Mesorhizobium huakuii ZK1 T and application thereof

Abstract

The present application relates to the field of microbial technology, and discloses a new Aeschynomene indica rhizobium ZK1 T and application thereof, the new Aeschynomene indica rhizobium ZK1 T was preserved in the China General Microbiological Culture Collection Center on December 30, 2024, and the preservation number is CGMCC No.33228. The strain provided by the present application grows fast and has good stability, can grow and pass on the rhizobium culture medium YMA containing 2.0% NaCl at pH 4-10, and shows unique acid, alkali and salt nitrogen fixation capacity. The Aeschynomene indica rhizobium ZK1 T provided by the present application can degrade phosphorus elements on the organic phosphorus plate culture medium within 3d, and the phosphorus dissolution ring diameter is about 0.5 cm after about 5d. The Aeschynomene indica rhizobium ZK1 T provided by the present application can significantly improve the average plant height of different Aeschynomene indica variety materials by 106.81%, and the Aeschynomene indica rhizobium ZK1 T bacterial liquid can also improve the fresh weight of the aboveground part of the plant by 345.11%. The discovery of the strain provides strain resources and scientific support for the development and utilization of Aeschynomene indica rhizobium agent and the development of high-efficiency planting technology of marginal land Aeschynomene indica.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Rhizobium sesbaniae ZK1. T And its applications. Background Technology

[0002] Tian Jing ( Sesbania cannabina ( Retz. ) Poir. Sesbania sesbania is an annual green manure crop belonging to the genus Sesbania in the legume family. It is salt-tolerant, alkali-tolerant, acid-tolerant, flood-tolerant, high-yielding, and adaptable to poor soil conditions, making it a pioneer plant for improving saline-alkali land. Sesbania plants are rich in nutrients, with a large underground root biomass, which can significantly improve soil structure and fertility. Rhizobia are Gram-negative rod-shaped bacteria that live in the soil. Under suitable conditions, rhizobia can infect legumes and form symbiotic nodules to fix nitrogen, converting free nitrogen from the atmosphere into usable nitrogen. Compared to industrial nitrogen fixation, biological nitrogen fixation not only fixes a huge amount of nitrogen but is also inexpensive, pollution-free, reduces fertilizer use, and saves energy. It is an effective means of cost reduction and efficiency improvement in future agricultural production and one of the main nitrogen sources for modern agriculture. Numerous studies both domestically and internationally have reported the excellent performance of sesbania in improving soil fertility and reducing salinity. Inoculation with rhizobia can promote the growth of sesbania and enhance its effect on improving saline-alkali land. The sesbania-rhizobia symbiotic system has broad application prospects in the improvement of saline-alkali land. At present, the development and utilization of soybean rhizobia are very mature and continue to be applied on a large scale. At the same time, the rhizobia of peanut, alfalfa, and leguminous forage grasses such as clover, alfalfa, and saxaul are also widely used. However, existing research has limited exploration and utilization of sesbania rhizobia resources.

[0003] Sesbania scoparia exhibits strong stress tolerance and adaptability. Previous studies have found that it can grow well in Northeast China's soda-alkali soils with a pH of 9-9.5 and coastal saline-alkali soils with a pH of 0.4-0.6%, producing nitrogen-fixing root nodules. Its effects on reducing soil erosion and improving soil quality are significant after being plowed into the soil. Due to its outstanding stress tolerance and potential as a high-protein forage, sesbania scoparia has received considerable attention in recent years as a valuable tool for developing marginal land. However, its rhizobium resources, especially rhizobium strains that can efficiently match superior germplasm resources, are scarce.

[0004] Given that biological nitrogen fixation using rhizobia can meet 60%-70% of the nitrogen requirements of leguminous plants, and its nitrogen fixation efficiency is the highest known to date, research on rhizobia in sesquiterpenes has significant practical application value. Providing highly efficient rhizobium strains and their applications for superior sesquiterpenes germplasm is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Senecio scandens* rhizobium (… Rhizobium sesbaniaesp.), and its application in agricultural production was described. Further identification revealed that this strain is a new species of *Senecio scandens* rhizobium and it was named (sp.). Rhizobium Sesbania ZK1 T The sesbania rhizobium ZK1 mentioned above. T It can tolerate high levels of acid, alkali and salt stress, and after re-inoculation, it can effectively promote nodulation and growth of different sesbania varieties. It has broad application prospects in the use of the sesbania-rhizobium system for marginal land improvement.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0007] The first aspect of this invention provides a strain of *Senecio scandens* rhizobium (… Rhizobium sesbaniae ZK1 T The sessilia rhizobium ( Rhizobium sesbaniae ZK1 T It was deposited on December 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33228. The 16S rRNA sequence is shown in SEQ ID No. 1.

[0008] The second aspect of the present invention provides a bacterium containing the aforementioned sesquiterpenoid ZK1. T Fermentation broth, bacterial suspension, or its sterile supernatant or preparation thereof.

[0009] The third aspect of the present invention provides the aforementioned sesbania rhizobium ZK1 T Or the application of the fermentation broth, bacterial suspension, or its sterile supernatant or its preparation in the degradation of organophosphorus compounds.

[0010] The fourth aspect of the present invention provides the aforementioned sesbania rhizobium ZK1 T Or the application of the fermentation broth, bacterial suspension or its sterile supernatant or its preparation in the acid, alkali and salt tolerance of plants.

[0011] The application includes using the sesquiterpenoid rhizobium ZK1. T It grows in a medium with a pH of 4-10.

[0012] The culture medium is YMA culture medium, which includes YMA solid culture medium and YMA liquid culture medium (also known as YEM). The YMA solid culture medium includes 0.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of sodium chloride, 10.0 g of mannitol, 1.0 g of yeast extract, 15.0 g of agar, and 1000 mL of distilled water. The YMA liquid culture medium is obtained by removing the agar from the YMA solid culture medium, and the remaining components are the same.

[0013] The application includes growing the *Rhizobium guarense* in a culture medium containing 0-2% NaCl.

[0014] The culture medium is YMA medium, including YMA solid medium and YMA liquid medium (also known as YEM), with only the NaCl concentration adjusted (0.5-4%).

[0015] The fifth aspect of the present invention provides the aforementioned sesbania rhizobium ZK1 T Or the application of the fermentation broth, bacterial suspension, or its sterile supernatant or its preparation in promoting plant growth. The plants mentioned include, but are not limited to, sesbania.

[0016] A sixth aspect of the present invention provides a microbial agent for promoting the growth of sesame, wherein the active ingredient of the microbial agent includes the sesame rhizobium ZK1. T .

[0017] The seventh aspect of the present invention provides the aforementioned sessilia rhizobium ZK1 T Or the application of the plant growth-promoting microbial agents in the production of bio-organic fertilizers.

[0018] The eighth aspect of the present invention provides a bio-organic fertilizer containing the *Sinonovacula sibirica* ZK1. T Or the aforementioned microbial agents that promote plant growth.

[0019] The beneficial effects of this invention are:

[0020] Compared with the prior art, the present invention has the following advantages: the sesquiterpenoid rhizobium of the present invention ( Rhizobium Sesbania ZK1 T It grows rapidly and has good stability, and can be grown and passaged on YMA rhizobium medium with pH 4-10 and 2.0% NaCl, demonstrating its unique acid, alkali, and salt-fixing tolerance. This invention relates to the *Zok1* rhizobium species. T It can degrade phosphorus on organic phosphorus plates within 3 days, and the diameter of the phosphorus-solubilizing zone is about 0.5 cm after about 5 days. The *ZK1* rhizobium provided by this invention... T It can significantly increase the average plant height of seedlings of different sesbania varieties by 106.81%, while the sesbania rhizobium ZK1 T The bacterial solution can also increase the fresh weight of the above-ground parts of the plant by 345.11%. The discovery of this strain provides strain resources and scientific support for the development and utilization of sesbania rhizobium agents and for accelerating the development of efficient sesbania planting technology on marginal land. Attached Figure Description

[0021] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The present invention relates to the sessilia rhizobium ZK1. T Colony morphology on agar plates;

[0023] Figure 2 The present invention relates to the sessilia rhizobium ZK1. T Phylogenetic tree constructed based on 16S rRNA gene;

[0024] Figure 3 The present invention relates to the sessilia rhizobium ZK1. T Phylogenetic tree constructed based on whole genome sequence;

[0025] Figure 4 The present invention relates to the sessilia rhizobium ZK1. T OD during growth on YMA liquid medium (YEM) with different pH and NaCl concentrations 600 ;

[0026] Figure 5 The present invention relates to the sessilia rhizobium ZK1. T Growth was carried out on nitrogen-free (NFM), potassium-solubilized (K), organic phosphorus (P), and YMA media with different pH and NaCl concentrations.

[0027] Figure 6 The present invention relates to the sessilia rhizobium ZK1. T Results of growth-promoting experiments on different sesame varieties (CK is blank liquid culture medium, R is sesame rhizobium ZK1 inoculated according to this invention). T (bacterial solution);

[0028] Figure 7 The present invention relates to the sessilia rhizobium ZK1. T Results of the effects of re-inoculation of different sesbania varieties on plant height, biomass, and root nodule number (CK is blank liquid culture medium, R is sesbania rhizobium ZK1 of this invention). T Bacterial suspensions; ns showed no significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001). Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments.

[0030] This invention isolated a strain of *Senecio scandens* rhizobium (… Rhizobium sesbaniae Based on comprehensive analysis of data including colony and cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequencing, this strain was identified as a new species of *Rhizobium sesquiterpenoides* and named ZK1. T The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 30, 2024, with accession number CGMCC No. 33228.

[0031] The sesquiterpenoids ( Rhizobium sesbaniae ZK1 T It can tolerate high levels of acid, alkali and salt stress, and after re-inoculation, it can effectively promote nodulation and growth of different sesbania varieties. It has broad application prospects in the use of the sesbania-rhizobium system for marginal land improvement.

[0032] The following are specific embodiments of the present invention:

[0033] Example 1: Sesbania rhizobia ( Rhizobium sesbaniae ZK1 T Separation and identification

[0034] I. Isolation and Preservation of Rhizobium

[0035] The seeds of *Zhongke Jing 1* were disinfected twice with 75% alcohol and planted in sterilized vermiculite. Plants that had grown for 55 days were taken, rinsed thoroughly with tap water, and the exposed, plump, light pink or pink root nodules were collected for isolation, purification, and culture. In a clean bench, the root nodules were treated with 75% ethanol for 30 seconds, followed by 2.5% NaClO for 10 minutes, then rinsed 3-4 times with sterile water, and dried. The nodules were then ground into a homogenate using a sterile grinding rod, and finally 1 ml of sterile water was added. After serial dilution, the nodules were plated on YMA agar plates and incubated upside down at 28 ℃ for 2-4 days. After multiple purifications, milky white, round or oval, well-defined, moist, and glossy monoclonal colonies were obtained, preserved and identified using the glycerol method (Figure 1).

[0036] II. Identification and Phylogenetic Analysis of Rhizobium 16S rRNA Gene

[0037] Colonies were scraped and placed in 100 μL of TE buffer. After mixing, the mixture was incubated at 100 °C for 10 min, followed immediately by an ice bath at −20 °C for 30 min. The mixture was centrifuged at 12000 r / min for 10 min, and the supernatant was used as a template for PCR. Bacterial DNA was extracted using the Ezup column-based bacterial genomic DNA extraction kit. The 16S rRNA gene (sequence shown in SEQ ID NO: 1) was amplified by PCR using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The specific PCR reaction system was: 95 °C for 5 min; 94 °C for 30 s, 57 °C for 30 s, 72 °C for 90 s, 30 cycles; 72 °C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0038] Based on the comparison results from the EzBioCloud database, similar strains were selected and their corresponding 16S rRNA gene sequences were downloaded. A phylogenetic tree was constructed using MEGA 11.0 combined with the Negotiator-Joining (NJ) method, with the bootstrap value set to 1000. This study selected... Bradyrhizobium japonicum LMG 6138 T As an outgroup (Figure 2).

[0039] III. Whole-genome sequencing analysis of rhizobia

[0040] The extracted bacterial DNA was sent to the PacBio platform of Wuhan Hope Group Biotechnology Co., Ltd. for sequencing. FastP v0.23.1 was used for quality control of the raw reads to remove low-quality data and obtain clean reads. Canu software was used to assemble the quality-controlled data. Simultaneously, the quality-controlled FastQ format read files were aligned to the assembled genome using Minimap2 software, and the sequencing depth of each locus was calculated using the depth function of Samtools software. Prokka was used for gene annotation and prediction of the assembled genome. tRNAscan-SE and RNAmmer were used to predict tRNA and rRNA genes, respectively.

[0041] IV. Digital DNA-DNA hybridization values, average nucleotide identity, and whole-genome phylogenetic analysis

[0042] Download the whole genome sequences of similar strains from the NCBI and JGI (Joint Genome Institute) databases. (Note: No results were found in the databases.) Rhizobium yanglingense SH 22623 T , Rhizobium sullae IS123 T and Rhizobium acid soil FH13 T The whole genome sequences of these species were used, therefore the whole genome sequences of non-type strains of these species were selected as replacements. A whole genome phylogenetic tree was constructed using FastME software; JspeciesWS software was used to calculate the phylogenetic tree of *Rhizobium stenoptera* (…). Rhizobium sesbaniae ZK1 T The average nucleotide identity (ANI) between the strain and the reference strain was calculated; the digital DNA-DNA hybridization (dDDH) value was calculated using the GGDC (Genome to Genome Distance Calculator) to further identify the rhizobium of *Senecio scandens*. Rhizobium sesbaniae ZK1 T The classification status (Table 1, Figure 3).

[0043] Table 1. Rhizobium in sesquiterpenoids ( Rhizobium sesbaniae ZK1 T Comparison of ANI and dDDH values ​​with reference strain

[0044]

[0045] Example 2: Sesbania rhizobia ( Rhizobium sesbaniae ZK1 T biological characteristics

[0046] I. Testing of Acid, Alkali and Salt Tolerance Range

[0047] The well-growing rhizobia ( Rhizobium sesbaniae ZK1 T The strain was inoculated at a volume fraction of 5% into LB liquid medium with NaCl concentration gradients of 0.5%, 1%, 1.5%, 2%, 3%, and 4%, respectively, and cultured at 37℃ and 200 r / min for 24 h. Its OD value was then measured. 600 Value. Simultaneously, the same inoculum size and salt concentration gradient were used to inoculate into YEM solid medium and cultured at 37 ℃ for 24 h. Rhizobium sesquiterpenoids (Rhizobium sesquiterpenoids) were observed in the YEM medium. Rhizobium Sesbania ZK1 T OD 600The strain was largely unaffected by NaCl concentrations below 1.5%, but its growth decreased sharply at 2%, and it essentially failed to grow at 4% NaCl concentration (Figure 4A). Under extreme acid-base conditions (pH 2, 3, 10, and 11) in YEM medium, the rhizobium ZK1... T It was basically unable to grow, and was not significantly affected compared to the control at pH 5 and 6. It was somewhat affected at pH 4, but still showed good growth (Figure 4B).

[0048] Purified sesquiterpenoids ( Rhizobium sesbaniae ZK1 T The strain was inoculated at a volume fraction of 5% into LB medium adjusted to pH 2, 3, 4, 5, 6, 7, 10, and 11 with concentrated hydrochloric acid, and cultured at 37 ℃ and 200 r / min for 24 h. Its OD600 value was then measured. Simultaneously, the same inoculation volume and pH gradient were used to inoculate into YEM solid medium, and cultured at 37 ℃ for 24 h. Results consistent with those observed in the liquid medium were observed in YEM solid medium. *Rhizobium sesquiterpenoides* ( Rhizobium sesbaniae ZK1 T It can tolerate a NaCl concentration of 2% and a pH range of 4-10 (Figure 5).

[0049] II. Testing of Nitrogen Fixation, Potassium Solubilization, and Organophosphorus Solubilization Capabilities

[0050] The well-growing rhizobia ( Rhizobium sesbaniae ZK1 T The strains were inoculated into nitrogen-free (NFM), potassium-free (K), and Monkina organic phosphorus (P) medium, respectively, and cultured at 37 ℃ for 6 days. *Senecio scandens* rhizobium (… Rhizobium Sesbania ZK1 T It can grow in Monkina organophosphate (P) medium but cannot grow in nitrogen-free medium (NFM) or potassium-solubilizing medium (K). *Senecio scandens* rhizobium (*Senecio scandens*) was observed to grow on organophosphate medium after 3 days of growth. Rhizobium Sesbania ZK1 T A transparent halo forms around the colony of the strain, and the diameter of the phosphate-solubilizing ring is about 0.5 cm after 5 days of growth.

[0051] Example 3: Sesbania rhizobium ( Rhizobium sesbaniae ZK1 T Growth-promoting characteristics of different sesbania varieties

[0052] Sesbania rhizobia ( Rhizobium sesbaniae ZK1 TAfter streaking onto YEM solid medium and culturing at 28 ℃ for 2-3 days, single colonies were picked and cultured in 300 ml of YEM liquid medium for 1 day until turbidity was observed. 600 When the value is around 1.045, centrifuge at 14000 rpm for 10 min twice, discard the supernatant, rinse twice with sterile water, and then resuspend the bacterial block with sterile water. Calculate the OD value of the bacterial solution. 600 Simply set the value to 0.1.

[0053] Seeds of sesbania sesquiterpenes, including Zhongke Jing 1 (ZK1), Zhongke Jing 2 (ZK2), Zhongke Jing 6 (ZK6), Zhongke Jing 10 (ZK10), and Lu Jing 5 (LJ5), were pre-treated with concentrated sulfuric acid for 8-10 minutes, then washed three times with sterile water, and sterilized overnight with chlorine (100 mL sodium hypochlorite + 5 mL concentrated hydrochloric acid) for 12-15 hours. The seeds were then removed and air-dried in a clean bench for 30-60 minutes. After sterilization of vermiculite, the seeds were placed in sterilized trays and filled with sterilized Hoagland's modified liquid nutrient solution. Sowing was carried out after the vermiculite had fully absorbed the solution. Ten days after sowing, the isolated rhizobia were inoculated, and phenotypic data such as plant height and above-ground fresh weight were observed. Twenty-four days after inoculation, phenotypic data such as plant height and above-ground fresh weight were recorded.

[0054] To verify the isolated sesquiterpenoid rhizobia ( Rhizobium sesbaniae ZK1 T The effects of different sesbania cultivars on growth and nodulation ability were investigated using a pot inoculation experiment conducted under sterile conditions (Figures 6-7). The results showed that 24 days after inoculation, the sesbania rhizobium (…) of this invention… Rhizobium sesbaniae ZK1 T It can significantly increase the average plant height of seedlings of different sesbania varieties by 106.81%, while sesbania rhizobium ( Rhizobium sesbaniae ZK1 T The bacterial solution can also increase the fresh weight of the above-ground parts of the plant by 345.11% and significantly promote the nodulation of Zhongkejing No. 1, Zhongkejing No. 10 and Lujing No. 5.

[0055] In summary, the sessilia rhizobium (Sesamum indicum) of the present invention Rhizobium sesbaniae ZK1 T CGMCC No. 33228 exhibits rapid growth and good stability, and can be grown and passaged on YMA rhizobium medium with pH 4-10 and 2.0% NaCl, demonstrating its unique acid, alkali, and salt-tolerance nitrogen-fixing abilities. This invention relates to the *Senecio scandens* rhizobium (…). Rhizobium sesbaniae ZK1 T It can degrade phosphorus on organic phosphorus plates within 3 days, and the diameter of the phosphorus-solubilizing zone is about 0.5 cm after about 5 days. This invention relates to *Senecio scandens* rhizobium (…). Rhizobium sesbaniae ZK1 TIt can significantly increase the average plant height of seedlings of different sesbania varieties by 106.81%, while sesbania rhizobium ( Rhizobium sesbaniae ZK1 T The bacterial solution can also increase the fresh weight of the above-ground parts of the plant by 345.11%. The discovery of this strain provides strain resources and scientific support for the development and utilization of sesbania rhizobium agents and for accelerating the development of efficient sesbania cultivation technology on marginal land.

[0056] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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.

Claims

1. A strain of Rhizobium huanghuantiao (R. huanghuantiao) ZK1 Rhizobium sp. ) ZK1 T characterized in that, Rhizobium shibendae ZK1 Rhizobium sp. ) ZK1 T On December 30, 2024, it was preserved in the China General Microbiological Culture Collection Center, and the preservation number was CGMCC No. 33228.

2. A fermentation broth, bacterial suspension or preparation thereof comprising the new Aeschynomene root nodule bacteria (Ensifer adhaerens) of claim 1 Rhizobium sp. ) ZK1 T .

3. Azorhizobium caulinodans (Ito 1 Rhizobium sp. ) ZK1 T or the use of the fermentation broth, the bacterial suspension or the preparation thereof according to claim 2 for the degradation of organophosphorus compounds.

4. Bradyrhizobium yamatoense according to claim 1, Rhizobium sp. ) ZK1 T or use of the fermentation broth, the bacterial suspension or the preparation thereof according to claim 2 for plant growth promotion, the plant being Sesbania.

5. A bacterial agent for promoting growth of Sesbania herbacea, characterized by, The active ingredient of the bacterial agent includes the new Aeschynomene root nodule bacteria (Rhizobium Aeschynomene) of claim 1 Rhizobium sp. ) ZK1 T .

6. The Azorhizophilus ingrahamii (ATCC 29735) of claim 1. Rhizobium sp. ) ZK1 T or the use of the bacteria agent for promoting growth of the Sesbania in the production of the bio-organic fertilizer suitable for the Sesbania.

7. A bio-organic fertilizer, characterized by, The bio-organic fertilizer contains the new Aeschynomene indica root nodule bacteria of claim 1 Rhizobium sp. ) ZK1 T or the bacteria agent for promoting growth of Aeschynomene indica of claim 5.

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