Kosakia sp. ly-1 and application thereof in promoting rice growth

Abstract

The application discloses a kossakia sp. LY-1 and application thereof in promoting rice growth, which is preserved in China Center for Type Culture Collection and has a preservation number CCTCC NO: M 20242320. The strain carries complete nifHDKY-ENX-BQ-VZW nitrogen fixation gene cluster, and has a nitrogenase content of 160 ng / mL. Meanwhile, the strain has four growth promoting functions of dissolving inorganic phosphorus (57 mg / L), producing indole acetic acid (19.76 mg / L) and producing siderophore (48%). Under the condition of nitrogen-free water culture, the strain can significantly promote the growth of rice seedlings, and the stem length is increased by 25.7% and the biomass is increased by 45.6% compared with a control, so that the strain provides a functional strain resource for green production of rice.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to Cossackie bacterium LY-1 and its application in promoting rice growth. Background Technology

[0002] Rice is my country's most important food crop, accounting for about one-third of the country's total grain output annually. Its stable and high yields are of strategic significance for ensuring national food security. However, my country's rice production currently faces a prominent contradiction between excessive nitrogen fertilizer application and low utilization rates. Statistics show that my country's annual nitrogen fertilizer application for rice reaches 6.3 million tons, accounting for about one-third of the global total, but the nitrogen fertilizer utilization rate is only 43.3% (Ministry of Agriculture and Rural Affairs, 2025), which is 8-24 percentage points lower than the international advanced level (52%-67%). A large amount of unabsorbed nitrogen is emitted into the environment through ammonia volatilization, leaching, and runoff, with annual emissions exceeding 1.69 million tons. The resulting environmental costs, such as air pollution (e.g., PM2.5 formation), water eutrophication, and greenhouse gas emissions, are equivalent to 52% of the economic benefits of increased nitrogen fertilizer production (Nature, 2023), severely restricting sustainable agricultural development. To this end, the Ministry of Agriculture and Rural Affairs has clearly put forward the strategic goal of "promoting the control of fertilizer use and increasing efficiency". Developing biological growth-promoting technologies that can effectively replace or reduce the use of chemical nitrogen fertilizers is not only an urgent need to break through the current technical bottlenecks, but also an important technical support for achieving the green transformation of agriculture and the national "dual carbon" goals.

[0003] Among the various forms of biological nitrogen fixation in paddy field ecosystems, endophytic diazotrophs have the greatest potential for development and application. Compared with rhizosphere diazotrophs, endophytic diazotrophs have the following unique advantages: (1) Microenvironmental protection effect: Endophytic diazotrophs colonize the tissues of rice roots, stems, leaf sheaths, etc. The plant endophytic microenvironment can provide a natural low-oxygen protection barrier for the nitrogenase complex, effectively avoiding irreversible inactivation of nitrogenase by oxygen, thereby maintaining stable nitrogenase activity (Reinhold-Hurek & Hurek, 2011). (2) Resource competition advantage: Endophytic colonization allows the strains to physically avoid fierce competition with rhizosphere soil microorganisms, without having to compete with soil microorganisms for limited root exudates. (3) High nitrogen conversion efficiency: The nitrogen fixed by endophytic nitrogen-fixing bacteria can be directly transported to the host cells through the plant vascular bundle system for utilization, avoiding intermediate nitrogen loss during soil nitrogen fixation. It has a higher nitrogen conversion efficiency than soil-free nitrogen-fixing bacteria (Reinhold-Hurek & Hurek, 1998). (4) Multifunctional synergistic growth promotion: Studies have shown that endophytic nitrogen-fixing bacteria generally have multiple plant growth promotion (PGP) functions, such as producing indoleacetic acid (IAA), dissolving inorganic phosphorus, producing siderophores, and synthesizing ACC deaminase. They can synergistically promote rice growth from multiple dimensions, such as nitrogen supply, hormone regulation, phosphorus and iron nutrition, and stress relief, and significantly improve the plant's absorption and utilization efficiency of mineral ions. The growth promotion effect is particularly prominent under nitrogen-deficient conditions (Shrestha & Ladha, 1996). Therefore, rice endophytic nitrogen-fixing bacteria are an ideal strain resource for developing new bio-fertilizers with both biological nitrogen fixation and multifunctional growth promotion characteristics. In recent years, endophytic nitrogen-fixing bacteria in rice, represented by Azoarcus, Herbaspirillum, and Gluconacetobacter, have attracted widespread attention. These strains mainly colonize the inside of plant tissues and have significant intraplant nitrogen-fixing activity, showing good application prospects.

[0004] Despite significant progress in research on endophytic nitrogen-fixing bacteria in rice, existing technologies still have obvious shortcomings in the following aspects, which restrict their widespread application in production practice:

[0005] First, their functions are limited and their growth-promoting mechanisms are incomplete. Most of the reported endophytic nitrogen-fixing bacteria in rice possess only one or a few growth-promoting functions. Rice growth has complex requirements for multiple nutrients such as nitrogen, phosphorus, and iron. Strains with limited functions cannot achieve synergistic effects through multiple pathways, thus limiting their actual growth-promoting effects.

[0006] Second, the complex structure of endophytic nitrogen-fixing bacteria communities makes the screening of highly efficient strains difficult. The community structure of rice endophytic nitrogen-fixing bacteria is influenced by multiple factors, including plant tissue type, growth stage, variety genotype, and soil nitrogen status. Furthermore, different isolates within the same bacterial community may either promote or inhibit overall nitrogen fixation activity, making the targeted screening of highly efficient strains with both high nitrogen fixation activity and stable colonization ability a significant challenge (Prakamhang et al., Applied SoilEcology, 2009). The lack of stable, highly efficient strains introduces additional difficulties for large-scale preparation and application.

[0007] Fourth, the application verification system is incomplete. Existing functional studies of endophytic nitrogen-fixing bacteria are mostly conducted under in vitro conditions or in greenhouse pots, lacking standardized hydroponic evaluation systems. There is insufficient systematic research on their root colonization dynamics in rice, nutrient stress response, and synergistic effects with chemical fertilizers, making it difficult to accurately assess their application potential in controlled environments. Summary of the Invention

[0008] The technical problem to be solved by this invention is: to address the technical bottlenecks of existing rice endophytic nitrogen-fixing bacteria with single function and imperfect application verification system, to provide a rice endophytic bacterium carrying a complete nitrogen-fixing gene cluster and possessing four growth-promoting functions of nitrogen fixation, phosphorus solubilization, IAA production, and siderophore production, as well as its application technology in a standardized hydroponic system.

[0009] The technical solution of the present invention is: Kosakonia sacchari LY-1, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20242320.

[0010] Microbial agents containing Kosakonia sacchari LY-1 as described above, such as live cells, fermentation broth, or lyophilized powder thereof.

[0011] The application of Kosakonia sacchari LY-1 or microbial agents mentioned above in promoting rice growth.

[0012] Furthermore, the application is to promote rice growth in nitrogen-free or low-nitrogen environments.

[0013] Kosakonia sacchari LY-1 has the following microbiological characteristics:

[0014] Taxonomic characteristics: Based on 16S rDNA, rpoB gene and physiological and biochemical identification, it has 99% similarity to the Kosakonia sacchari type strain;

[0015] Genome characteristics: It carries the complete nifHDKY-ENX-BQ-VZW nitrogen fixation gene cluster (23.5 kb), which is highly homologous to the classic nitrogen fixation gene cluster of Klebsiella oxytoca (similarity >85%), and contains nifA (positive regulator) and nifL (negative regulator).

[0016] Nitrogen fixation characteristics: The nitrogenase content is 160 ng / mL, and it can grow autonomously in nitrogen-free Ashby medium;

[0017] Growth-promoting properties: It simultaneously possesses the ability to dissolve inorganic phosphorus (57 mg / L), produce indoleacetic acid (19.76 mg / L), and produce siderophores (48% active units);

[0018] Physiological characteristics: Optimal growth temperature 37℃, optimal pH 7.0, NaCl tolerance range 0%–5.0% (optimal 2.0%).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The strain LY-1 of this invention is a rice endophytic nitrogen-fixing bacterium belonging to the Sacchariidae family, carrying the complete nitrogen-fixing gene cluster nifHDKY-ENX-BQ-VZW. It contains 160 ng / mL of nitrogenase and can provide nitrogen nutrition to rice through biological nitrogen fixation under nitrogen-free hydroponic conditions. Simultaneously, this strain possesses the ability to solubilize phosphorus (57 mg / L), produce IAA (19.76 mg / L), and produce siderophores (48%). It can synergistically promote rice growth from multiple dimensions, including nitrogen supply, hormone regulation, and phosphorus and iron nutrition. After 14 days of hydroponic cultivation, the stem length increased by 25.7% and the biomass increased by 45.6% compared to the control, significantly outperforming the growth-promoting effects of existing single-function strains. This provides a strain resource with a clear genetic background, well-defined function, and verifiable effects for rice hydroponic seedling cultivation and bio-fertilizer development.

[0021] Preservation Information

[0022] Kosakonia sacchari LY-1 was deposited at the China Center for Type Culture Collection on October 24, 2024, with accession number CCTCC NO: M 20242320, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0023] Figure 1 Colony morphology of strain LY-1 (A: LB plate, 37℃, 20 hours; B: 48 hours).

[0024] Figure 2 Phylogenetic tree of strain LY-1 based on 16S rDNA sequence.

[0025] Figure 3 PCR amplification results of nitrogenase core gene (M: DL2000 Marker; 1: nifK 1560 bp; 2: nifD 380 bp; 3: nifH 360 bp).

[0026] Figure 4 Growth validation of strain LY-1 on nitrogen-free Ashby medium (left: LY-1; right: negative control).

[0027] Figure 5 Effects of environmental factors on the growth of strain LY-1 (A: temperature; B: NaCl; C: pH).

[0028] Figure 6 Structure of nitrogen-fixing gene clusters in strain LY-1 and comparison with Klebsiella acidogenic bacteria.

[0029] Figure 7 ELISA results of nitrogenase content determination in strain LY-1 (standard curve and sample determination).

[0030] Figure 8 Growth-promoting effect of strain LY-1 on hydroponic rice (A: phenotype; B: stem length; C: root length).

[0031] Figure 9 Verification of endophytic colonization of strain LY-1 in rice (fluorescence micrograph). Detailed Implementation

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0033] The culture medium components involved in the examples are as follows:

[0034] (1) Preparation of NB medium: 3 g beef extract, 10 g peptone, 10 g NaCl, adjust pH to 7.2, add water to 1L.

[0035] (2) Preparation of NA medium: Add 17g of agar to NB medium to make NA medium.

[0036] (3) Preparation of LB medium: 10g tryptone, 5g yeast extract, 10g NaCl, adjust pH to 7.2, add water to 1L.

[0037] (4) Preparation of inorganic phosphorus solid culture medium: 1 L of culture medium contains 0.5 g yeast extract, 10 g glucose, 5 g Ca3PO4, 0.5 g (NH4)2SO4, 0.3 g KCl, 0.3 g NaCl, 0.03 g MgSO4·7H2O, 0.03 g MnSO4, 0.003 g FeSO4·7H2O, and 18 g agar. Adjust the pH to 7.0-7.5.

[0038] (5) Preparation of CAS solid culture medium: 0.0605 g of Chromium Azurite S (CAS), 0.0729 g of hexadecyltrimethylammonium bromide (HDTMA), 0.002645 g of FeCl·6H2O, 9 g of agar, 50 ml of 0.1 mol / L phosphate buffer (0.29525 g of NaH2PO4·2H2O, 1.213.5 g of Na2HPO4·12H2O, 0.125 g of NH4Cl, 0.0375 g of KH2PO4, 0.0625 g of NaCl, pH 6.6-7.0)

[0039] (6) Preparation of Ashby nitrogen-free medium: mannitol 10g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O 0.1g, CaCO3 5.0g, agar 15g, pH 7.2~7.4, add water to 1L. Autoclave at 121℃ for 20 min, then store at 4℃.

[0040] (7) VF iron-deficient medium: sucrose 20.0 g, (NH4)2SO4 3.5 g, L-aspartic acid 1.5 g, L-methionine 0.02 g, L-histidine 0.01 g, KH2PO4 1.0 g, MgSO4 0.5 g, NaCl 0.5 g

[0041] (8) JNFb medium: L-malic acid 5 g, K2HPO4 0.6 g, KH2PO4 1.8 g, MgSO4·7H2O 0.2 g, NaCl 10.1 g, CaCl2·2H2O 0.02 g, trace element solution 2.0 mL, (Na2MoO4·2H2O 0.2 g, MnSO4·H2O 0.235 g, H3BO3 0.28 g, CuSO4·5H2O 0.008 g, ZnSO4·7H2O 0.024 g, water 1000 mL), bromothymol blue 2.0 mL (0.5% aqueous solution dissolved in 0.2 mol / L KOH), Fe-EDTA 4.0 mL (1.64% aqueous solution), vitamin solution 1.0 mL. (Biotin 0.01 g, VB 0.02 g, water 1000 mL), KOH 4.5 g, adjust pH to 5.8 with KOH, add water to 1 L. JNFb solid medium is made by adding 1.5% agar to JNFb medium, autoclaving at 121℃ for 20 min, and storing at 4℃.

[0042] Example 1: Isolation and screening of LY-1, an endophytic nitrogen-fixing bacterium in rice.

[0043] (1) Acquisition and initial screening of endophytic bacteria in rice

[0044] Healthy rice seedlings (variety: Jinxiangyu 1) growing normally in the field were selected, and their roots, stems, and leaves were collected as isolation materials. The surface disinfection procedure was as follows: first, the sample surface was rinsed with sterile water until clean; then, it was disinfected with 0.1% mercuric chloride solution (2 min for leaves, 3 min for roots and stems); subsequently, it was rinsed 4 times with sterile water; the last wash solution was spread onto NA plates and incubated overnight at 37°C for sterility testing. If no colonies grew, the surface disinfection was thorough, and subsequent operations could proceed; if colonies grew, the disinfection steps had to be repeated.

[0045] After removing the withered and yellowed tissues, grind the roots, stems, and leaves separately into a homogenate in a sterile mortar, and dilute with sterile water at a ratio of 10:1. 4 Take 100 μL of the culture medium and spread it onto NA plates. Incubate at 37°C upside down for 3–7 days. Pick single colonies with different morphologies and inoculate them into 50 mL of NB medium. Incubate at 37°C and 220 r / min for 24 h with shaking to prepare seed culture.

[0046] (2) Rescreening and strain preservation

[0047] Seed culture was inoculated at a ratio of 1:100 (v / v) into the following selection medium and cultured at 37°C and 220 r / min for preliminary evaluation of growth-promoting characteristics:

[0048] Ashby Nitrogen-Free Medium: Nitrogen Fixation Capacity

[0049] Inorganic phosphorus solid culture medium: phosphorus solubility

[0050] CAS solid culture medium: siderophore production capacity

[0051] King's B medium containing L-tryptophan: IAA production capacity

[0052] A strain exhibiting multiple growth-promoting characteristics was selected for streak purification. After morphological observation and preliminary identification using 16S rDNA, the target strain was designated LY-1. The purified LY-1 bacterial culture was mixed with sterile glycerol (final glycerol concentration 30%, v / v) and stored at –80℃.

[0053] Example 2: Identification of Kosakonia sacchari LY-1, an endophytic nitrogen-fixing bacterium in rice.

[0054] (1) Colony morphology characteristics

[0055] LY-1 was streaked onto LB agar plates and incubated at 37°C. After 20 h of incubation, the colonies were approximately 1–2 mm in diameter, regularly round, slightly convex, with smooth and regular edges, a moist and glossy surface, and a milky white to translucent color (see...). Figure 1 (A). After incubation extended to 48 h, the colony diameter increased to 3-4 mm, the color changed to opaque pale yellow, and the texture became viscous and easily picked up. Figure 1 (B)

[0056] (2) Physiological and biochemical identification of bacteria

[0057] Micro-biochemical reactions and enzyme activities of LY-1 were performed using API 20NE and API ZYM reagent strips (bioMérieux, France). The results are shown in Table 1. Its biochemical characteristics showed approximately 95% similarity to the reported Kosakonia sacchari type strain.

[0058] Table 1. API biochemical identification results of strain LY-1

[0059] characteristic LY-1 potassium nitrate + L-tryptophan - Dextrorotatory + L-arginine + urea - Ferric aescin citrate + Gel (Bovine Source) - 4-Nitrophenyl-β-D-galactopyranoside + Dextrorotatory - L-arabinose + Dextrorotatory mannan + Dextrorotatory mannitol - N-acetylglucosamine + D-maltose + Potassium gluconate + caprylic acid - adipic acid - malic acid + Sodium citrate + Phenylacetic acid -

[0060] (3) Antibiotic susceptibility testing

[0061] Paper disc diffusion method: 12-hour culture of LY-1 (OD) 600≈0.8 μg of the sample was evenly spread on a nanoplatelet (NA plate), and filter paper discs containing different antibiotics (spectinomycin, neomycin sulfate, chloramphenicol, streptomycin, kanamycin, tetracycline, gentamicin, and carbenicillin, all 10 μg / disc) were attached. After incubation at 37°C for 12 h, the diameter of the inhibition zone was measured. The results showed that LY-1 was resistant to carbenicillin (inhibition zone <6 mm) and sensitive to the other 7 antibiotics.

[0062] Liquid culture method: LY-1 was inoculated at a ratio of 1:100 into NB medium containing carbenicillin (0, 10, 25, 50 μg / mL), and cultured at 37℃ and 220 r / min for 12 h to determine OD. 600 The results showed that LY-1 could tolerate up to 50 μg / mL of carbenicillin.

[0063] (4) Molecular biological identification

[0064] Genomic DNA extraction: Genomic DNA of LY-1 was extracted using a bacterial genomic DNA extraction kit (TIANGEN) and stored at -20℃ for later use.

[0065] 16S rDNA gene amplification and sequencing:

[0066] The primers were: F27: 5'-AGAGTTTGATCATGGCTCAG-3' and R1492: 5'-ACGGTTACCTTGTTACGACTT-3';

[0067] PCR system (50 μL): Template DNA 2 μL, forward and reverse primers (10 μmol / L) 2 μL each, 2×Taq PCRMaster Mix 25 μL, ddH2O 19 μL

[0068] Reaction program: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, 34 cycles; 72℃ final extension for 5 min.

[0069] Product detection: 0.8% agarose gel electrophoresis, target fragment approximately 1.5 kb.

[0070] Sequencing: After purification, the PCR product was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing, and a 1330 bp sequence was obtained.

[0071] rpoB gene amplification and sequencing:

[0072] Primers: FCM81 (5'-CAGTTCCGCGTTGGCCTG-3') and RCM32b (5'-CGGACCGGCCTGACGTTGCAT-3')

[0073] The reaction conditions were the same as for 16S rDNA: annealing temperature 58℃, extension time 60 s.

[0074] A 598 bp sequence was obtained.

[0075] Sequence alignment and phylogenetic analysis: The 16S rDNA and rpoB sequences were BLAST-aligned in the NCBI database, showing 99% similarity to the Kosakonia sacchari type strain. A Neighbor-Joining phylogenetic tree was constructed using MEGA 11 software (see [link to MEGA 11]). Figure 2 ), confirming that LY-1 and K. sacchari clustered in the same branch.

[0076] Comprehensive identification conclusion: Based on the analysis of colony morphology, physiological and biochemical characteristics and 16S rDNA / rpoB gene sequence, LY-1 was identified as a species of Kosakonia sacchari.

[0077] Example 3: Verification of the nitrogen-fixing capacity of endophytic nitrogen-fixing bacterium Kosakonia sacchari LY-1

[0078] (1) Growth verification in nitrogen-free medium

[0079] LY-1 was inoculated onto nitrogen-free Ashby solid medium and cultured at 37°C for 48 hours, after which regular colonies formed; the negative control strain Cupriavidus metallidurans CML2 showed no growth (see...). Figure 4 This indicates that LY-1 can maintain its growth by utilizing autonomous nitrogen fixation under conditions without exogenous nitrogen.

[0080] (2) Molecular detection of nitrogenase gene

[0081] Gene-specific primers for nifH / nifD / nifK were designed, and PCR amplification was performed using LY-1 genomic DNA as a template. Agarose gel electrophoresis showed that all three genes appeared as clear single bands at the expected locations (nifH 360 bp, nifD 380 bp, nifK 1560 bp, see...). Figure 3 Sequencing results showed a 99% similarity to the nitrogenase gene of Kosakonia sacchari, confirming that LY-1 carries the complete nitrogenase core gene system.

[0082] (3) Quantitative determination of nitrogenase activity

[0083] The nitrogenase content of LY-1 bacterial culture was determined to be 160 ng / mL using a microbial nitrogenase ELISA kit. Figure 7 OD 450Substituting into the standard curve for calculation), it was significantly higher than the negative control (< detection limit).

[0084] Example 4: Genomic analysis and identification of nitrogen-fixing gene clusters of endophytic nitrogen-fixing bacterium Kosakonia sacchari LY-1

[0085] This embodiment verifies the nitrogen fixation genetic basis of strain LY-1 at the genomic level through whole-genome sequencing and bioinformatics analysis.

[0086] (1) Genome sequencing and assembly

[0087] Genomic DNA was extracted from LY-1 and whole-genome sequencing was performed using the Illumina NovaSeq platform. After quality control, the raw data was assembled using SPAdes v3.15.0. The final genome size was approximately 5.2 Mb, with a GC content of 56.8%, a contig N50 of 185 kb, and a CheckM integrity score of 99.2%, indicating good assembly quality.

[0088] (2) Gene function annotation

[0089] The predicted gene sequences were submitted to the Swiss-Prot database for functional annotation. The results showed that a complete nitrogen fixation gene cluster (nif cluster) was successfully located in the LY-1 genome, containing 13 nitrogen fixation-related genes, including nifH, nifD, nifK, nifE, nifN, nifX, nifB, nifQ, nifV, nifW, nifZ, nifA, and nifL (see [link to relevant documentation]). Figure 6 ).

[0090] (3) Nitrogen fixation gene cluster structure analysis

[0091] This nitrogen-fixing gene cluster is approximately 23.5 kb in length and contains two operons:

[0092] nifHDKY operon: Encodes core components of the nitrogenase complex (ferritin, molybdenum-ferritin α / β subunits).

[0093] The nifENX-BQ-VZW operon encodes accessory proteins and electron transport components required for nitrogenase maturation.

[0094] The gene sequence is: nifH-nifD-nifY-nifK-nifE-nifN-nifX-nifB-nifQ-nifV-nifW-nifZ, which is highly homologous (>85%) to the classic nitrogen-fixing gene cluster of the nitrogen-fixing model bacterium Klebsiella oxytoca. Furthermore, nifA (positive regulator) and nifL (negative regulator) are intact, indicating that this gene cluster has the potential to be regulated by oxygen concentration and nitrogen source level.

[0095] The integrity of the nitrogen-fixing gene cluster in the LY-1 genome, the structure of its operon, and its high homology with classical nitrogen-fixing bacteria fully demonstrate the molecular basis of its nitrogen-fixing ability from a genetic perspective, providing a stable genetic guarantee for its application as a nitrogen-fixing agent.

[0096] Example 5: Growth-promoting ability of endophytic nitrogen-fixing bacterium Kosakonia sacchari LY-1

[0097] This embodiment quantitatively detects the phosphorus-solubilizing, IAA-producing, and siderophore-producing abilities of strain LY-1.

[0098] (1) Determination of phosphorus solubility

[0099] Methods: The molybdenum-antimony colorimetric method was used. LY-1 was inoculated at a ratio of 1:100 into inorganic phosphorus liquid medium (containing 25 g / L Ca3(PO4)2) and cultured at 37℃ and 220 r / min for 7 days. The fermentation broth was centrifuged at 12,000 r / min for 10 min, and the supernatant was collected. Molybdenum-antimony colorimetric reagent was added, and the mixture was incubated at room temperature for 30 min. The OD was then measured. 700 A standard curve was plotted using KH₂PO₄ standard solution (0–100 mg / L), and the soluble phosphorus content was calculated.

[0100] Results: The phosphorus solubility of LY-1 was 57.0 ± 0.13 mg / L (see Table 2), indicating that this strain can effectively activate insoluble inorganic phosphorus.

[0101] (2) Determination of indoleacetic acid (IAA) production capacity

[0102] Methods: The Salkowski colorimetric method was used. LY-1 was inoculated into King's B medium containing L-tryptophan (500 mg / L) and cultured at 37℃ and 220 r / min for 3 days. The supernatant was collected by centrifugation of the fermentation broth, and Salkowski colorimetric reagent (1 mL) was added. The reaction was carried out at room temperature in the dark for 30 min, and the OD was measured. 530 Plot a standard curve using IAA standards (0–100 mg / L) and calculate IAA yield.

[0103] Results: The yield of IAA by LY-1 was 19.76 ± 0.08 mg / L (see Table 2), which significantly promoted the elongation of plant roots.

[0104] (3) Measurement of ferrocarrier generation capacity

[0105] Methods: A combination of the Chrome Azurol S (CAS) plate method and liquid quantification method was used. LY-1 was inoculated into VF iron-deficient liquid medium and cultured at 30℃ and 220 r / min for 3 days. The supernatant of the fermentation broth was collected by centrifugation and mixed with an equal volume of CAS detection solution. The mixture was reacted at room temperature for 1 h, and the OD was measured. 630 Formula for calculating siderophore activity units (SU%):

[0106] SU( % ) = (Ar - As) / Ar × 100%

[0107] Ar represents the absorbance at 630 nm of the supernatant of each strain after mixing with the CAS detection solution; As represents the absorbance at 630 nm of the mixture of uninoculated VF medium and CAS detection solution.

[0108] Results: The siderophore activity of LY-1 was 48.04% (Table 2), indicating that the strain has a strong ability to chelate and supply iron ions.

[0109] Table 2 Results of growth-promoting function assay of strain LY-1

[0110]

[0111] Note: Values ​​are mean ± standard deviation (n=3)

[0112] Conclusion: Strain LY-1 possesses triple growth-promoting functions of phosphorus solubilization, IAA production, and siderophore production. Combined with the nitrogen fixation ability verified in Example 3, this strain exhibits a synergistic effect of quadruple growth-promoting functions.

[0113] Example 6: Optimization of growth conditions for endophytic nitrogen-fixing bacterium Kosakonia sacchari LY-1

[0114] To determine the optimal culture conditions and environmental tolerance range of strain LY-1, its growth temperature, NaCl tolerance concentration, and pH range were systematically optimized.

[0115] (1) Optimal growth temperature

[0116] LY-1 seed culture (12 h, OD) 600≈0.8) was inoculated at a ratio of 1:100 (v / v) into 5 mL of NB medium and incubated at 22℃, 30℃, 37℃ and 42℃ with shaking at 220 r / min for 12 h, respectively. OD was then measured. 600 Values, with 3 replicates for each temperature.

[0117] The results are as follows Figure 5 As shown in Figure A: LY-1 can grow in the temperature range of 22–42℃, and its OD value is [missing value] at 37℃. 600 The value was the highest (1.35 ± 0.08), significantly higher than other temperatures (P < 0.05), indicating that the optimal growth temperature was 37℃.

[0118] (2) NaCl tolerance concentration

[0119] The seed culture was inoculated at a ratio of 1:100 into NB medium containing different NaCl concentrations (0, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, w / v), and cultured at 37℃ and 220 r / min for 12 h. The OD was then measured. 600 .

[0120] The results are as follows Figure 5 As shown in Figure B: LY-1 can grow in the range of 0–5.0% NaCl, with optimal growth at 2.0% NaCl (OD). 600 =1.12 ± 0.06); when NaCl ≥ 4.0%, growth was significantly inhibited (OD). 600 <0.8). The tolerance range for NaCl was determined to be 0–5.0%, with an optimal concentration of 2.0%.

[0121] (3) Optimal pH

[0122] The seed culture was inoculated at a ratio of 1:100 into NB medium (adjusted with 1 mol / L HCl or NaOH) at pH levels of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, respectively, and cultured at 37℃ and 220 r / min for 12 h. OD was then measured. 600 .

[0123] The results are as follows Figure 5 As shown in Figure C: LY-1 grows well in the pH range of 5.0–10.0, with an OD of [value missing] at pH 7.0. 600 The highest value was (1.42 ± 0.10); growth was significantly inhibited at pH < 5.0 or pH > 10.0 (OD). 600 <0.1). The suitable pH range for growth was determined to be 5.0–10.0, with the optimum pH being 7.0.

[0124] Example 7: Application of endophytic nitrogen-fixing bacterium Kosakonia sacchari LY-1 in promoting growth in hydroponic rice

[0125] This embodiment verifies the growth-promoting effect of strain LY-1 on rice seedlings using a hydroponic system.

[0126] Rice seedlings (variety: Jinxiangyu 1) with uniform germination and growth were selected as experimental materials. The nutrient solution used was a modified Kimura B formula, with two nitrogen levels: a nitrogen-free treatment group (complete removal of NH4NO3) and a low-nitrogen treatment group (containing 1 / 4 standard nitrogen concentration, 0.71 mmol / L NH4NO3). The inoculum was LY-1 bacterial suspension (4.1 × 10⁻⁶). 8 cfu / mL, OD 600 ≈1.0), the control group was set as blank control (equal volume of sterile water) and negative control (non-nitrogen-fixing bacteria Cupriavidus metallidurans CML2, 4.1×10). 8 (cfu / mL).

[0127] Rice seedlings were transferred to black 96-well hydroponic boxes (200 mL nutrient solution per well). The nutrient solution was changed and 15 mL of bacterial culture was inoculated every 3 days. The culture conditions were: constant temperature of 28℃, photoperiod of 14 h / 10 h (light / dark), light intensity of 200 μmol / m² / s, and relative humidity of 70%. Each treatment was replicated in triplicate, with 20 seedlings per replicate. After 14 days of culture, stem length, root length, aboveground fresh weight, and dry weight (dried at 105℃ to constant weight) were measured.

[0128] The results are shown in Table 3 and Figure 8 As shown in the table. In the nitrogen-free treatment groups, the stem length of the LY-1 treatment group was 15.14 ± 0.85 cm, significantly higher than that of the blank control (12.05 ± 0.72 cm) and the CML2 control (12.18 ± 0.68 cm), an increase of 25.7% compared to the blank control (P<0.01); the root length was 19.25 ± 0.90 cm, an increase of 6.8% compared to the blank control. In the low-nitrogen treatment groups, the stem length and root length of the LY-1 treatment group increased by 18.5% and 5.2% respectively compared to the blank control. Fresh weight and dry weight indices increased by 45.6% and 38.9% respectively under nitrogen-free conditions (Table 3). Figure 8 The results from the A-value show that the rice seedlings in the LY-1 treatment group have well-developed root systems and robust stems and leaves; Figure 8 Box plots in groups B and C showed that the stem and root length data of group LY-1 were significantly higher than those of the control group (P<0.05, t test).

[0129] The above results indicate that under nitrogen-free and low-nitrogen hydroponic conditions, LY-1 can significantly promote the vegetative growth of rice seedlings through the synergistic effects of biological nitrogen fixation, phosphorus solubilization, IAA production, and iron carrier production. The growth-promoting effect is particularly prominent under nitrogen-free conditions, providing a functional verification basis for subsequent field applications.

[0130] Table 4 Effects of different treatments on the growth of hydroponic rice seedlings (n=20, x±SD)

[0131]

[0132] Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's method).

Claims

1. Kosakonia sacchari LY-1, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20242320.

2. A microbial inoculant containing Kosakonia sacchari LY-1 as described in claim 1.

3. The application of Kosakonia sacchari LY-1 as described in claim 1 or the microbial agent as described in claim 2 in promoting rice growth.

4. The application according to claim 3, characterized in that, The application is to promote rice growth in nitrogen-free or low-nitrogen environments.

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