Phosphorus-dissolving nitrogen-fixing recarburization functional bacterium and application thereof in fertile soil growth promotion

Abstract

The invention provides a phosphorus-solubilizing nitrogen-fixing recarburization functional bacterium and application thereof in fertile soil growth promotion, and belongs to the technical field of agricultural microorganisms. The rahnella maple ZC-7 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20252883, the preservation time is December 15, 2025, and the preservation address is Wuhan University. The strain has the effects of dissolving phosphorus, fixing nitrogen, producing IAA and the like. In addition, the strain is wide in carbon source utilization spectrum, has resistance to various chemical substances and can adapt to various soil environments. The strain can obviously promote the growth of corn plants and increase the corn yield, and field tests show that the use of chemical fertilizers can be reduced and the corn yield can be increased by 27% by applying a microbial agent containing the strain. The strain has comprehensive functions and strong adaptability, provides technical support for green agriculture development, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a phosphorus-solubilizing, nitrogen-fixing, and carbon-enhancing functional bacterium and its application in promoting growth in fertile soil. Background Technology

[0002] Nitrogen and phosphorus, as essential mineral elements for plants, are crucial components of various structural and functional substances within plant cells, including proteins, nucleic acids, phospholipids, enzymes, and plant hormones. They play vital roles in plant energy metabolism, organic matter synthesis and metabolism, substance transport, gene expression and regulation, and cell signal transduction. Nitrogen and phosphorus deficiencies can inhibit plant growth and development, causing deficiency symptoms and even preventing the completion of the plant's life cycle. The application of chemical fertilizers not only increases agricultural production costs but also leads to the selective accumulation of certain elements in the soil and the deterioration of soil physicochemical properties, causing environmental pollution. Developing and utilizing microbial agents with multiple growth-promoting functions is an effective strategy for improving soil and promoting crop growth.

[0003] Microorganisms play a crucial role in soil nutrient cycling. Existing technologies already possess many microbial resources with single functions. For example, phosphorus-solubilizing microorganisms can convert insoluble phosphorus in the soil into its available form; nitrogen-fixing microorganisms can fix atmospheric nitrogen into a usable nitrogen source for plants. Furthermore, some strains also possess the ability to secrete plant growth hormones or antagonize pathogens. However, improving soil fertility is a systematic project requiring the synergistic effect of multiple nutrient elements, such as nitrogen fixation, phosphorus solubilization, and carbon enrichment. Nevertheless, existing commercially available microbial agents generally suffer from poor environmental adaptability and limited functionality, and their actual application effectiveness is constrained by various external factors, including soil type and climate conditions. Summary of the Invention

[0004] In view of this, the present invention provides a phosphorus-solubilizing, nitrogen-fixing, and carbon-enhancing functional bacterium and its application in promoting growth in fertile soil. This strain has the effects of phosphorus solubilizing, nitrogen fixing, carbon enhancement, and promoting growth in fertile soil. Moreover, this strain has strong environmental adaptability, providing technical support for the development of green agriculture and has important application value.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a strain of Rahnella sp. ZC-7, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252883, deposited on December 15, 2025, at Wuhan University.

[0007] The present invention also provides a microbial inoculant, wherein the active ingredient of the inoculant is the aforementioned *Laenia maculata* ZC-7.

[0008] Preferably, the microbial agent is a liquid agent, and the number of effective viable bacteria in the liquid agent is 1~9×10⁻⁶. 9 CFU / mL.

[0009] The present invention also provides a method for preparing the microbial agent, wherein the *Laenia natans* ZC-7 is inoculated into a fermentation medium and fermented to obtain the microbial agent.

[0010] Preferably, the fermentation culture temperature is 28~32℃ and the rotation speed is 180~220 rpm.

[0011] The present invention also provides the application of the aforementioned Raenella asiatica ZC-7 in any one or more of the following:

[0012] (1) Application in phosphorus solubilization;

[0013] (2) Application in the production of nitrogenase;

[0014] (3) Application in nitrogen fixation;

[0015] (4) Application in the production of indoleacetic acid;

[0016] (5) Application in promoting plant growth and / or increasing crop yield;

[0017] (6) Application in soil fertilization;

[0018] (7) Application in the preparation of phosphate-solubilizing bacteria agents;

[0019] (8) Application in the preparation of microbial agents for nitrogenase production;

[0020] (9) Application in the preparation of nitrogen-fixing bacterial agents;

[0021] (10) Application in the preparation of microbial agents for the production of indoleacetic acid;

[0022] (11) Application in the preparation of microbial agents that promote plant growth and / or increase crop yield;

[0023] (12) Application in the preparation of microbial agents with soil fertilization effect.

[0024] Preferably, the soil enrichment effect is to increase the content of available phosphorus, ammonium / nitrate nitrogen, dissolved organic carbon / nitrogen and total organic carbon in the soil.

[0025] The present invention also provides a method for promoting plant growth by applying the aforementioned *Laenia natans* ZC-7 or the aforementioned microbial agent to the soil in the rhizosphere of the plant.

[0026] Preferably, after applying the microbial inoculant, the effective viable count of *Laenia natans* ZC-7 in the rhizosphere soil of the plant is 1 × 10⁻⁶. 6 CFU / g soil.

[0027] Preferably, the plant includes corn.

[0028] By adopting the above technical solution, the present invention has the following beneficial effects: The Rahnella sp. ZC-7 strain of the present invention is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20252883, deposited on December 15, 2025, at Wuhan University. This strain was isolated from the rhizosphere soil of maize and has the functions of phosphorus solubilization, nitrogen fixation, and IAA production. Furthermore, this strain has a broad carbon source utilization spectrum, capable of utilizing 49 different carbon sources, and also exhibits broad resistance to various chemical substances, enabling it to adapt to diverse soil environments. This strain can significantly promote maize plant growth and increase maize yield. Field trials have shown that applying microbial preparations containing this strain can increase maize yield by 27%. This strain is comprehensive in function and highly adaptable, providing technical support for the development of green agriculture and possessing significant application value. Attached Figure Description

[0029] Figure 1 The image shows the phosphate-solubilizing zones of *Laenia natans* ZC-7 on phosphate-solubilizing plates with different phosphorus sources.

[0030] Figure 2 The figure shows the results of the IAA secretion performance measurement of the Laenella mapleensis ZC-7 strain.

[0031] Figure 3 The growth curves of *Laenia natans* strain ZC-7 under different pH (A) and temperature (B) conditions are shown.

[0032] Figure 4 Figure 1 shows the results of soil physicochemical index determination for different treatments; A represents the content of ammonium nitrogen and nitrate nitrogen in soils under different treatments; B represents the content of available phosphorus in soils under different treatments; C represents the content of dissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soils under different treatments; D represents the content of total organic carbon (TOC) in soils under different treatments.

[0033] Figure 5 A graph showing maize yields under different treatments.

[0034] Figure 6 Figures showing maize plant height under different treatments.

[0035] Figure 7 Image showing the weight of maize ears under different treatments.

[0036] Figure 8The growth status of maize ears under the treatments of 100N (A), 80N (B), and 80N+ZC-7 (C) is shown in the figure.

[0037] Biological Preservation Instructions

[0038] The taxonomic name of the Rahnella sp. ZC-7 strain of this invention is Rahnella sp., which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252883, deposited on December 15, 2025, at Wuhan University. Detailed Implementation

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Monkina Inorganic Phosphorus Medium (Calcium Phosphate): Glucose 10.00 g / L, Yeast Extract 0.50 g / L, Ammonium Sulfate 0.50 g / L, Sodium Chloride 0.30 g / L, Potassium Chloride 0.30 g / L, Magnesium Sulfate 0.30 g / L, Ferrous Sulfate 0.03 g / L, Manganese Sulfate 0.03 g / L, Calcium Phosphate 5.0 g / L, Agar 15 g / L.

[0041] Monkina Organic Phosphorus Medium (Lecithin): Glucose 10.00 g / L, Yeast Extract 0.50 g / L, Ammonium Sulfate 0.50 g / L, Sodium Chloride 0.30 g / L, Potassium Chloride 0.30 g / L, Magnesium Sulfate 0.30 g / L, Ferrous Sulfate 0.03 g / L, Manganese Sulfate 0.03 g / L, Calcium Carbonate 1.00 g / L, Lecithin 0.20 g / L, Agar 15 g / L.

[0042] Organic phosphorus medium (calcium phytate): glucose 10.00 g / L, calcium phytate 5.00 g / L, magnesium chloride 5.00 g / L, magnesium sulfate 0.25 g / L, potassium chloride 0.20 g / L, ammonium sulfate 0.10 g / L, agar 15 g / L.

[0043] Example 1. Screening and isolation of ZC-7 strain

[0044] Take 5 g of black soil from the top 20 cm of a cornfield and place it in a centrifuge tube. Add 45 mL of sterile water to prepare 10... -1 The bacterial suspension was placed in a shaker at 180 rpm and shaken for 10 min, then removed and allowed to stand for 1 h.

[0045] Take 1 mL of the settled soil suspension and add 9 mL of sterile water to prepare 10... −2 The bacterial suspension was diluted sequentially to 10...−3 10 −4 10 −5 10 -6 10 -7 Take 100 μL of each of the following 10 −5 10 -6 10 -7 Diluted bacterial suspensions were spread on Mongkina inorganic and organic phosphorus culture media. After colony growth, single colonies with phosphate-solubilizing zones were selected for species identification.

[0046] Genomic DNA was extracted from the selected single bacteria and amplified by PCR using the universal primers 27F / 1492R for bacterial 16S rRNA. The amplified sequence is shown in SEQ ID NO.1.

[0047] The 16S rRNA sequence of strain ZC-7:

[0048]

[0049] After comparing the effective sequences obtained from the sequencing with known sequences in the NCBI database, the results showed that strain ZC-7 had 100% similarity to Rahnella aceris, and the strain was named Rahnella aceris ZC-7.

[0050] Example 2. Determination of phosphorus solubilization performance of Raenella asiatica ZC-7

[0051] (1) Molecular biological assay

[0052] To verify the phosphorus-solubilizing potential of *Laenia natans* ZC-7, the genomic DNA sequence of *Laenia natans* ZC-7 was sequenced in NCBI. The results showed that *Laenia natans* ZC-7 contains phosphorus-solubilizing genes phoH, phoR, phoB, phoA, phoU, pqqA, pqqB, pqqC, pqqD, pqqE, and pqqF, indicating that this strain has potential phosphorus-solubilizing ability.

[0053] (2) Determination of phosphorus solubility

[0054] Medium plates containing organic phosphorus (lecithin), inorganic phosphorus (calcium phosphate), and calcium phytate were prepared. Each plate was divided into four regions. 2 μL of the medium was aspirated and placed at the center of each region. The plates were incubated at 28°C for 3 days. The growth of the strain and the formation of phosphate-solubilizing zones were observed. The diameter of the phosphate-solubilizing zone (D / cm) and the colony diameter (d / cm) were recorded. The D / d ratio was calculated to determine the strain's phosphate-solubilizing ability and efficiency for different insoluble phosphorus compounds. The phosphate-solubilizing ability and efficiency of strain ZC-7 for insoluble phosphorus are shown in Table 1 and [Table data missing]. Figure 1 .

[0055] Table 1. Ratio of phosphorus-solubilizing zone to growth zone on plates with different phosphorus sources

[0056]

[0057] From Table 1 and Figure 1 It can be seen that *Laenia natans* ZC-7 has the ability to solubilize insoluble phosphorus, with the solubilizing ability being phytate > lecithin > calcium phosphate.

[0058] (3) Prepare inorganic phosphorus (calcium phosphate) and organic phosphorus (calcium phytate) culture medium for Monkina. After the strain is revived and rejuvenated, prepare seed culture and adjust to OD. 600 =1.0, and was inoculated into the fermentation medium at a ratio of 1%. Samples were taken on days 1, 3, 5 and 7, and centrifuged. The effective phosphorus content of the supernatant was determined by the molybdenum antimony colorimetric method to characterize the phosphorus solubility of the strain. The results are shown in Table 2.

[0059] Table 2. Phosphorus solubility of strain ZC-7 in liquid culture medium with calcium phosphate and calcium phytate as phosphorus sources.

[0060] Phosphate solubility (mg / L) Day 1 Day 3 Day 5 Day 7 Calcium phosphate 113.76 129.69 188.49 231.65 Calcium phytate 14.58 31.19 49.10 64.64

[0061] The results in Table 2 show that on day 7, the amount of calcium phosphate solubilized by strain ZC-7 was 231.65 mg / L and the amount of calcium phytate solubilized was 64.64 mg / L, indicating that the strain has the ability to solubilize phosphate, and its ability to solubilize calcium phosphate is higher than that of calcium phytate.

[0062] Example 3. Determination of nitrogen fixation and carbon enrichment performance of *Laenia natans* ZC-7

[0063] (1) Molecular biological assay

[0064] To verify the nitrogen fixation and carbon enrichment potential of *Laenia natans* ZC-7, the genomic DNA sequence of *Laenia natans* ZC-7 was compared with the whole genome in NCBI. The results showed that *Laenia natans* ZC-7 contains the nifH nitrogen fixation gene and the accA, accB, accC, and accD carbon enrichment genes, indicating that this strain has potential nitrogen fixation and carbon enrichment capabilities.

[0065] (2) Nitrogen fixation capacity determination

[0066] The nitrogenase activity of this strain was determined using a microbial nitrogenase (NITS) ELISA kit. The nitrogenase activity of *Laenia natans* ZC-7 was 102.31 ng / mL, indicating that *Laenia natans* ZC-7 has nitrogen-fixing activity.

[0067] Example 4. Determination of IAA production capacity of Raenella asiatica ZC-7

[0068] The Salkowski colorimetric method was used to determine the IAA secretion performance of the strain. A suspension of strain ZC-7 was inoculated into 5 mL of L-tryptophan growth medium and incubated in the dark at 28 °C for 48 h. Then, 200 μL was transferred to a white porcelain plate, and 180 μL of Salkowski's reagent (0.5 mmol / L FeCl3, 35% HClO4) was immediately added. The plate was then incubated at 30 °C for 30 min. The results are shown below. Figure 2 As shown in the figure. The results indicate that *Raenella aceta* ZC-7 has the ability to produce IAA.

[0069] Example 5. Determination of optimal growth conditions for Raenella asiatica ZC-7

[0070] 1. Screening based on optimal pH and optimal temperature

[0071] Seed culture (OD) of strain ZC-7 600=1.0) was inoculated into the fermentation medium at a ratio of 1%, and cultured at different pH values ​​(pH=5, 6, 7, 8, 9) and different temperatures (15, 20, 25, 28, 30℃). The growth curves of the strain are shown below. Figure 3 As shown. Figure 3 The results showed that the optimal pH for the growth of Raenella acetabulae ZC-7 was 7, and the optimal temperature was 30℃.

[0072] 2. Carbon source utilization of the strain

[0073] The utilization of 71 different carbon sources and the sensitivity to 23 sensitive substances by strain ZC-7 were analyzed using the BIOLOG automated bacterial identification system. The results are shown in Tables 3 and 4.

[0074] Table 3 Carbon source utilization of strain ZC-7

[0075] carbon source ZC-7 carbon source ZC-7 beta-glucan w glycine-L-proline w D-maltose + L-alanine w D-trehalose + L-arginine - D-Cellobiose w L-Aspartic acid w Gentian disaccharide + L-glutamic acid w sucrose + L-histidine w D-Melalose w L-pyroglutamic acid - Stachyose w L-Serine w D-Melato + pectin w α-D-lactose + Galacturonic acid + D-Melfibre + L-galactonic acid + β-methyl-D-glucoside + D-gluconic acid + D-Salicin + Glucuronic acid + N-acetyl-D-glucosamine + glucuronid + N-acetyl-β-D-mannosamine + mucus acid w N-acetyl-D-galactosamine - Quinic acid w N-acetyl-mannosamine pyruvate - D-glucaric acid w α-D-glucose + p-Hydroxyphenylacetic acid - D-mannose + Pyruvate w D-fructose + D-methyl lactate - D-galactose + L-lactic acid w 3-Methylglucose - Citric acid w D-fucose - α-Ketoglutarate - L-fucose w D-hydroxysuccinic acid - L-Rhamnose w L-hydroxysuccinic acid + Inosine w Bromosuccinic acid - D-sorbitol + Twain 40 - D-Mannitol + γ-aminobutyric acid - D-Aliquotol - α-Hydroxybutyric acid - Inositol - β-hydroxy-D,L-butyric acid - Glycerol + α-Keto-butyric acid - <![CDATA[D-glucose-6-PO4]]> w Acetoacetic acid w <![CDATA[D-fructose-6-PO4]]> w propionic acid - D-Aspartic acid - acetic acid w D-Serine - Formic acid w gelatin -

[0076] Note: "+" indicates a positive test, "w" indicates a weak positive test, and "-" indicates a negative test.

[0077] Table 4. Sensitivity of ZC-7 strain to chemically sensitive substances

[0078] chemical substances ZC-7 chemical substances ZC-7 pH 6 + Guanidine hydrochloride w pH 5 + Sodium tetradecyl sulfate + 1% NaCl + Vancomycin + 4% NaCl w Tetrazolium violet + 8% NaCl w Tetrazolyl blue + 1% Sodium Lactate + Naphthylpyridinone acid + fusobionic acid w Lithium chloride w D-Serine - Potassium tellurite - Acid-derived basilin - Aztreonam w Rifamycin SV + Sodium butyrate w Dimethylaminetetracycline - Sodium bromate w Lincomycin w

[0079] Note: "+" indicates a positive result and the test is not sensitive to the substance; "w" indicates a weak positive result and the test is weakly sensitive to the substance; "-" indicates a negative result and the test is sensitive to the substance.

[0080] The results in Tables 3 and 4 show that the ZC-7 strain has a wide range of available carbon sources, including 49 different carbon sources, and exhibits broad resistance to chemical substances, enabling it to survive under various adverse conditions.

[0081] Example 6. Verification of the Fertile Soil Function of ZC-7 Strain

[0082] The pot experiment was conducted in the greenhouse of the Agricultural Experiment Station at Zhejiang University's Zijingang Campus. The test plant was maize, variety "Jinboshi 825". Three treatments were set up: 100N, 80N, and 80N+ZC-7, with three replicates for each treatment. Each pot was filled with 5 kg of air-dried, sieved black soil. After applying basal fertilizer, three seeds were sown in each pot. After emergence, seedlings were thinned, leaving one plant per pot that showed similar growth and good condition. Topdressing was applied at the jointing stage. Throughout the cultivation process, the soil moisture content in the pots was adjusted to 60% of field capacity, and watering was done by spraying. The basal and topdressing fertilizer settings for each treatment are as follows:

[0083] The 100N treatment is the normal fertilization treatment: 3 g of basal fertilizer per pot, and 0.5 g of urea per pot as top dressing during the jointing stage;

[0084] The 80N treatment was a 20% reduction in fertilizer application: 2.4 g / pot as basal fertilizer and 0.4 g / pot as topdressing urea at the jointing stage;

[0085] The 80N+ZC-7 treatment involves reducing fertilizer application by 20% and applying ZC-7 microbial agent: basal fertilizer application rate is 2.4 g / pot, topdressing with 0.4 g / pot of urea at the jointing stage, and additional application of ZC-7 microbial solution during the seedling and jointing stages, with the solution added at a rate of 10... 6 CFU / g soil was added, and equal amounts of sterile water were applied at 100N and 80N.

[0086] The preparation method of ZC-7 bacterial culture is as follows:

[0087] The strain was inoculated into LB agar for resuscitation and activation. After activation, it was inoculated into LB liquid medium at a 1% inoculum and cultured until OD. 600 =3.0, centrifuge at 10000 rpm for 10 min, discard the supernatant and resuspend in sterile water until the effective viable count is 5×10. 9 CFU / g.

[0088] In all the above treatments, the base fertilizer was a chemical compound fertilizer with an N:P:K ratio of 23:18:7; the top dressing was urea.

[0089] Soil samples were collected 50 days after planting, and soil physicochemical properties were measured. The results are as follows: Figure 4 As shown. Figure 4 The results showed that applying ZC-7 strain could increase the content of available phosphorus, ammonium / nitrate nitrogen, dissolved organic carbon / nitrogen, and total organic carbon in the soil.

[0090] Example 7. Verification of the growth-promoting function of strain ZC-7

[0091] A field experiment was conducted on maize at the Zijingang Campus of Zhejiang University, using the variety "Jinboshi 825". Sowing took place on April 18, 2025, and harvesting on July 16, 2025. Three treatments were established, similar to the potted plant treatment. Compound fertilizer (1000 kg / mu, N:P:K = 23:18:7) was applied during the seedling stage, and urea (150 kg / mu) was applied as topdressing during the jointing stage. Inoculum solution (10% concentration) was also applied during both the seedling and jointing stages. 6 (CFU / g soil addition), estimated yield as follows: Figure 5 . Figure 5 The results showed that under the condition of reducing fertilizer application by 20%, the yield of the treatment with ZC-7 strain (80N+ZC-7) was higher than that of the normal fertilization treatment, with an increase of 27%. Figure 6 , Figure 7 and Figure 8 The results showed that the corn plants treated with 80N+ZC-7 were taller, had larger ears, fuller ears, and higher shelling percentage.

[0092] As can be seen from the above embodiments, the present invention provides a phosphorus-solubilizing, nitrogen-fixing, and carbon-enhancing functional bacterium and its application in promoting growth in fertile soil. The *Laenia natans* ZC-7 of the present invention has the effects of phosphorus solubilizing, nitrogen fixing, carbon-enhancing, growth promotion, and fertile soil.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of *Lahn's bacterium* from the maple tree ( Rahnella sp.) ZC-7, characterized in that, It is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252883, on December 15, 2025, at Wuhan University.

2. A microbial inoculant, characterized in that, The active ingredient of the bacterial agent is *Laenia natans* ZC-7 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a liquid agent, and the effective viable bacteria count in the liquid agent is 1~9×10⁻⁶. 9 CFU / mL.

4. The method for preparing the microbial inoculant according to claim 3, characterized in that, The *Laenia maculata* ZC-7 was inoculated into a fermentation medium and fermented to obtain the microbial agent.

5. The preparation method according to claim 4, characterized in that, The fermentation culture temperature is 28~32℃, and the rotation speed is 180~220 rpm.

6. The use of the *Laenia natans* ZC-7 as described in claim 1 in any one or more of the following: (1) Application in phosphorus solubilization; (2) Application in the production of nitrogenase; (3) Application in nitrogen fixation; (4) Application in the production of indoleacetic acid; (5) Application in promoting plant growth and / or increasing crop yield; (6) Application in soil fertilization; (7) Application in the preparation of phosphate-solubilizing bacteria agents; (8) Application in the preparation of microbial agents for nitrogenase production; (9) Application in the preparation of nitrogen-fixing bacterial agents; (10) Application in the preparation of microbial agents for the production of indoleacetic acid; (11) Application in the preparation of microbial agents that promote plant growth and / or increase crop yield; (12) Application in the preparation of microbial agents with soil fertilization effect.

7. The application according to claim 6, characterized in that, The soil fertilization effect is to increase the content of available phosphorus, ammonium / nitrate nitrogen, dissolved organic carbon / nitrogen, and total organic carbon in the soil.

8. A method for promoting plant growth, characterized in that, Apply the *Laenia natans* ZC-7 of claim 1 or the microbial agent of claim 2 to the soil in the rhizosphere of the plant.

9. The method according to claim 8, characterized in that, After applying the microbial inoculant, the effective viable count of *Laenia natans* ZC-7 in the rhizosphere soil was 0.8–1.2 × 10⁻⁶. 6 CFU / g soil.

10. The method according to claim 8 or 9, characterized in that, The plant mentioned includes corn.

Images