A novel rhizobium strain, Neorhizobium glycine EC2-8, and its applications.

Abstract

The application discloses a Neorhizobium rhizobium strain isolated and purified from soybean root nodules and application thereof. The strain is a new species of the Neorhizobium genus through physiological and biochemical determination and phylogenetic analysis, and is named as Neorhizobium glycine EC2-8. The strain re-inoculation experiment shows that the rhizobium strain can invade the soybean roots and form effective nodulation, and can promote the growth of soybeans.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a new species belonging to the genus Neorhizobium and its applications. Background Technology

[0002] The formation of the soybean symbiotic nitrogen fixation system is a complex process involving the joint participation, dialogue, recognition, and synergistic effects of rhizobia and soybean-related genes, regulated by environmental conditions and intracellular physiological changes. This legume-rhizobium co-symbiotic process not only provides the necessary nitrogen for its own growth and development but also enhances soil nitrogen supply capacity through litter decomposition. Biological nitrogen fixation can reduce the application of chemical nitrogen fertilizers, mitigating the risks of soil inorganic nitrogen accumulation, soil pH imbalance, and eutrophication caused by excessive nitrogen fertilizer application, thus promoting and improving the healthy development of farmland ecosystems. Simultaneously, legume nodulation and nitrogen fixation can improve phosphorus use efficiency, indirectly regulating the stoichiometric ratio of carbon, nitrogen, and phosphorus in the soil. Currently, in soybean-producing countries such as Brazil, the United States, and Argentina, the inoculation area of ​​rhizobia reaches over 80%, while in my country, the inoculation area is less than 5%. Therefore, isolating and purifying highly efficient rhizobium resources can, on the one hand, provide candidate strains for enriching the rhizobium strain resource bank and developing commercial rhizobium inoculants; on the other hand, the application of rhizobia can optimize soil carbon, nitrogen, and phosphorus cycling, promoting crop growth. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Neorhizobium EC2-8 isolated and purified from soybean root nodules and its application, which supplements the existing rhizobium strain resources. This rhizobium can infect soybean roots and form effective nodules, while promoting soybean growth.

[0004] This invention is achieved through the following technical solution:

[0005] This invention discloses a Neorhizobium rhizobium strain isolated and purified from soybean root nodules and its applications. Physiological and biochemical assays and phylogenetic analysis confirmed that this bacterium is a new species of the Neorhizobium genus, and it has been named Neorhizobium glycine EC2-8. This strain has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, China; deposit date: May 24, 2022; accession number: CCTCC NO: M2022709.

[0006] Based on morphological characteristics, physiological and biochemical tests, and phylogenetic analysis, this strain was identified as a new species belonging to the genus *Neorhizobium* sp., and its closest relative is *Neorhizobium toomejilensis* T17_20. T The similarity was 98.57%. Reinoculation experiments showed that this rhizobium could infect soybean roots and form effective nodules, while also promoting soybean growth.

[0007] The rhizobium Neorhizobium glycine EC2-8 described herein has a 16S rDNA sequence as shown in SEQ ID NO:1. This bacterium is a new species belonging to the genus Neorhizobium sp. in systematic classification.

[0008] The rhizobium Neorhizobium glycine EC2-8 described above can infect soybean roots and form effective root nodules, and can also significantly increase soybean root length, root surface area, root volume, as well as aboveground fresh weight and dry weight. Therefore, this bacterium can be used to promote soybean growth.

[0009] When the rhizobium Neorhizobium glycine EC2-8 is used to promote soybean growth, the rhizobium is fermented to obtain a fermentation liquid. Soybean seeds are then soaked in the fermentation liquid, or the fermentation liquid is used to irrigate the roots of soybean seedlings.

[0010] The advantages and beneficial effects of this invention are as follows:

[0011] This invention can reduce nitrogen fertilizer production input and reduce resource waste. At the same time, it can also reduce the amount of nitrogen fertilizer applied in the crop production process, save farmers' production costs, meet green and organic food production standards, improve crop yield and quality, promote agricultural production and farmers' income, ensure the quality of grain products and straw feed, and ensure the quality and safety of downstream products, resulting in significant social benefits.

[0012] Preservation Information

[0013] The rhizobium Neorhizobium glycine EC2-8 has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan, on May 24, 2022, with accession number CCTCC NO: M2022709. Attached Figure Description

[0014] Figure 1The present invention describes the growth morphology of Neorhizobium glycine EC2-8, wherein: a is the growth morphology of the strain on YMA medium, b is the morphology of the strain as captured by transmission electron microscopy, and c is the morphology of the strain as captured by scanning electron microscopy.

[0015] Figure 2 This is the 16S rDNA phylogenetic tree of Neorhizobium glycine EC2-8 of this invention;

[0016] Figure 3 This is a mass spectrum of the whole-cell protein of Neorhizobium glycine EC2-8 of this invention;

[0017] Figure 4 This is the polar ester spectrum of Neorhizobium glycine EC2-8 of this invention;

[0018] Figure 5 This is the nodule reconnection experiment of Neorhizobium glycine EC2-8 of the present invention;

[0019] For those skilled in the art, other related figures can be obtained from the above figures without any creative effort. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described below with reference to specific embodiments.

[0021] Example 1: Isolation and purification of Neorhizobium glycine EC2-8

[0022] Sample source: Soybean root nodules

[0023] Sampling location: Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin, Heilongjiang Province, China;

[0024] YMA medium (solid): K2HPO4 0.25g / L, KH2PO4 0.25g / L, MgSO4·7H2O 0.2g / L, NaCl 0.1g / L, yeast extract 0.8g / L, mannitol 10g / L, agar 18g / L, pH 6.8-7.2, sterilized at 121℃ for 30min.

[0025] YMA medium (liquid): K2HPO4 0.25g / L, KH2PO4 0.25g / L, MgSO4·7H2O 0.2g / L, NaCl 0.1g / L, yeast extract 0.8g / L, mannitol 10g / L, pH 6.8-7.2, sterilized at 121℃ for 30min.

[0026] Isolation and purification steps: Fresh soybean root nodules were collected during the vigorous formation period. Soil particles on the surface of the nodules were rinsed with tap water. The nodules were then immersed in 70% ethanol, sodium hypochlorite, and 70% ethanol for 7 minutes each, followed by rinsing four times with sterile water. The nodules were then ground in a sterile mortar and pestle, and a series of diluted bacterial solutions were prepared with sterile water. Each gradient of bacterial solution was spread onto petri dishes containing YMA solid medium and incubated at 28°C. Single colonies from different dilutions were repeatedly picked and streaked onto plates, and subcultured for 4-5 generations to obtain pure, morphologically consistent single colonies. The purified strains were stored at 4°C.

[0027] Example 2: Phenotypic identification of Neorhizobium glycine EC2-8

[0028] Neorhizobium glycine EC2-8 was cultured on YMA solid medium plates at 28°C for 3 days. The strain grew well on YMA medium, with white, thick colonies. The strain was rod-shaped, Gram-negative, and no flagella were observed under transmission electron microscopy. The strain was non-motile. Figure 1 ).

[0029] Example 3: Systematic classification and identification of Neorhizobium glycine EC2-8

[0030] First, bacterial DNA was extracted using a bacterial genome extraction kit (Tiangen, 50 tests / kit), following the kit's instructions. Using the bacterial DNA as a PCR template, PCR amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGCTACCTTGTTACGACT T-3'). The PCR reaction system was a 10 μL system, as follows: 0.1 μL each of 27F and 1492R primers (50 pmol), 0.3 μL DNA template, 1 μL dNTPs (2.5 mM TaKaRa), 1 μL buffer (TaKaRa), and 5 U rTaq polymerase (5 μL). -10.3 μL of TaKaRa and 7.2 μL of sterile water were added as a negative control for PCR amplification. The specific amplification program was: 94℃ for 3 min, 94℃ for 1 min, 55℃ for 1 min, 72℃ for 1 min (28 cycles), and 72℃ for 5 min. After amplification, 3 μL of PCR product was aspirated and analyzed on a 1.0% agarose gel to observe the presence or absence of the target band. PCR product purification was performed according to the magnetic bead purification kit (10 mL / bottle, Somei). The purified PCR product was then sent to BGI Genomics for sequencing. The 16S rDNA sequence is shown in SEQ ID NO:1. The obtained sequencing sequence was compared for homology in the EzBioCloud database (https: / / www.ezbiocloud.net). The results showed that Neorhizobium glycine EC2-8 belongs to a new species of Neorhizobium sp. (Neorhizobium genus), and is most closely related to the strain Neorhizobium tomejilensis T17_20. T The similarity was 98.57%. A phylogenetic tree was constructed using MOLECULAR EVOLUTIONARY GENETIC ANALYSIS software (MEGA 5.0). Figure 2 (), and sequences from the genus Neorhizobium can form stable evolutionary branches.

[0031] Example 4: Detection of Physiological and Biochemical Characteristics of Neorhizobium glycine EC2-8

[0032] (1) Using the "common identification methods for general bacteria", and referring to the physiological and biochemical characteristics of the strain with the highest similarity, the enzyme activity, optimal growth temperature, ability to hydrolyze Tween 80, oxygen requirement, optimal pH and salt concentration range of the strain were determined. The specific results are shown in Table 1:

[0033] Table 1. Physiological and biochemical characteristics of strain Neohizobium glycine EC2-8

[0034]

[0035] (2) Antibiotic susceptibility testing was performed on the strain most closely related to Neorhizobium glycine EC2-8, and it was found that Neorhizobium glycine EC2-8 was sensitive to tetracycline, kanamycin, streptomycin and ampicillin, but not sensitive to erythromycin.

[0036] (3) Referring to the strain most closely related to Neorhizobium glycine EC2-8, the enzyme activity and carbon source assimilation capacity of the strain were detected using API 20E, API20NE, API ZYM and API 50CHB kits. The specific results are shown in Tables 2-5:

[0037] Table 2. Physiological and biochemical characteristics of strain Neorhizobium glycine EC2-8 - enzyme activity and carbon source assimilation (API 20NE)

[0038]

[0039]

[0040] +: Positive reaction; -: Negative reaction;

[0041] Table 3. Physiological and biochemical characteristics of strain Neorhizobium glycine EC2-8 - enzyme activity (API ZYM)

[0042]

[0043]

[0044] +: Positive reaction; -: Negative reaction; W: Weak positive reaction

[0045] Table 4. Physiological and biochemical characteristics of strain Neorhizobium glycine EC2-8 - enzyme activity and carbon source oxidation (API 20E)

[0046]

[0047] +: Positive reaction; -: Negative reaction

[0048] Table 5. Physiological and biochemical characteristics of strain Neorhizobium glycine EC2-8 – Acid production using carbon sources (API CHB)

[0049]

[0050]

[0051] +: Positive reaction; -: Negative reaction

[0052] Example 5: Biolog Identification of Neorhizobium glycine EC2-8

[0053] The utilization of 95 different carbon sources by strain Neorhizobium glycine EC2-8 was detected using Biolog microanalysis, as shown in Table 6.

[0054] Table 6. Physiological and biochemical characteristics of strain Neorohizobium glycine EC2-8 - carbon source utilization

[0055]

[0056]

[0057] +: Positive reaction; -: Negative reaction; W: Weak positive reaction

[0058] Example 6: Whole-cell protein mass spectrometry identification of Neorhizobium glycine EC2-8

[0059] Based on whole-cell protein (Matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry analysis of the strain, the mass spectra of the strain's whole-cell proteins are obtained as follows: Figure 3 As shown, this is used to further illustrate the taxonomic status of Neorhizobium glycine EC2-8.

[0060] Example 7: Analysis of fatty acid composition in strain cells

[0061] The strain was cultured in TAB liquid medium at 180 rpm and 30℃ for 2 days. The cells were collected by centrifugation at 8000 rpm, washed twice with distilled water, and then freeze-dried under vacuum to obtain dried bacterial cells. The dried bacterial cells were sent to the China General Microbiological Culture Collection Center for whole-cell fatty acid composition determination. The fatty acid composition of Neorhizobium glycine EC2-8 is shown in Table 7 below.

[0062] Table 7 Fatty acid composition of Neorhizobium glycine EC2-8

[0063]

[0064]

[0065] Example 8: Quinone component analysis in strain cells

[0066] Collect approximately 150 mg of lyophilized bacterial cells, add 40 mL of a chloroform:methanol = 2:1 (v / v) solution, and stir magnetically in the dark for about 10 hours; filter with filter paper and collect the filtrate; distill under reduced pressure at 40–45 °C using a rotary evaporator until dry, discarding the distillate; redissolve the dried material with 1–2 mL of a chloroform:methanol = 2:1 (v / v) solution, and spot the solution in long strips onto a GF254 silica gel plate (100×200 mm, Qingdao Marine Chemical Plant Branch); Hexane:ethyl ether = 34:6 (v / v) was used as the developing solvent for approximately 20 minutes, and the mixture was then air-dried. Under a 254 nm UV lamp, the dark brown band against a green fluorescent background represents the position of methylnaphthoquinone (Rf = 0.8), and the band at Rf = 0.4-0.5 represents ubiquinone. The band at Rf = 0.4 was scraped off and placed in a 2 mL centrifuge tube, dissolved in 1 mL of chloroform, and then filtered through a bacterial filter to remove silica gel. The filtrate was collected to obtain a chloroform solution of ubiquinone. This solution was stored at 4 °C in the dark. The quinone components were determined using reversed-phase high-performance liquid chromatography (RP-HPLC). The high-performance liquid chromatography (HPLC) system used was a Yilit HPLC system. The reversed-phase HPLC column was octadecylsilane (ODS 5 mm, 150 × 4.6 mm, I, d). The mobile phase was methanol (spectrally pure):isopropyl ether (chromatographically pure) = 3:1 (v / v) solution. The flow rate was 1 mL / min, the column temperature was 40℃, and UV detection was 275 nm. The determination and data were recorded using Yilit HPLC. The quinone types of the experimental strains were analyzed based on the relationship between ubiquinone components and elution time in the reference strain. The reference strain was *Sphingopyxis witflariensis* (CGMCC 1.09093). Analysis using the above method revealed that the ubiquinone in strain EC2-8 cells was mainly CoQ-8 (4.2%) and CoQ-10 (95.8%).

[0067] Example 9: Determination and Analysis of Polar Lipid Components in Strains

[0068] (1) Extraction method of polar lipids: The total lipid components were extracted by chloroform-methanol filtration. The polar lipid components were identified by thin-plate two-phase chromatography, referring to the method used by Minnikin et al. (1977).

[0069] (2) Preparation of polar lipid samples:

[0070] 1) 100 mg of dried bacterial cells were suspended in 9.5 mL of chloroform:methanol:0.3% NaCl (2.5:5:2).

[0071] 2) Incubate in a water bath at 80℃ for 15 minutes. After cooling, filter through filter paper into a 50mL centrifuge tube. Add 2.5mL chloroform and 2.5mL 0.3% NaCl, centrifuge at 4000rpm for 5 minutes, and collect the lower layer. Carefully separate the lower chloroform phase into a clean rotary evaporator flask, and remove the chloroform by rotary evaporation under reduced pressure (water bath temperature not exceeding 40℃). If the sample contains a small amount of water, add a small amount of benzene and then rotary evaporate under reduced pressure until a dry total lipid sample is obtained.

[0072] 3) Collect the organic phase, evaporate it by rotary evaporation, dissolve it in 250 μL of chloroform-methanol (2:1, v / v), and store it in a refrigerator at 4°C for later use.

[0073] (3) TLC detection of polar lipids:

[0074] 1) Activation of TLC sheets: Place a 10cm×10cm silicone sheet (Merck, 25TLC aluminum sheets)

[0075] Activate 20cm×20cm Silica gel (60F254) in an oven at 110℃ for 1 hour, then remove and cool.

[0076] 2) Spotting and development: Use a 10μL pipette to pick up 2μL of total lipid sample and spot it onto the TLC plate. Repeat the spotting 3 times.

[0077] Two closed chromatography tanks were used. First, the TLC plate was placed in the first chromatography tank for development. The developing solvent for the first dimension was chloroform.

[0078] Methanol:water (65:25:4, v / v), after the solvent reaches the top, remove the plate and dry it. Then place the plate into a second chromatography tank, the developing solvent for the second dimension is chloroform:methanol:acetic acid:water (80:12:15:4, v / v), ascend in a direction perpendicular to the first dimension, after the solvent reaches the top, remove the plate and dry it for later use.

[0079] 3) Thin plate color development:

[0080] Detection of total lipids: Spray phosphomolybdic acid reagent onto the TLC plate until completely wetted, heat at 100℃ for 5-8 minutes, and once clear spots appear, immediately scan the TLC plate and record the results. For example... Figure 4 As shown, the strain's polar lipids include: diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), lecithin (PC), phosphatidylethanolamine (PE), phosphatidyldimethylethanolamine (PDE), and an unknown phospholipid (PL).

[0081] Example 10: Verification of Neorhizobium glycine EC2-8 nodulation re-inoculation and its promoting effect on soybean growth

[0082] The *Neorhizobium glycine* EC2-8 strain was inoculated into YMA liquid medium and cultured at 28°C with shaking for 48 hours to obtain a bacterial suspension. The suspension was centrifuged, and sterile water was added. Under OD600 conditions, a bacterial suspension with an OD value of 1 was prepared. Soybean seeds were soaked in alcohol for 7 minutes, rinsed twice with sterile water, and then soaked overnight in the bacterial suspension with an OD value of 1. Seeds soaked in sterile water served as a control. The next day, both the control seeds and the seeds soaked in the bacterial suspension were sown in sterilized vermiculite. This process was repeated three times. During seedling growth, the seedlings were irrigated with sterile water and a nitrogen-free nutrient solution (CaCl2·2H2O 0.13 g / L, MgSO4·7H2O 0.12 g / L, KH2PO4 0.1 g / L, Na2HPO4 0.3 g / L, Fe citrate). (0.005 g / L, H3BO3 0.002 g / L, MnSO4 0.001 g / L) During the second week of growth, 50 mL of a bacterial solution with an OD600 of 1 was poured into the seedling pots after the seed soaking treatment. Soybean plants were harvested on the 24th day of growth, and their fresh and dry weights were measured. Roots were scanned, and root length, volume, and surface area were measured. The results showed that the strain *Neorhizobium glycine* EC2-8 could infect soybean roots and form effective root nodules (see attached). Figure 5 It can also significantly increase soybean root length, root surface area and root volume, as well as above-ground fresh weight and dry weight (Table 8).

[0083] Table 8. Effects of Neorhizobium glycine EC2-8 on soybean growth.

[0084]

[0085] Example 11: Whole genome sequencing and tumor gene analysis of Neorhizobium glycine EC2-8

[0086] The strain Neorhizobium glycine EC2-8 was inoculated into YMA liquid medium and cultured at 28°C with shaking for 72 hours to obtain a bacterial suspension. The suspension was centrifuged, the supernatant was discarded, and the cells were washed with sterile water and centrifuged again, discarding the supernatant to obtain pure bacterial cells. Then, according to… Genomic DNA was extracted from the bacteria using the Promega genomic DNA purification kit. The purified genomic DNA was quantified using a TBS-380 fluorescence analyzer (Turner BioSystems Inc., Sunnyvale, CA). High-quality DNA (OD260 / 280 = 1.8–2.0, total DNA ≥ 5 μg, concentration ≥ 60 ng / μL) was used for library construction and sequencing, which was outsourced to BGI Genomics. After sequencing, the raw data underwent quality control to filter out low-quality reads, retaining only high-quality reads. The valid data after quality control was assembled using Canu (https: / / canu.readthedocs.io / en / latest / ). Reads were aligned to the assembled genome sequences, and the sequencing depth distribution of the assembled results was analyzed to evaluate the quality of the assembly. Then, the raw data was aligned to the assembled sequences, and the assembly results were optimized using Arrow software to correct erroneous assembly regions. The results showed that the genome size of strain Neorhizobium glycine EC2-8 was 6.77 Mbp and the GC content was 61.34%. After gene function comparison and annotation in the Database of Clusters of Orthologous Genes (COGs), 6819 genes could be annotated (Table 9).

[0087] The average nucleotide identity (ANI) of strain Neorhizobium glycine EC2-8 with its most similar strain was calculated using the EzBioCloud website (https: / / www.ezbiocloud.net / tools / ani). The results are shown in Table 10. The ANI values ​​of Neorhizobium glycine EC2-8 with its most similar strain range from 80.62% to 88.67%. Based on the threshold that the ANI for the same species must be greater than 95%, this further confirms that Neorhizobium glycine EC2-8 belongs to a new species of Neorhizobium.

[0088] Table 9. Genomic characteristics of Neorhizobium glycine EC2-8.

[0089]

[0090] Table 10. ANI values ​​of Neohizobium glycine EC2-8 and similar strains

[0091]

[0092]

[0093] Based on phylogenetic analysis, the morphological, physiological and biochemical characteristics, cytochemical composition analysis, and molecular analysis of the above-mentioned Neorhizobium glycine EC2-8 were combined with... Figure 2 Comparison with the most closely related strains on the phylogenetic tree shown further demonstrates that Neorhizobium glycine EC2-8 does indeed differ from different species at the same genus level (Table 11), and can be identified as a new species of this genus.

[0094] Table 11 Comparison of properties of Neorhizobium glycine EC2-8 with different species within the same genus at the taxonomic level

[0095]

[0096]

[0097] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A rhizobium Neorhizobium glycine EC2-8, characterized in that, Its accession number is CCTCC NO: M2022709.

2. The application of the rhizobium according to claim 1 in promoting soybean growth.

3. The application of the rhizobium according to claim 1 in increasing soybean root length, root surface area and root volume, as well as increasing the fresh weight and dry weight of soybean aboveground parts.

4. A culture comprising the rhizobium of claim 1.

5. A soybean growth regulator comprising the rhizobium of claim 1 and the culture of claim 4.

6. A method for promoting soybean growth, characterized in that, The rhizobium of claim 1 is fermented to obtain a fermentation broth, and soybean seeds are pretreated by immersing them in the fermentation broth.

7. A method for promoting soybean growth, characterized in that, The rhizobium of claim 1 is fermented to obtain a fermentation broth, which is then poured onto the roots of soybeans.

8. The method for promoting soybean growth according to claim 7, characterized in that, The fermentation liquid was poured onto the roots of soybean seedlings.

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