Pantoea agglomerans g6 and application thereof

Abstract

This invention discloses a *Pantotheca acuminata* strain G6 with rhizosphere growth-promoting functions in crops and its applications, belonging to the field of microbial technology. This invention isolated a multifunctional growth-promoting strain from the rhizosphere soil of japonica rice in cold-region black soil and, after reorganization, obtained *Pantotheca acuminata* G6, whose preservation number is CGMCC No. 38430. The *Pantotheca acuminata* G6 provided by this invention has the functions of decomposing inorganic phosphorus, decomposing organic phosphorus, producing iron carriers, producing IAA, fixing nitrogen, and decomposing potassium. This invention also discloses the application of *Pantotheca acuminata* G6 in improving seed germination and seedling growth in crops. *Pantotheca acuminata* G6 has a strong growth-promoting ability, providing an excellent growth-promoting bacterial resource for crops such as japonica rice, corn, and soybeans in cold regions, and has promotional value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a clustered pantothecin G6 strain with rhizosphere growth-promoting function in crops and its application. Background Technology

[0002] Plant growth-promoting rhizobacteria (PGPR) are a class of beneficial soil bacteria that can improve soil quality, enhance soil fertility, strengthen plant stress resistance, and increase plant yield.

[0003] Wild-type rhizosphere growth-promoting bacteria isolated directly from the natural environment possess basic growth-promoting and biocontrol potential. However, due to the specificity of their living environment and their own biological characteristics, they have many natural shortcomings, making it difficult to meet the requirements of modern agricultural production for efficient, stable, and multifunctional PGPR inoculants. Therefore, strain improvement is a necessary prerequisite for achieving industrial application and efficient field performance. Genome tamping technology is a novel method for improving microbial strains. Through recursive recombination at the genome level, it can efficiently achieve directed evolution of the entire organism. This technology has advantages in increasing strain yield, enhancing strain tolerance, and improving strain substrate utilization.

[0004] Plant rhizosphere growth-promoting bacteria, as a green alternative to chemical fertilizers, have broad prospects in sustainable agriculture. Currently, rice growth-promoting bacteria agents are generally mature in application and have stable effects, but resources and products specifically adapted to japonica rice grown in cold black soil are indeed scarce and development is lagging behind. Summary of the Invention

[0005] Based on the above-mentioned technological needs, the purpose of this invention is to provide a *Pantotheca acuminata* strain G6 with rhizosphere growth-promoting function for crops and its applications. This invention provides a *Pantotheca acuminata* strain G6 with rhizosphere growth-promoting function for crops. The *Pantotheca acuminata* strain G6 possesses multiple functions, including inorganic phosphorus solubilization, organic phosphorus solubilization, iron carrier production, IAA synthesis, nitrogen fixation, and potassium solubilization. The *Pantotheca acuminata* strain G6 has a significant effect on promoting rhizosphere growth in cold-climate crops, promoting seed germination and seedling growth, and has promotional value.

[0006] To achieve the above objectives, the present invention is accomplished through the following: This invention provides a *Pantotheca cumulus* G6 strain with rhizosphere growth-promoting function in crops. *Pantotheca cumulus* G6 is deposited at the China General Microbiological Culture Collection Center and is classified as *Pantotheca cumulus*. Pantoea agglomerans Its accession number is CGMCC No.38430.

[0007] Furthermore, the colonies of the Pantotheca G6 clumps are pale yellow with neat edges and a moist surface; the bacteria are rod-shaped and are Gram-negative.

[0008] Furthermore, the aforementioned Pantotheca G6 has the functions of decomposing inorganic phosphorus, decomposing organic phosphorus, producing iron carriers, producing IAA, fixing nitrogen, and decomposing potassium.

[0009] This invention also provides the application of the aforementioned Pantotheca G6 in the preparation of rhizosphere growth promoters for crops.

[0010] Furthermore, the crop includes at least one of japonica rice, corn, and soybeans.

[0011] Furthermore, the applications include promoting seed germination, enhancing overall seed germination vigor, and improving seedling growth potential.

[0012] Furthermore, the application method includes: preparing a bacterial suspension by fermenting the aforementioned Pantotheca G6 agglomerates, wherein the OD of the bacterial suspension is... 600 The concentration is 0.4~0.6, and the viable count of the bacterial suspension is higher than 10. 8 The bacterial suspension was diluted with cfu / mL and used to immerse crop seeds for 22-28 hours. The seeds were then incubated in the dark at 25-30°C.

[0013] Furthermore, the application specifically includes the following steps: (1) Activation of the strain and expansion culture to obtain bacterial solution; (2) Preparation of inoculum suspension: Centrifuge the bacterial solution, discard the supernatant, add an equal volume of sterile water to resuspend the bacterial cells, repeat centrifugation and washing, and finally resuspend the bacterial cells in sterile water to OD. 600 = 0.4~0.6, which is the bacterial suspension used for soaking seeds; (3) Disinfection treatment of rice seeds: First, select rice seeds that are uniform in size, intact and plump. Rinse them with sterile water, disinfect them with 3.5% sodium hypochlorite solution for 5 minutes, rinse, disinfect, and rinse with sterile water. (4) Rice seed germination treatment: Add an appropriate amount of bacterial suspension to the disinfected rice seeds, enough to submerge the seeds, and soak for 22-28 hours; then incubate in the dark at 25-30℃. After germination, the seeds can be sown and planted normally.

[0014] Furthermore, the crops include crops grown in cold, black soil regions, including at least one of japonica rice, corn, and soybeans.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial technical effects: 1. This invention first isolated a highly efficient, multifunctional growth-promoting bacterial strain from the rhizosphere soil of Japonica rice grown in cold-region black soil. Based on the wild strain, rapid gene modification was carried out using genome shuffling technology, and key shuffling parameters were optimized. While ensuring high performance, genetic stability, and safety of the strain, its growth-promoting effects on seed germination and seedling growth of Japonica rice in cold-region were studied. This invention not only tapped into the rhizosphere growth-promoting bacterial resources in cold-region black soil but also significantly improved the performance of the strain through biotechnology. It provides a highly suitable source of strains for the development of growth-promoting bacterial agents suitable for Japonica rice grown in cold-region black soil, effectively solving the problem of strain adaptability to cold-region environments and Japonica rice plants.

[0016] 2. The Pantotheca agglomeratus strain J1 and the modified Pantotheca agglomeratus strain G6 isolated and screened in this invention were identified by Columbia blood agar plate testing. These strains showed no hemolytic activity and were non-toxic and harmless to humans, animals, crops, and the ecological environment. Furthermore, the modified strains can rapidly proliferate in inexpensive and readily available fermentation media. The fermentation process is simple and easy to control, low in cost, and highly safe, meeting the needs of large-scale production.

[0017] 3. The fermentation broth of Pantotheca agglomerata J1 provided by this invention has a soluble phosphorus content of 379.55 mg / L, a siderophore expression level of 69.09%, and an IAA production content of 15.23 mg / L. The modified Pantotheca agglomerata strain G6 obtained through two rounds of genome shuffling has a soluble phosphorus content of 435.14 ± 0.51 mg / L, a siderophore expression level of 78.11 ± 0.11%, and an IAA production content of 25.87 ± 0.08 mg / L, respectively, which are 14%, 13%, and 70% higher than the original strain, respectively. This indicates that the wild-type isolate and the modified strain contain core growth-promoting substances and exhibit strong growth-promoting capabilities.

[0018] 4. This invention experimentally confirmed that *Pantotheca acuminata* J1 and *Pantotheca acuminata* G6 can significantly enhance the early germination ability of rice seeds. The germination potential of the treated groups reached over 80% on the second day, significantly higher than the control group. The germination index of both cold-region japonica rice varieties was significantly increased, the average germination time was shortened, and the vigor index showed a significant improvement. In summary, rhizosphere growth-promoting bacteria can significantly improve rice seed germination-related indicators and have a significant promoting effect on early rice seed germination. Attached Figure Description

[0019] Figure 1 The image shows the morphology of the clustered pantothecin J1 colony (A) and the Gram staining image under a microscope (B). Figure 2 A phylogenetic tree of the clustered pantothenic bacterium J1 constructed based on the 16S rDNA sequence; Figure 3 Optimization of preparation and regeneration conditions for pantothenic acid J1 protoplasts; Figure 4 Optimization of protoplast inactivation conditions for clustered pantothenia J1; Figure 5 To optimize the fusion conditions of protoplasts of the clustered pantothenic bacteria J1; Figure 6 Qualitative determination results for Pantotheca clumps J1 and Pantotheca clumps G6; Figure 7 Quantitative analysis of soluble phosphorus, siderophores, and IAA production in *Plasmodium clumpacans* J1 and G6; Note: Different lowercase letters in the figure indicate significant differences in morphology between different strains. P < 0.05), the same below; Figure 8 Genetic stability analysis of pantothenic acid G6; Figure 9 Hemolytic activity of Pantotheca clumps J1 and G6 on Columbia blood agar medium was analyzed. Figure 10 The effects of Pantotheca aggregata J1 and Pantotheca aggregata G6 on the germination potential of rice seeds; Figure 11 The effects of Pantotheca acuminata J1 and Pantotheca acuminata G6 on the average germination days of rice seeds; Figure 12 The effects of Pantotheca agglutinosa J1 and Pantotheca agglutinosa G6 on the germination index of rice seeds; Figure 13 The effects of Pantotheca acuminata J1 and Pantotheca acuminata G6 on the germination vigor index of rice seeds. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are merely illustrative and are not intended to limit the scope of protection of this invention.

[0021] The experimental materials and reagents involved in this invention include the following: LB medium: 10 g tryptone, 10 g NaCl, 5 g yeast extract, distilled water to a final volume of 1 L, pH 7.2-7.5, autoclaved at 121℃ for 20 min. LB solid medium is prepared by adding 20 g agar to the LB liquid medium.

[0022] Inorganic phosphorus-releasing liquid medium (NBRIP): 10 g glucose, 5 g Ca3(PO4)2, 5 g MgCl2, 0.25 g MgSO4, 0.2 g KCl, 0.1 g (NH4)2SO4, diluted to 1 L with distilled water, pH 7.2-7.5, autoclaved at 121℃ for 20 min. Inorganic phosphorus-releasing solid medium is prepared by adding 20 g agar to the NBRIP liquid medium.

[0023] Organic phosphorus-dissolving liquid medium: 10 g glucose, 0.5 g yeast extract, 0.3 g NaCl, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4, 0.03 g FeSO4, 0.03 g MnSO4·4H2O, 0.2 g lecithin, 5 g CaCO3, diluted to 1 L with distilled water, pH 7.2-7.5, autoclaved at 121℃ for 20 min. Organic phosphorus-dissolving solid medium is prepared by adding 20 g agar to the organic phosphorus-dissolving liquid medium.

[0024] Siderophore medium (CAS): 0.0605 g chromaine, 0.0729 g hexadecyltrimethylammonium bromide (HDTMA), 0.0026 g FeCl3·6H2O, 0.2952 g NaH2PO4·2H2O, 1.2135 g Na2HPO4·12H2O, 0.125 g NH4Cl, 0.0375 g KH2PO4, 0.0625 g NaCl, 15 g agar, diluted to 1 L with distilled water, pH 7.2-7.5, autoclaved at 121℃ for 20 min.

[0025] Ashby's nitrogen-free solid medium: KH2PO4 0.2 g, MgSO4 0.2 g, NaCl 0.2 g, CaCO3 5 g, mannitol 10 g, CaSO4 0.1 g, agar 20 g, distilled water to a final volume of 1 L, pH 7.2-7.5, autoclaved at 121℃ for 20 min.

[0026] Potassium-solubilizing medium: Na2HPO4 2.0 g, MgSO4·7H2O 0.5 g, FeCl3 0.005 g, CaCO3 0.1 g, sucrose 5 g, potassium feldspar powder 1 g (washed 5 times with deionized water, dried at 60℃ for about 6 h, and sieved through 180 mesh), agar 20 g, distilled water to a final volume of 1 L, pH 7.2-7.5, autoclaved at 121℃ for 20 min.

[0027] KMB liquid culture medium: 5 g casein amino acids, 15 mL glycerol, 2.5 g K2HPO4, 2.5 g MgSO4·7H2O, distilled water to a final volume of 1 L, pH 7.2-7.5, autoclaved at 121℃ for 20 min.

[0028] Iron-enriched KMB liquid medium: 0.0278 g of FeSO4·7H2O was added to the MKB iron-free liquid medium, pH 7.2-7.5, and autoclaved at 121℃ for 20 min.

[0029] LB liquid medium containing L-tryptophan: Add sterile L-tryptophan filtered through a 0.22 µm microporous membrane to sterile LB liquid medium to achieve a final concentration of 100 mg / L.

[0030] Hyperosmolar regeneration bacterial culture medium (SMR upper layer): 0.5 mol / L sucrose, 20 mmol / L MgCl2, 15 g agar, pH 7.2-7.5, autoclaved at 121℃ for 20 min.

[0031] Hypertonic regeneration bacterial culture medium (lower layer of SMR): Add 0.5 mol / L sucrose, 20 mmol / L MgCl2, and 15 g agar to the bacterial culture medium, set the pH to 7.2-7.5, and autoclave at 121℃ for 20 min.

[0032] Molybdenum-antimony anti-storage solution: First, measure 163 mL of concentrated sulfuric acid (analytical grade, density 1.84 g / mL) and slowly add it to 400 mL of distilled water, stirring continuously with a glass rod until the solution cools. Separately, weigh 10 g of ammonium molybdate and dissolve it in 300 mL of distilled water at approximately 60°C, then cool. Next, slowly pour the concentrated sulfuric acid solution into the ammonium molybdate solution while stirring. Then add 100 mL of 0.5% potassium antimony tartrate solution, and dilute to 1000 mL with distilled water. Shake well and transfer to a brown reagent bottle for storage away from light.

[0033] Molybdenum-antimony colorimetric reagent: Weigh 1.5 g of ascorbic acid and dissolve it in 100 mL of molybdenum-antimony colorimetric reagent storage solution. Note that this solution has a short shelf life and should be prepared fresh for use.

[0034] 2,4-Dinitrophenol indicator: Weigh 1 g of 2,4-dinitrophenol and dissolve it in 1000 mL of anhydrous ethanol.

[0035] 100 mg / L Phosphorus Standard Stock Solution: Weigh 0.4394 g of potassium dihydrogen phosphate (dried in an oven at 60℃ for 6 h), dissolve it in 100 mL of distilled water, add 5 mL of concentrated sulfuric acid (for preservation), and dilute to 1 L with distilled water to obtain a phosphorus standard stock solution with a concentration of 100 mg / L.

[0036] 5 mg / L Phosphorus Standard Solution: Take 10 mL of phosphorus standard stock solution into a 200 mL volumetric flask, add distilled water to make up to the volume, which is a phosphorus standard solution with a concentration of 5 mg / L.

[0037] CAS Blue Detection Solution: Add 6 mL of HDTMA solution (10 mM) to a 100 mL volumetric flask and dilute appropriately with distilled water. Mix 1.5 mL of 1 mmol / L FeCl3 (prepared using 10 mmol / L HCl) with 7.5 mL of 2 mmol / L CAS solution and slowly add the mixture to the volumetric flask along a glass rod. Weigh 4.307 g of anhydrous dimethylamine (anhydrous piperazine) and dissolve it in 30 mL of distilled water. Slowly add 6.25 mL of concentrated hydrochloric acid (12 mmol / L) along the wall of the flask. Transfer this solution to the volumetric flask and dilute to 100 mL with distilled water to obtain the CAS detection solution.

[0038] Salkowski colorimetric solution: Weigh 13.5 g FeCl3 and dissolve it in 100 mL of water to obtain a 0.5 mol / L FeCl3 solution. Take 1 mL of the 0.5 mol / L FeCl3 solution and add it to 50 mL of distilled water. Then add 30 mL of concentrated sulfuric acid, cool, and make up to 100 mL.

[0039] Phosphate buffer: 0.1 mol / L Na2H(PO)4, 0.1 mol / L NaH2(PO)4, pH = 7.0, sterilized by moist heat at 121℃ for 20 min.

[0040] Protoplast Stabilizing Solution (SMM): 0.5 mol / L sucrose, 20 mmol / L MgCl2, 20 mmol / L maleic acid, pH = 7.0, sterilized by moist heat at 121℃ for 20 min.

[0041] 1 mg / mL lysozyme solution: prepared with SMM solution.

[0042] 10 mg / mL penicillin: Dissolve 0.96 g of penicillin sodium salt in 96 mL of sterile water.

[0043] Hyperosmolar crystal violet staining solution: Dissolve 0.2 g crystal violet and 0.08 g ammonium oxalate in 100 mL SMM solution.

[0044] Example 1 1. Strains screening This embodiment provides the isolation process of multifunctional rhizosphere growth-promoting bacteria, the details of which are as follows: On October 15, 2021, soil samples from the rhizosphere of japonica rice were collected in the cold black soil region of Jiamusi City, Heilongjiang Province. Phosphorus-solubilizing strains were isolated and screened using NBRIP medium and organic phosphorus-solubilizing medium. Strains capable of decomposing both organic and inorganic phosphorus were obtained. Based on this, the siderophore production, nitrogen fixation, and potassium-solubilizing abilities of the strains were further verified using siderophore medium, Assumption nitrogen-free solid medium, and potassium-solubilizing medium.

[0045] Multifunctional growth-promoting bacteria with the ability to solubilize inorganic phosphorus, organic phosphorus, produce siderophores, produce IAA, fix nitrogen, and solubilize potassium were screened and fermented. The soluble phosphorus content, siderophore expression level, and IAA yield in the fermentation broth of the strains were quantitatively determined.

[0046] From the five multifunctional rhizosphere growth-promoting bacteria obtained in the initial screening, which have the ability to solubilize inorganic phosphorus, organic phosphorus, produce iron carriers, produce IAA, fix nitrogen, and solubilize potassium, strain J1 was determined as the starting strain of the revamp after comprehensive consideration.

[0047] The starting strain (strain J1) was streaked onto LB medium and cultured at 28°C. The colony morphology of strain J1 was observed. Gram staining of wild-type strain J1 was performed, and the cell morphology and staining results were observed under an oil immersion microscope. Physiological and biochemical identification was carried out using bacterial physiological and biochemical identification tubes. Molecular biological identification was performed based on the bacterial 16S rDNA sequence, and a phylogenetic tree was constructed to finally determine its species status.

[0048] Strain J1 colonies were observed to be pale yellow with regular edges and a moist surface on LB plates. Figure 1 (A). Under a microscope, the bacteria are rod-shaped and stain red with Gram stain, thus identifying them as Gram-negative bacteria. Figure 1 (B)

[0049] Strain J1 belongs to the same genus as Pantotheca, and strain J1 was identified as belonging to the genus Pantotheca. Pantoea sp. The strain, designated as Pantothenia J1, has a 16S rDNA sequence submitted to NCBI with the sequence number PP064067. Figure 2 The nucleotide sequence of the 16S rDNA of the pantothenic J1 is shown in SEQ ID NO.1.

[0050] Example 2 This embodiment provides the preparation of pancreatic J1 protoplasts and the mass / volume concentration of lysozyme, enzymatic hydrolysis temperature, and enzymatic hydrolysis time. Effects on the preparation and regeneration of pantotheca J1 protoplasts.

[0051] 1. Preparation of Protoplast Stabilizing Solution (SMM) After activating and culturing the aforementioned Pantotheca J1, it was transferred to LB liquid medium for overnight culture. The bacterial suspension obtained from the overnight culture was then transferred to fresh LB liquid medium at a concentration of 1-5%, and cultured with shaking to allow the OD to rise. 600 = 0.4, obtain OD 600 =0.4 bacterial solution.

[0052] OD 600 = 0.4% bacterial suspension was added to lysozyme solution, mixed evenly, and then placed in a water bath at 42℃ for 50 min. The mixture was then stained with hyperosmolar crystal violet to prepare protoplast stabilization solution (SMM).

[0053] Take 5 mL of each Pantotheca J1 bacterial suspension into a 10 mL sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, discard the supernatant, resuspend the bacterial cells in 5 mL of phosphate buffer, centrifuge again at 4000 r / min for 10 min, discard the supernatant, resuspend the bacterial cells in 5 mL of protoplast stabilization medium (SMM), centrifuge at 4000 r / min for 10 min, and resuspend in 5 mL of protoplast stabilization medium (SMM). Store at 4°C for later use. Take 1 mL of each of the above bacterial suspensions into a 10 mL centrifuge tube, and perform serial dilutions with physiological saline. Take 10 mL of each of the above bacterial suspensions into a 10 mL centrifuge tube. -5 10 -6 10 -7 Each dilution gradient of 100 µL was spread on LB solid medium and incubated at 37°C for 24 h. Plate counts were performed after each dilution gradient was set up in triplicate.

[0054] 2. The Influence of Different Factors on Protoplast Formation (1) Effect of lysozyme concentration on protoplast formation Take OD respectively 600 = 0.4% bacterial culture 1 mL into 10 mL centrifuge tubes, and add 20 mg / mL lysozyme (Solarbio lysozyme (1 g / bottle), enzyme activity > 20000 U / mg) solution to the final concentrations of 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL and 2.5 mg / mL respectively. After mixing evenly, place in a water bath at 42℃ for 50 min. Take samples every 10 min, stain with hyperosmolar crystal violet solution, and observe the protoplast formation under a microscope.

[0055] (2) Effect of enzymatic hydrolysis temperature on protoplast formation Take OD respectively 600= 0.4% bacterial culture 1 mL into 10 mL centrifuge tubes, add 20 mg / mL lysozyme solution to the final concentration of 1 mg / mL, mix well and place in water baths at 36℃, 39℃, 42℃, 45℃ and 48℃ for 50 min. Take samples every 10 min, stain with hyperosmolar crystal violet solution and observe the protoplast formation under a microscope.

[0056] (3) Effect of enzymatic hydrolysis time on protoplast formation Take OD respectively 600 = 0.4% bacterial culture 1 mL into 10 mL centrifuge tubes, add 20 mg / mL lysozyme solution to the final concentration of 1 mg / mL, mix well and place in a 42℃ water bath for 10 min, 20 min, 30 min, 40 min and 50 min respectively, stain with hyperosmolar crystal violet staining solution, and observe the protoplast formation under a microscope.

[0057] When microscopic examination showed that more than 90% of protoplasts were present, the enzymatic digestion reaction was stopped, centrifuged at 4000 r / min for 10 min, the supernatant was discarded, the bacterial cells were resuspended in hypertonic buffer, washed to remove lysozyme, centrifuged again at 4000 r / min for 10 min, the protoplasts were resuspended in 5 mL of protoplast stabilization solution (SMM) and stored at 4℃ for later use.

[0058] (4) Determination of residual bacterial count Take 1 mL of each of the above protoplast suspensions, perform serial dilutions with sterile water, mix by inverting, and wait 10 min for complete lysis and death of the protoplasts. Take 10 mL of each of these solutions. -5 10 -6 10 -7 Each dilution gradient of 100 µL was spread on LB solid medium and incubated at 37°C for 24 h. Plate counts were performed after each dilution gradient was set up in triplicate.

[0059] (5) Regeneration of protoplasts (determination of the number of regenerated bacteria) Using the double-layer plate method, a pre-pouring hypertonic regeneration medium layer was prepared as the bottom layer and incubated overnight at 37°C for sterility testing. 1 mL of each of the above protoplast suspensions was then serially diluted using protoplast stabilizing medium (SMM). 10 mL of each solution was pipette-diluted. -5 10 -6 10 -7 Dilute each of the dilution gradients by 300 µL to 15 mL of the upper hypertonic regeneration medium, mix well, and pour into the lower hypertonic regeneration medium solid plates. Incubate at 37°C for 24 h, then count the plates. Set up 3 replicates for each dilution gradient.

[0060] Calculation of protoplast formation rate and regeneration rate: Protoplast formation rate (%) = (A - B) / A × 100% ; Protoplast regeneration rate (%) = (C - B) ∕ (A - B) × 100% ; In the formula A —Total bacterial count (the number of colonies on LB plates that have not been treated with lysozyme). B —Remaining bacterial count (number of colonies on LB plates that have died after enzymatic digestion). C —Regeneration bacterial count (the number of colonies on the regeneration plate after enzymatic hydrolysis).

[0061] When the enzymatic hydrolysis concentration was 1 mg / mL, the protoplast formation rate and regeneration rate were 91.2% and 28.7%, respectively. When the enzymatic hydrolysis treatment lasted 30 min, the protoplast formation rate and regeneration rate were 91.3% and 28.8%, respectively. When the enzymatic hydrolysis temperature was 42℃, the protoplast formation rate and regeneration rate were 91.3% and 28.7%, respectively. Figure 3 ).

[0062] Based on the above experimental results, the optimal process for preparing the optimized protoplast stabilizing solution is as follows: After activating and culturing Pantotheca J1, it was transferred to LB liquid medium for overnight culture. The bacterial suspension obtained overnight was then transferred to fresh LB liquid medium at a concentration of 1-5%, and cultured with shaking to allow OD to rise. 600 = 0.4, obtain OD 600 = 0.4% bacterial culture; OD 600 = 0.4% bacterial suspension was added to lysozyme solution to a final concentration of 1 mg / mL, mixed evenly, and then placed in a water bath at 42℃ for 30 min to prepare protoplast stabilization solution (SMM).

[0063] Example 3 This embodiment provides optimized conditions for inactivating and fusing the protoplast stabilizing solution prepared from Pantotheca J1 (obtained from the optimal process in Example 2). This embodiment provides two inactivation methods, specifically including the following: 1. Optimization of protoplast inactivation conditions prepared from pantothenic acid J1 (1) Investigation of ultraviolet inactivation conditions Take 1 mL of the protoplast suspension stored at 4℃ into a 10 mL sterile centrifuge tube, add 9 mL of protoplast suspension for serial dilution, and take 10 mL of the solution. -5 10 -6 10 -7A series of gradient protoplast suspensions were transferred to 9 cm sterile petri dishes. The distance between the petri dish and the UV lamp was adjusted to 30 cm. The petri dish lid was opened, and UV inactivation was performed for 25 min. Samples were taken at 1 min, 5 min, 10 min, 15 min, 20 min, and 25 min. The samples were then serially diluted using protoplast stabilizing solution (SMM) and poured into plates. Uninactivated protoplast suspensions were used as controls. Each gradient was repeated 3 times. The plates were wrapped in black cloth and incubated at 37°C for 24 h. Colony growth was observed and counted, and the inactivation rate was calculated. An inactivation rate of 100% was selected as the optimal inactivation time.

[0064] (2) Investigation of thermal inactivation conditions Take 0.5 mL of the protoplast suspension stored at 4℃ and place it in a 10 mL sterile centrifuge tube. Add 4.5 mL of protoplast suspension for serial dilution. Take 10 mL of the solution and dilute. -5 10 -6 10 -7 A series of protoplast suspensions were placed in a constant temperature water bath and inactivated at temperatures of 50℃, 55℃, 60℃, 65℃ and 70℃ for 25 min. After serial dilution with protoplast stabilizing solution (SMM), the suspensions were poured into plates and the subsequent procedures were the same as for UV inactivation. The effect of different temperatures on the heat inactivation effect was observed.

[0065] In addition, based on the optimal heat inactivation temperature, samples were taken at 1 min, 5 min, 10 min, 15 min, 20 min and 25 min respectively. After serial dilution with protoplast stabilizing solution (SMM), the samples were poured into plates and the subsequent operations were the same as those for UV inactivation. The effect of different heat inactivation times on the heat inactivation effect was observed.

[0066] Calculation of protoplast inactivation rate: Protoplast inactivation rate (%) = (Number of colonies in the control group on the regeneration plate - Number of colonies in the regeneration plate after inactivation) / Number of colonies in the regeneration plate after inactivation × 100%.

[0067] (3) Experimental results After 10 minutes of UV inactivation, the inactivation rate of protoplasts reached 100%. Figure 4 (As shown in Figure B); at a temperature of 60℃, the protoplast inactivation rate reaches 100% ( Figure 4 As shown in Figure A); after 20 min of heat inactivation treatment, the protoplast inactivation rate reached 100% ( Figure 4 (As shown in C).

[0068] 2. Optimization of protoplast fusion conditions prepared from pan-bacterium J1 The protoplast fusion conditions were explored using PEG solutions of different concentrations and pH values. The fusion rate was calculated, and the condition with the highest fusion rate was selected as the optimal fusion condition.

[0069] (1) The process of protoplast fusion of pan-bacteria J1 Take 1 mL of the inactivated protoplast suspension into a 10 mL sterile centrifuge tube, centrifuge at 6000 r / min for 10 min, discard the supernatant, add 0.4 mL of phosphate buffer to resuspend, add 3.6 mL of PEG solution with different mass concentrations (30%, 35%, 40%, 45%, 50%) and pH 9, mix well, place it in a water bath at 42℃ for 5 min, centrifuge at 4000 r / min for 10 min, discard the supernatant, and obtain the fusion solution.

[0070] Based on the optimal concentration of the PEG solution, the pH values ​​of the PEG solution were adjusted to 7, 8, and 9 to investigate the effect of the pH value of the PEG solution on the fusion conditions.

[0071] Based on the optimal concentration and pH value of the PEG solution, the water bath treatment time was set to 1 min, 3 min, 5 min, 7 min, and 10 min, respectively, to investigate the effect of different water bath treatment times on the fusion conditions.

[0072] The fusion solutions obtained under the different conditions described above were washed twice, resuspended in 2 mL of phosphate buffer, poured into regeneration plates, and incubated at 37°C for 1-2 days. Colony growth was observed. The number of colonies in the regeneration plates of the inactivated protoplast suspension was used as a control. The fusion rate was calculated, and the condition with the highest fusion rate was selected as the optimal fusion condition.

[0073] Protoplast fusion rate (%) = Number of colonies of fusion molecule in regeneration plate / Number of colonies of uninactivated parent in regeneration plate × 100%.

[0074] Experimental results: The highest protoplast fusion rate was 8.89% when the PEG concentration was 40%; the highest fusion rate was 8.83% when the fusion time was 5 min; and the highest fusion rate was 8.89% when the pH of the co-solvent was 9.0. Figure 5 ).

[0075] Based on the above experimental results, the optimal fusion process for inactivated Pantotheca J1 protoplasts is as follows: Take 1 mL of the inactivated pantothecin J1 protoplast suspension and place it in a sterile centrifuge tube. Mix well and centrifuge at 6000 r / min for 10 min. Discard the supernatant, add 0.4 mL of phosphate buffer to resuspend, then add 3.6 mL of PEG solution with a mass concentration of 40% and a pH of 9. Mix well and place it in a water bath at 42℃ for 5 min. Centrifuge at 4000 r / min for 10 min and discard the supernatant to obtain the fusion solution.

[0076] Example 4 In this embodiment, the multifunctional rhizosphere growth-promoting bacterium J1 obtained from previous screening was selected as the starting strain. Protoplasts were obtained using the optimal preparation conditions provided in Example 2. The protoplasts were then inactivated using the optimal heat inactivation conditions provided in Example 3. After inactivation, recursive fusion was performed. The first round of fusions was prepared using the optimal fusion conditions provided in Example 3. The fusions selected in the first round were then used as the starting strain, and the above method was repeated for the next fusion. The functional characteristics of the modified strains obtained after multiple fusions were investigated, specifically including the following: 1. Recursive protoplast fusion The multifunctional rhizosphere growth-promoting bacterium J1 obtained from the previous screening was selected as the starting strain. The optimal conditions for preparing protoplasts were explored and the protoplasts were heat-inactivated using the optimal heat inactivation conditions. Recursive fusion was then performed, and the optimal fusion conditions were selected for the first round of screening of fusions. The fusions obtained from the first round of screening were used as the starting strains for the next fusion in the same way. The regenerated strain from the first round of fusion was denoted as the first round regenerated strain G1, and the regenerated strain from the second round of fusion was denoted as the regenerated strain G6.

[0077] 2. Screening of fusion units (1) Initial screening The recombined strains that grew from each round of fusion regeneration plates were inoculated into inorganic phosphorus solubilizing, organic phosphorus solubilizing, and iron-producing culture medium plates, respectively. The original strain was used as a control. The changes in the plates were observed. Colonies with a ratio of phosphorus solubilizing ring diameter (D) to colony diameter (d) (D / d) and a ratio of orange-yellow halo diameter (H) to colony diameter (h) (H / h) that were all greater than those of the original strain were recorded and preserved. The recombined strains with multiple functions were preliminarily screened and recorded and preserved.

[0078] The obtained modified strains were screened using a plate assay to determine their abilities to solubilize inorganic phosphorus, organic phosphorus, produce siderophores, produce IAA, fix nitrogen, and solubilize potassium. Results are shown below. Figure 6 Compared with the original strain, the modified strain showed significant improvements in its ability to solubilize inorganic phosphorus, organic phosphorus, produce siderophores, produce IAA, and fix nitrogen.

[0079] (2) Secondary screening Qualitative screening of the modified strain G6 was carried out on various selective plates. For strains that showed better performance than the starting strain, quantitative rescreening was performed.

[0080] The modified strain G6 was inoculated into LB liquid medium to prepare seed culture. The seed culture was then transferred at a concentration of 1% to LB liquid fermentation media containing inorganic phosphorus, siderophores, and L-tryptophan, respectively. Soluble phosphorus content was determined by the molybdenum-antimony colorimetric method; siderophore expression levels of different strains were determined by CAS assay; the type of siderophore produced was verified using ferric perchlorate and FeCl3 assays; and the Salkowski colorimetric reaction was used to determine the strain's IAA production capacity.

[0081] Soluble phosphorus content was determined using the molybdenum-antimony colorimetric method. Seed culture was transferred at a concentration of 1% to inorganic phosphorus-free liquid medium and cultured at 28℃ and 140 r / min for 10 days. Every 2 days, 5 mL of bacterial suspension was collected, centrifuged at 4000 r / min for 15 min, and 100 µL of the supernatant was transferred to a 25 mL graduated test tube. Deionized water was added to a final volume of 15 mL, along with one drop of 2,4-dinitrophenol indicator. The solution was adjusted to a slightly yellow color with 1 mol / L NaOH solution, and 5 mL of molybdenum-antimony colorimetric solution was added. The volume was then adjusted to 25 mL with deionized water, mixed thoroughly by inverting, and allowed to react at room temperature for 30 min. Colorimetric analysis was performed at 700 nm using a spectrophotometer, with the uninoculated solution used as a reference for zeroing. The absorbance value of the test solution was measured and recorded. This value was then substituted into the phosphorus standard curve formula to calculate the soluble phosphorus content. Simultaneously, every 2 days... d. Measure the pH value of the remaining supernatant using a pH meter and record its pH value changes.

[0082] The expression levels of siderophores in different bacterial strains (recombined strain G6 and original strain J1) were determined using CAS detection solution. Seed cultures of different strains were transferred at a concentration of 1% to KMB liquid medium and iron-enriched KMB liquid medium, respectively. The cultures were incubated at 28℃ and 140 r / min for 72 h with shaking. 5 mL of the bacterial suspension was centrifuged at 4000 r / min for 15 min, and the supernatant was retained. The supernatant was mixed with CAS detection solution at a ratio of V(supernatant):V(CAS detection solution) = 1:1 and inverted for 10 min. Using deionized water as a control, the absorbance of the reaction solution was measured at 630 nm using a spectrophotometer. The siderophore expression level was calculated using the following formula: Su = (Ar - A) / Ar × 100%; where Su is the expression level of the siderophore; Ar is the OD value of the supernatant of the iron-rich KMB medium; and A is the OD value of the supernatant of the KMB liquid medium.

[0083] Experiment to determine the type of siderophore-producing strain: The ferric perchlorate test was used for verification, which included the following: Take 0.5 mL of the retained supernatant and add 2.5 mL of ferric perchlorate solution. Observe the color change. If the color turns red or orange, it indicates that the supernatant contains isohydroxamic acid siderophores.

[0084] FeCl3 experiment: Take 1 mL of the retained supernatant and add 1-5 mL of 2% FeCl3 solution. If the color turns red or purple, it indicates the presence of siderophores. If 1 mL of supernatant is added to 1 mL of 2% FeCl3 solution and the color immediately turns red, it indicates the presence of isohydroxamic acid siderophores in the supernatant. If more than 1 mL of 2% FeCl3 solution is added to 1 mL of supernatant and the color turns red or purple, it indicates the presence of catechol siderophores. If there is no color change, it indicates other types of siderophores.

[0085] Experimental results: (1) After the reaction with ferric perchlorate solution, the strains with the ability to produce iron carriers did not turn red or orange, indicating that the supernatant of the strains did not contain isohydroxamic acid iron carriers.

[0086] (2) After adding 1-5 mL of 2% FeCl3 solution, the strains with siderophore production ability did not turn red or purple, indicating that the supernatant of the strains did not contain isohydroxamic acid siderophores or catechol siderophores.

[0087] The Salkowski chromogenic reaction was used to determine the IAA production capacity of bacterial strains. Strains that reacted with the Salkowski chromogenic reaction to produce a pink color were selected. Their seed culture was transferred at a concentration of 1% to LB liquid medium containing L-tryptophan, with three replicates for each strain. The cultures were incubated at 30℃ and 140 r / min for 72 h with shaking. 100 µL of the bacterial suspension was mixed with 100 µL of the Salkowski chromogenic solution, and the experiment was repeated three times. The reaction was carried out under dark conditions for 30 min, and colorimetric measurements were performed at 530 nm using a spectrophotometer. A mixture of 100 µL of deionized water and 100 µL of the Salkowski chromogenic solution was used as a reference solution for zeroing the instrument. The absorbance of the test solution was measured and recorded, and then substituted into the IAA standard curve formula to calculate the IAA production content of different strains.

[0088] Figure 7The results showed that compared with the starting strain J1, the contents of soluble phosphorus, siderophore expression, and IAA production in the fermentation broth of the reshuffled strain G6 were 435.14 ± 0.51 mg / L, 78.11 ± 0.11%, and 25.87 ± 0.08 mg / L, respectively, which were 1.14-fold (379.55 mg / L), 1.13-fold (69.09%), and 1.70-fold (15.23 mg / L) higher than those of the starting strain J1, respectively.

[0089] The screened reshuffled strain G6 was preserved. The preservation unit of the reshuffled strain G6: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Preservation date: April 24, 2026; Pantoea agglomerans Pantoea agglomerans The preservation number is: CGMCC No. 38430.

[0090] The strain obtained by protoplast fusion reshuffle screening did not change the genetic characteristics of the strain. The nucleotide sequence of the 16S rDNA of the strain G6 is also shown in SEQ ID NO.1. It belongs to the genus Pantoea together with the strain J1.

[0091] The method for preparing the protoplasts of the reshuffled strain G6 is as follows: After activating and culturing Pantoea agglomerans G6, it is transferred to LB liquid medium for overnight culture. The overnight cultured bacterial suspension is transferred to fresh LB liquid medium at a content of 1-5%, and shaken culture is carried out to obtain a bacterial solution with an OD 600 = 0.4-0.6; The bacterial solution is added to the lysozyme solution to a final concentration of 1 mg / mL, and after uniform mixing, it is placed in a water bath at 42°C for 30 min to prepare protoplasts.

[0092] Example 5 This example mainly explores the genetic stability of the reshuffled strain G6, which specifically includes the following content: The reshuffled strain G6 screened in Example 4 was subcultured five times continuously, and the relevant properties of each generation of the strain were measured. The measurement method was the same as that in "Example 4" to verify the genetic stability of the reshuffled strain.

[0093] A stability test was carried out on the phosphate-solubilizing ability, siderophore-producing ability, and IAA-producing ability of the reshuffled strain G6. The results are shown in Figure 8 . After continuous subculture for 5 times, the differences in the content of soluble phosphorus, siderophore expression, and IAA content in the fermentation broth of the reshuffled strain G6 were not significant. Therefore, it was determined that the reshuffled strain G6 had good genetic stability.

[0094] Example 6 This embodiment mainly explores the safety of the modified strain G6, specifically including the following: The modified strain G6 was activated, and single colonies were picked and placed on Columbia blood agar medium. The culture was carried out at 37°C for 48 h. The safety of the strain was determined by the presence or absence of a hemolytic zone on the plate. An α-hemolytic zone is grass-green, while a β-hemolytic zone is colorless and transparent. In this example, Staphylococcus aureus was used as a positive control strain.

[0095] Figure 9 As shown, neither the original strain J1 nor the modified strain G6 showed a hemolytic zone on Columbia blood agar medium, while Staphylococcus aureus did, indicating that both the original strain J1 and the modified strain G6 are safe.

[0096] Example 7 This embodiment investigates the growth-promoting effect of strain G6 on the germination of cold-region japonica rice seeds, specifically including the following: 1. Activation and scale-up culture of the strain First, the rhizosphere growth-promoting bacteria J1 and the isolated modified strain G6 were activated and cultured on LB solid medium. After culturing at 28°C for 24 h, single colonies were picked and transferred to LB liquid medium and cultured overnight in a shaker at 28°C for expansion.

[0097] 2. Preparation of inoculum suspension The bacterial culture, incubated overnight at 28°C, was centrifuged at 10,000 r / min for 10 min at room temperature. The supernatant was discarded, and the cells were resuspended in an equal volume of sterile water. The centrifugation and washing were repeated twice. Finally, the cells were resuspended in sterile water to OD200. 600 = 0.5, which is the bacterial suspension used for soaking.

[0098] 3. Disinfection treatment of rice seeds First, select rice seeds that are uniform in size, intact and plump. Rinse them twice with sterile water, then disinfect them with 3.5% sodium hypochlorite solution for 5 minutes, rinse them 6 times, then disinfect them with 75% ethanol for 1 minute, and rinse them once with sterile water.

[0099] 4. Rice seed germination treatment Disinfected Longjing 57, Longjing 3024, and Zhongjia Longjing 18 rice seeds were placed into 12 beakers, divided into 3 treatment groups and 1 control group, with 100 seeds in each treatment. An appropriate amount of bacterial suspension (10...) was added to each of the 3 treatment group beakers. 8The seeds were immersed in a bacterial suspension (cfu / mL) for 24 hours. A control group was prepared by immersing the treated rice seeds in the same volume of sterile water in a beaker. After treatment, the seeds were removed and air-dried. Using sterilized tweezers, 50 rice seeds were evenly placed in each glass petri dish lined with double-layered filter paper, and an appropriate amount of sterile water was added. The seeds were then placed in a sterile incubator and incubated in the dark at 28°C. Germination potential was measured on day 3 of germination, and germination rate, root length, shoot length, fresh weight, and dry weight were measured on day 7. The average germination time and germination index were calculated.

[0100] Germination potential = (Number of seeds germinated on day 3 / Number of seeds tested) × 100%; Germination rate = (Number of seeds germinated on day 7 / Number of seeds tested) × 100%.

[0101] Mean Germination Time (MGT) = ∑(Di × Ni) / ∑Ni, where Di is the number of germination days, Ni is the number of newly germinated seeds on day i, and ∑Ni is the final total number of normally germinated seeds.

[0102] Germination Index (GI) = ∑(Ni / Di, where Ni is the number of newly germinated seeds on day i, and Di is the corresponding number of germination days.

[0103] Vigor Index (VI) = Germination Index × Average Root Length

[0104] Compared with the control group (CK), rice seeds treated with suspensions of the original strain J1 and the modified strain G6 showed stable and slightly improved germination potential and germination rate, significantly increased germination index, significantly shortened average germination time, and significantly improved vigor index. P < 0.05).

[0105] Longjing 57 rice seeds were treated with the starting strain J1 and the modified strain G6. The germination potential of the seeds on the second day was above 80%, which was significantly higher than that of the CK group (about 70%). The germination potential of the seeds on the third day was above 95%. There was no significant difference in germination potential among the treatment groups. Figure 10 These results demonstrate that the strain not only enhances overall germination capacity but also significantly improves germination uniformity and speed.

[0106] Treatment of Longjing 57 and Longjing 3024 rice seeds with the modified strain G6 significantly shortened the average germination time, by 0.34 days compared to the control group. Figure 11 ).

[0107] Rice seeds of Longjing 57 and Longjing 3024 were treated with the modified strain G6, and their germination indices were 55.52 and 52.28, respectively, which were 21.2% and 5.6% higher than those of the control group. This result further confirms that the modified strain G6, as a rhizosphere growth promoter, can significantly accelerate the overall germination rate of rice seeds and comprehensively improve the seed germination process.

[0108] The seed vigor indices of Longjing 57 treated with the original strain J1 and the modified strain G6 reached 5767.5 and 4464.7, respectively, which were 47.6% and 14.3% higher than those of the CK group (3907.6). The seed vigor indices of Longjing 3024 treated with the original strain J1 and the modified strain G6 were 5780.2 and 5361.1, respectively, which were 23.2% and 13.4% higher than those of the CK group (4725.6). These results indicate that rhizosphere growth-promoting bacteria can not only promote rapid seed germination but also effectively enhance overall seed germination vigor and seedling growth potential.

[0109] In view of the relative scarcity of dedicated rhizosphere growth-promoting bacteria resources for japonica rice grown in cold black soil, this invention isolates and screens multifunctional rhizosphere growth-promoting bacteria from the rhizosphere of japonica rice grown in cold black soil. These bacteria possess the ability to solubilize inorganic phosphorus, organic phosphorus, produce iron carriers, produce IAA, fix nitrogen, and solubilize potassium. Using genome shuffling technology, two rounds of targeted shuffling of this strain were performed to obtain the clustered pan-bacteria G6, which significantly improved the yield of active substances and verified the genetic stability of the shuffling strain. This provides an excellent improved strain for the growth-promoting application of japonica rice grown in cold black soil.

[0110] Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, and are not intended to limit them. 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. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A clustered pantothecin G6 strain with rhizosphere growth-promoting function in crops, characterized in that, The aforementioned *Pantotheca cum Cuboides* G6 is deposited at the China General Microbiological Culture Collection Center, and its classification name is *Pantotheca cum Cuboides*. Pantoea agglomerans Its accession number is CGMCC No.38430.

2. The Pantotheca agglomerata G6 according to claim 1, characterized in that, The colonies of the Pantotheca G6 clumps are pale yellow with neat edges and a moist surface; the bacteria are rod-shaped and are Gram-negative.

3. The Pantotheca agglomerata G6 according to claim 1, characterized in that, The aforementioned Pantotheca G6 has the functions of decomposing inorganic phosphorus, decomposing organic phosphorus, producing iron carriers, producing IAA, fixing nitrogen, and decomposing potassium.

4. The protoplast of the clustered pantothecin G6 as described in claim 1, characterized in that, The preparation method of the protoplast is as follows: after the activation culture of Pantoea agglomerans G6, the bacteria suspension obtained by overnight culture is transferred into fresh LB liquid medium at a content of 1-5%, and is subjected to oscillation culture to obtain bacteria liquid with OD 600 = 0.4-0.6; the bacteria liquid is added into a lysozyme solution to a final concentration of 1 mg / mL, and after uniform mixing, it is placed in a water bath at 42 DEG C for treatment to obtain the protoplast.

5. The application of the Pantotheca G6 clusters as described in claim 1 in the preparation of rhizosphere growth promoters for crops.

6. The application according to claim 5, characterized in that, The crops include at least one of japonica rice, corn, and soybeans.

7. The application according to claim 5, characterized in that, The applications include promoting seed germination, enhancing overall seed germination viability, and improving seedling growth potential.

8. The application according to claim 7, characterized in that, The average germination time was significantly shortened, the vigor index was significantly improved, and the uniformity and speed of germination were significantly enhanced.

9. The application according to claim 5, characterized in that, The application method comprises: fermenting the Pantoea agglomerans G6 to obtain a bacterial suspension, wherein OD 600 of the bacterial suspension is 0.4-0.6, the viable bacterial count of the bacterial suspension is higher than 10 8 cfu / mL, and the bacterial suspension is used to immerse crop seeds, and the soaking time is 22-28 h, and the dark culture is carried out at 25-30 DEG C.

10. The application according to claim 5, characterized in that, The bacterial suspension has a bacterial content of 10. 8 ~10 10 cfu / mL.

Images