused by the variation of a single nucleotide at the genome level, including the conversion and transversion of a single base, and are an important type of heritable variation that drives species evolution; small fragment insertions and deletions (Indels, InDels), usually less than 50bp, are second only to SNPs in the genome; and structural variation (SVs), which are changes in the sequence and position relationship of large fragments on the genome, including insertions, deletions, inversions, translocations, and other complex structural changes of more than 50bp. The comparison results in Table 2 show that there are 44,701 total SNPs in the E1 genome, including 27,157 synonymous SNPs and 9,814 non-synonymous SNPs, accounting for about 22% of the total SNPs; 37,085 SNPs in the gene region and 7,616 SNPs in the intergenic region. Indels occurred in 48 CDS regions, accounting for about 0.74% of the total CDS, 28 insertion mutations in the middle of CDS, 32 deletion mutations in the middle of CDS, and 34 Indels mutations causing frameshift mutations. Figure 3 In the figure, the inner circle is the E1 genome, and the outer circle is the reference strain genome. Collinear indicates a synlinear region; Translocation indicates a translocation region; Inversion indicates an inversion region; Tran+Inver indicates a translocation and inversion region; Insertion indicates an insertion region greater than or equal to 50 bp in length; Deletion indicates a deletion region greater than or equal to 50 bp in length; ComplexInDel indicates a region that cannot be aligned but has a corresponding position; Forward chain indicates the forward chain of the genome sequence, in which case the gene coordinates increase in a clockwise direction; Reversechain indicates the reverse chain of the genome sequence, in which case the gene coordinates increase in a counterclockwise direction; Forward CDS indicates the CDS translated on the forward chain of the genome sequence; Reverse CDS indicates the CDS translated on the reverse chain of the genome sequence; SubjoinForward CDS indicates the CDS translated on the supplementary forward chain of the genome sequence; Subjoin Reverse CDS indicates the CDS translated on the supplementary reverse chain of the genome sequence, with the paired sequence of DeleteGene or InsertGene as the supplementary CDS.

[0043] Table 2. Statistics of SNPs and Indels in the E1 genome

[0044] E1 Total SNPs 44,701 Synonymous SNPs 27,157 Nonsynonymous SNPs 9，814(22％) Gene region SNPs 37，085 Intergenic region SNPs 7，616 Total CDS Indels 48(0.74％) Insertion mutation Indels 28 Deletion mutation Indels 32 Frameshift Indels 34

[0045] 3. Morphological identification

[0046] Gram cell staining and scanning electron microscopy were performed on the rhizobium of the present invention according to Bergey's Bacterial Identification Manual. The results showed that E. sesbaniae (E1) was a Gram-negative short bacillus ( Figure 4 ).

[0047] 4. Identification of culture characteristics

[0048] Polysaccharides are considered to be the main macromolecular components of the extracellular matrix (EPS), which contribute to coagulation, water retention, adsorption and stress resistance. As an effective binder of soil particles, exopolysaccharides can affect soil structure through the combined effects of hydrophilicity, water absorption and water retention, and have significant effects in improving soil quality. A large number of research results have confirmed that the yield of exopolysaccharides is significantly correlated with soil properties such as soil crust thickness and soil aggregate stability. When microorganisms with the ability to produce exopolysaccharides adhere to the surface of plant roots, they will secrete a variety of extracellular substances including exopolysaccharides to greatly enhance the adhesion between bacteria and between bacteria and plant roots, thereby achieving the effect of enriching the entire microbial community on the surface of plant roots. As the number of bacteria adhering to the surface of plant roots continues to increase, the content of exopolysaccharides also continues to increase. Excessive exopolysaccharides will form a layer of hydrogel genes on the surface of the bacteria, and finally form a biofilm covering the surface of the root system, thereby effectively isolating the stress factors in the environment and regulating the growth of crops.

[0049] The culture characteristics of rhizobia were detected using YMA medium containing 0.25% Congo Red (CR) dye and YMA medium containing 0.02% Calcofluor White (CFW) dye. Figure 5 The ethanol precipitation method was used to quantify the extracellular polysaccharide yield. E1 was inoculated into YMB liquid medium and YMB liquid medium with NaCl added to 200 mM to simulate adversity. The culture was carried out at 28°C and 220 rpm for 6 days. The supernatant after centrifugation to remove the bacteria was added with 3 times the volume of anhydrous ethanol overnight to precipitate the polysaccharide. The polysaccharide precipitate was collected by centrifugation at 10,000 rpm for 30 min, and the lyophilized and weighed. The yield was measured by the polysaccharide / bacterial mass ratio. The crystal violet staining method was used to quantify the biofilm formation ability. At OD 595The absorbance was measured at nm. The average OD value of the negative control plus 3 times the standard deviation was defined as ODc. By comparing the OD of the test strain with ODc, the biofilm formation ability can be divided into four categories: negative: no biofilm formation, OD ≤ ODc; weakly positive: ODc < OD ≤ 2ODc; moderately positive: 2ODc < OD ≤ 4ODc; strongly positive: OD > 4ODc.

[0050] As Figure 5 shown, the staining results showed that there was no color absorption reaction of E1 on the YMA Congo red medium. The colonies were blister-like, translucent, with a large circle, flowing colloid, and produced a large amount of polysaccharide substances. CFW is a polysaccharide dye that binds only to β-1,3 and β-1,4 glycosidic bonds, which mainly exist in cellulose and chitin, but not in the more common extracellular matrix (β-1,6-N-acetylglucosamine) of microorganisms. When excited by long-wave ultraviolet light, CFW will show the extracellular matrix containing β-1,3 and β-1,4 glycosidic bonds. The staining results showed that the E1 colonies could not show fluorescence, indicating that their extracellular matrix had no β-1,3 and β-1,4 glycosidic bonds. The extracellular polysaccharide quantification results showed that the polysaccharide production rate of E1 reached 93% under non-stress conditions and still reached 86% under NaCl stress conditions. In addition, according to the biofilm quantitative typing (OD > 4ODc), the results showed that E1 was a strong biofilm synthesis strain ( Figure 5 ).

[0051] Example 2: Nodulation characteristics of Rhizobium E1

[0052] I. Back-inoculation of peas

[0053] Seed germination: Pea seeds were sterilized with 10% NaClO for 10 min, washed thoroughly with sterile water, soaked in sterile water for 4 - 5 h to fully imbibe, then spread out in a water-draining container, covered with sterile gauze to keep moist, and germinated at a constant temperature of 25°C. When the radicle grew to 3 - 5 cm, the seedlings were transplanted into a seedling pot filled with sterilized vermiculite substrate and cultured in an artificial climate chamber at 25°C under a 16 h light / 8 h dark photoperiod for 7 days before inoculating with Rhizobium.

[0054] Nodule inoculation: The Rhizobium was cultured to the logarithmic growth phase using YMB medium, the cells were washed 3 times with sterile water to remove the medium, and the Rhizobium cells were diluted with sterile water to OD 600 = 1.0. Each seedling was inoculated with 5 mL of the Rhizobium suspension, and the blank control was inoculated with an equal amount of sterile water. The nodulation situation was detected after 21 days. The back-inoculation results ( Figure 6 A) showed that the Rhizobium strain E1 described in the present invention nodulated with peas.

[0055] II. Back-inoculation of Sesbania cannabina and Lotus corniculatus

[0056] Seed germination: Sesbania seeds were sterilized with 70% alcohol for 5 minutes, washed thoroughly with sterile water, sterilized with 10% NaClO for 20 minutes, washed thoroughly with sterile water, soaked in sterile water overnight for imbibition, and then spread in a drainage container, covered with sterile gauze to keep moist, and germinated at a constant temperature of 25°C. When the radicle grew to 2-3 cm, the seedlings were transplanted into a test tube filled with sterilized Fahraeus nitrogen-free plant nutrient solution, and cultured in an artificial climate room at 25°C with a 16h light / 8h dark photoperiod until the seedlings grew true leaves, and then inoculated with rhizobia.

[0057] Nodule inoculation: Use YMB medium to culture rhizobia to the logarithmic growth phase, wash the bacteria three times with sterile water to remove the medium, and use sterile water to dilute the rhizobia to OD 600 =1.0, 1 mL of rhizobium suspension was inoculated per seedling, and the blank control was inoculated with the same amount of sterile water. Sterile Fahraeus nitrogen-free plant nutrient solution was regularly supplemented, and the nodulation of the plants was tested after 21 days. Figure 6 B and 6C) show that the rhizobium strain E1 of the present invention can nodulate with Sesbania sesbania and Lotus japonicus.

[0058] Example 3: Biological characteristics of Rhizobium E1

[0059] 1. Growth curve determination

[0060] Take 10 μL and dilute it with sterile water to OD 600 = 0.6 rhizobium suspension was inoculated into a 96-well tissue culture plate, 200 μL YMB medium was added to each well, and the blank control well was inoculated with an equal amount of sterile water. The plate was shaken at 28°C for 96 h, and the OD was measured every 30 min. 600 The value was obtained, and the growth curve was drawn to determine the growth of rhizobia. Bradyrhizobium sp. was selected as the control. Figure 7 A results show that E1 is a fast-growing rhizobium, which enters the growth plateau phase after about 25 hours.

[0061] 2. Motility Measurement

[0062] Under the induction of root secretions of leguminous plants, rhizobia undergo chemotaxis, migrate to the plant roots through flagellar movement and ciliary swimming, enter the roots through the gaps at the root hairs or lateral roots, and then symbiotically fix nitrogen. Mobility and swimming provide important competitiveness for rhizobia to quickly occupy the root ecological niche.

[0063] Adjust the YMA medium so that the agar content of the medium is 1.5% (ordinary YMA), 0.5% (motility test), and 0.3% (mobility test), and take 5 μL and dilute it with sterile water to OD 600= 0.6 rhizobium suspension inoculated strains were inoculated on the culture medium and cultured upright for 5 days, and the colony size was recorded. Bradyrhizobium sp. was selected as a control. Figure 7 B results show that E1 is a strong motile and swimming rhizobium.

[0064] 3. Resistance test

[0065] Prepare NaCl concentration gradient YMA medium (300, 400, 450, 500 mM), and use HCl and KOH to prepare pH gradient YMA medium (pH = 4, 6, 10, 12). Wash the rhizobia in the logarithmic growth phase and resuspend them to OD 600 =0.6, and inoculated on sodium chloride concentration gradient YMA and pH gradient YMA plates, 5μL / plaque, and the plaque growth was counted after 3-5 days. The experimental results showed that E1 could tolerate salt stress up to 450mM, alkali stress up to pH=12, and acid stress up to pH=4 (Table 3).

[0066] Configuration H 2 O 2 YMA medium with concentration gradient (20, 50, 100 μM). The detection method is the same as the salt-alkali tolerance range detection. The experimental results show that E1 can tolerate 50 μM H 2 O 2 stress (Table 3). Common bacterial antibiotics kanamycin, chloramphenicol, gentamicin and carbenicillin were selected to detect rhizobia resistance. The YMA medium was adjusted to a working concentration of 75 μg mL-1 for Kanamycin, 25 μg mL-1 for Chloramphenicol, 15 μg mL-1 for Gentamicin and 100 μg mL-1 for Carbenicillin. The detection method was the same as the salt-alkali tolerance range detection. The experimental results showed that E1 had strong resistance to Kanamycin, but not to Chloramphenicol, Gentamicin and Carbenicillin (Table 3).

[0067] Table 3

[0068]

[0069]

[0070] +: normal growth; w: weak growth; -: no growth.

[0071] 4. IAA production capacity test

[0072] The precursor L-tryptophan (200 mg L -1 ) in YMB medium, cultured at 28°C, 220 rpm for 3 days, centrifuged 1 mL of culture solution at 12000 rpm for 5 min, added Salkowsi colorimetric solution in a ratio of 1:1 to the supernatant, incubated for 30 min in the dark, and the color turned red, indicating that IAA could be secreted. The OD was measured. 530 The higher the IAA production, the greater the absorbance value. The experimental results show that E1 has a certain ability to secrete IAA ( Figure 8 ).

[0073] 5. Suppress soil-borne plant diseases

[0074] The rhizosphere is the first barrier for plants to resist the invasion of pathogens. Using rhizosphere microorganisms to resist the invasion of pathogens can effectively reduce the occurrence of soil-borne diseases. The rhizobium strain E1 described in this study can be used as a biocontrol bacterium to inhibit bacterial wilt caused by the pathogen Ralstonia solanacearum. Take 10 μL and dilute it with sterile water to OD 600 = 0.6 of rhizobium E1 and solanacearum suspension, 1:1 inoculated into the root tip of col-0 Arabidopsis 7d-old seedlings, with the mock treatment group inoculated with sterile water, and other Ensifer strains (Ensifer sesbaniae E2, deposit number: CCTCC NO: M20241559, deposited by China Center for Type Culture Collection, deposited at Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: July 15, 2024) + solanacearum as control, and the disease was recorded after culturing at 28°C on 1 / 2MS medium for 7 days. Fig. 9 As shown, E1 can significantly alleviate the disease caused by Ralstonia solanacearum, especially in terms of leaf area and lateral root growth, while the application of other Ensifer strains is ineffective.

[0075] It can be concluded from the above examples that the rhizobium E1 described in the present application has good stress resistance, can widely adapt to adverse environments, synthesize rich extracellular polysaccharides, and can be used as a source of soil physical and chemical property improvers; in addition, E1 can not only nodulate and fix nitrogen with pea, sesbania, and lotus roots, but also alleviate plant diseases caused by soil-borne pathogenic bacteria Ralstonia solanacearum, and has good application prospects in agricultural production.

Claims

1. An Ensifer sesbaniae E1, characterized in that The accession number is CCTCC NO: M20241925.

2. Use of the Sesbania sesbania E1 as claimed in claim 1 in promoting plant growth.

3. Use of the Sesbania rhizobium E1 as claimed in claim 1 in the preparation of a bacterial agent for promoting plant growth.

4. The use according to claim 2 or 3, characterized in that The plant growth promotion performance includes at least any one of the following: symbiotic nitrogen fixation, production of nitrogenase, production of indoleacetic acid, salt resistance, alkali resistance, acid resistance, and kanamycin resistance.

5. The use according to claim 2 or 3, characterized in that: The plant is pea, lotus root or sesbania.

6. Use of the Sesbania rhizobium E1 as claimed in claim 1 in the preparation of a medicament for preventing and treating bacterial wilt.

7. The use according to claim 6, characterized in that The bacterial wilt disease is caused by Ralstonia solanacearum.

8. A bacterial agent for promoting plant growth, characterized in that: The active ingredient of the bacterial agent includes the Sesbania rhizobium E1 described in claim 1.

9. A drug for preventing and treating bacterial wilt, characterized in that: The active ingredient of the bacterial agent includes the Sesbania rhizobium E1 described in claim 1.

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