Plant growth-promoting rhizobacteria SPN5 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly provides a plant growth-promoting rhizobacteria SPN5 and application thereof. The strain is separated from rhizosphere soil of rubber trees in Hainan Province and is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 66466. Through identification, the SPN5 is a potential new species in pantoea, has various plant growth promoting capacities of phosphate solubilization, potassium solubilization, nitrogen fixation, siderophore production, cellulase production, indoleacetic acid production, ammonia production and the like, and can well grow in the salt concentration of 3% or below and the pH range of 5.0-9.0. Pot experiments show that the SPN5 has a remarkable growth promoting effect on lettuce and rubber tree seedlings, plant biomass, leaf chlorophyll content and crop quality can be improved, and growth of rubber trees can still be effectively promoted especially under the condition that application of chemical fertilizer is reduced by 20%-40%. The strain is suitable for preparing a microbial agent, and has a good application prospect in the aspects of promoting crop growth and realizing reduction and synergism of chemical fertilizers.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a plant rhizosphere growth-promoting bacterium SPN5 and its applications. Background Technology

[0002] Plant rhizosphere growth-promoting bacteria (PGPRs) colonize around plant roots, directly or indirectly promoting plant growth, increasing crop yield, and enhancing stress resistance. Their mechanisms of action include biological nitrogen fixation, dissolving insoluble phosphorus and potassium, secreting plant growth hormones (such as indoleacetic acid), producing siderophores, and inducing systemic resistance. Developing microbial agents using PGPRs is one of the important ways to promote green agricultural development and achieve reduced fertilizer and pesticide use while increasing efficiency.

[0003] Currently, the resources of PGPR strains used in agricultural production remain relatively limited, and many strains suffer from problems such as single function, strong host specificity, or narrow environmental adaptability. For example, some strains only possess single functions such as nitrogen fixation or phosphorus solubilization, making it difficult to consistently exert a comprehensive growth-promoting effect in the complex environment of actual farmland. Furthermore, PGPR strains isolated and screened from the rhizosphere of specific crops often exhibit superior colonization ability and growth-promoting effects due to their good adaptability to the native crop rhizosphere microenvironment. Therefore, systematically exploring specific, multifunctional rhizosphere growth-promoting microbial strains for important economic crops (such as rubber trees) is of great significance for developing efficient and specialized microbial fertilizers.

[0004] On the other hand, the long-term excessive application of chemical fertilizers has led to a series of soil degradation problems, such as soil compaction, acidification, salinization, and nutrient imbalance. Promoting the reduction of chemical fertilizer application has become a national strategy for the sustainable development of agriculture in my country. Against this backdrop, there is an urgent need to find microbial solutions that can still ensure crop nutrient supply and promote healthy crop growth through multiple biological pathways under conditions of lower chemical fertilizer input. However, most existing microbial fertilizer products, under conditions of significant reduction in chemical fertilizer application, often fail to comprehensively meet the nutritional needs of crops due to their limited functionality, resulting in unstable growth-promoting effects and limiting their widespread application in actual production.

[0005] Therefore, isolating and screening novel PGPR strains from the rhizosphere environment of specific crops that possess multiple growth-promoting functions, have broad environmental adaptability, and can stably exert growth-promoting effects, especially under conditions of reduced chemical fertilizer application, and developing them into corresponding microbial inoculants or biofertilizers, has important practical value and application prospects for enriching agricultural microbial germplasm resources, supporting the action of reducing chemical fertilizer use, and promoting the development of ecological agriculture. Summary of the Invention

[0006] To address the existing problems of a lack of specific growth-promoting bacteria agents for rubber trees and a shortage of highly efficient growth-promoting strains in scenarios involving reduced chemical fertilizer application, this invention provides a strain of the genus *Pantheraea* (…). PantoeaThe potential new species SPN5 has comprehensive plant growth-promoting functions, strong adaptability, and can effectively promote crop growth under medium and low fertility or reduced fertilizer application conditions, achieving the dual goals of increasing yield and quality and reducing fertilizer use.

[0007] This invention provides the following technical solution: (I) Basic information about the strain The plant rhizosphere growth-promoting bacteria are Pantotheca ( Pantoea SPN5 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510075, China. The telephone number is 020-37656629. It is classified and named as follows: Pantoea sp., with accession number GDMCC NO: 66466, accession date June 4, 2024, isolated from the rhizosphere soil of rubber trees in Hainan rubber plantation.

[0008] (ii) Characteristics of the strain 1. Morphological characteristics: After culturing at 30°C for 48 hours on LB solid medium, single colonies are pale yellow, round and raised, with a smooth and moist surface, and a diameter of 1.5~3.0 mm; the bacteria are short rod-shaped, about 0.8~1.2×1.2~2.5 μm in size, have flagella, and are Gram-negative.

[0009] 2. Physiological and biochemical characteristics: It can utilize multiple carbon sources such as glucose, maltose, rhamnose, and mannitol. It is positive for catalase and nitrate reduction reactions, and negative for oxidase and VP reactions. Specific physiological and biochemical characteristics are shown in Table 1.

[0010] 3. Genomic characteristics: Whole-genome sequencing showed that it contains two circular chromosomes and two plasmids with a total length of 5,915,721 bp and an average GC content of 52.95%; it is predicted to contain 5,450 coding sequences, 80 tRNAs and 22 rRNAs; its 16S rDNA sequence is 1,539 bp in length (SEQ ID NO:1), and its average nucleotide identity (ANI value) with the type strain of Pantotheca is lower than the 95% interspecies threshold, making it a potential new species within the Pantotheca genus.

[0011] 4. Growth adaptability: It can grow well in salt concentrations below 3% and pH ranges of 5.0 to 9.0, and is suitable for most farmland soil environments.

[0012] (III) Promoting function The pan-bacterium SPN5 possesses multiple plant growth-promoting abilities: 1. Phosphorus solubilization ability: It can hydrolyze solid Ca3(PO4)2 and form a stable hydrolysis zone on phosphorus-solubilizing medium; 2. Potassium solubilization ability: It can stably grow on potassium-solubilizing medium using insoluble potassium feldspar powder as the sole potassium source; 3. Nitrogen fixation ability: It can be continuously passaged on nitrogen-free medium (Assumption medium) to achieve the fixation and utilization of nitrogen in the air; 4. Siderophore production capacity: On CAS detection medium, it can competitively bind Fe. 3+ And it forms a fading halo; 5. Cellulase production capacity: On the cellulase production screening medium, obvious hydrolysis zones can be seen after destaining with Congo red; 6. Indoleacetic acid (IAA) production capacity: After culturing in a medium containing L-tryptophan, the IAA production reached 41.33 ± 1.91 μg / mL; 7. Ammonia production capacity: The fermentation supernatant reacts with Nessler's reagent to form a yellowish-brown precipitate, which has the function of ammonia production.

[0013] Based on the above capabilities, the present invention provides the application of the aforementioned strain SPN5 or its bacterial suspension, in any of the following ways: A1) Application in promoting the growth of lettuce or rubber tree seedlings; A2) Application in the preparation of products that promote the growth of lettuce or rubber tree seedlings.

[0014] The specific method of application is to apply the bacterial suspension to lettuce or rubber tree seedlings by root irrigation; The preparation method of the bacterial suspension is as follows: SPN5 strain was inoculated into LB liquid medium at a 1% inoculum size, cultured at 200 rpm and 30℃ for 48 h, then the fermentation broth was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, the bacterial precipitate was washed twice with sterile water, resuspended, and diluted with sterile water to a bacterial concentration of 1.0 × 10⁻⁶. 8 cfu / mL is the bacterial suspension used.

[0015] The present invention also provides a microbial agent / biophosphate fertilizer / biopotassium fertilizer / bionitrogen fertilizer and / or soil conditioner comprising the aforementioned strain SPN5.

[0016] (iv) Application effect 1. Growth-promoting effect on lettuce: After root irrigation with SPN5 bacterial solution, the lettuce plant height, stem diameter, and number of cotyledons increased by 12.52%, 31.33%, and 9.93% respectively compared with the control; the fresh weight of the whole plant, the fresh weight of the above-ground parts, the fresh weight of the underground parts, and the dry weight all increased significantly, with the highest increase reaching 45.45%; the soluble sugar content of the leaves increased by 22.61%, and the chlorophyll content increased by 5.58%, achieving growth promotion and quality improvement.

[0017] 2. Growth-promoting effect on rubber seedlings: Under the condition of reducing chemical fertilizer application by 20%~40%, the height growth of rubber tissue culture seedlings increased by up to 79.22% compared with the control; the fresh weight of the whole plant, the fresh weight of the aboveground parts, and the fresh weight of the underground parts increased by up to 23.40%, 30.68%, and 10.29%, respectively; the chlorophyll content of leaves increased by 4.56%~6.34%, and the growth-promoting effect was particularly significant under medium and low fertility conditions.

[0018] The beneficial effects of this invention are: (1) The strain is a potential new species of Pantotheca, which enriches the germplasm resource bank of growth-promoting bacteria for tropical crops and provides new strain support for the research and development of special bacterial agents for rubber trees; (2) It has comprehensive growth-promoting functions, integrating phosphorus solubilization, potassium solubilization, nitrogen fixation, production of growth-promoting substances and enzymes, and can enhance the plant's growth potential in multiple dimensions. (3) It is highly adaptable, tolerant of certain salt concentrations and pH ranges, and adaptable to different farmland soil environments; (4) It is suitable for fertilizer reduction scenarios. It can still promote growth efficiently when fertilizer usage is reduced by 20% to 40%, which can reduce agricultural production costs, alleviate soil degradation problems, and help the green transformation of agriculture. (5) It has a wide range of applications and has a good growth-promoting effect on crops of different families and genera, such as lettuce and rubber trees. It has broad application prospects in vegetable planting, economic crop cultivation and other fields. Attached Figure Description

[0019] Figure 1 The phosphorus solubilization effect of SPN5 strain on phosphorus-solubilizing medium.

[0020] Figure 2 The colony morphology of SPN5 strain on LB medium.

[0021] Figure 3 The cell morphology of SPN5 strain under a transmission electron microscope.

[0022] Figure 4 This is a diagram of chromosome A of the SPN5 strain genome.

[0023] Figure 5 This is a B-circle diagram of the genome chromosome of strain SPN5.

[0024] Figure 6 This is a diagram of the A-circle of the SPN5 strain genomic plasmid.

[0025] Figure 7 This is a diagram of the B-circle of the SPN5 strain's genomic plasmid.

[0026] Figure 8 Phylogenetic tree of SPN5 16S rDNA.

[0027] Figure 9A heatmap showing the calculated average nucleotide identity (ANI) value for the entire genome sequence.

[0028] Figure 10 The results show the salt (NaCl) concentration tolerance of the SPN5 strain.

[0029] Figure 11 The results show the pH tolerance test results for the SPN5 strain.

[0030] Figure 12 The colony morphology of SPN5 on potassium-solubilizing medium.

[0031] Figure 13 This describes the colony morphology of SPN5 on nitrogen-free medium.

[0032] Figure 14 The results show the siderogenic vector detection results for the SPN5 strain.

[0033] Figure 15 The results show the cellulase production detection results of SPN5 strain.

[0034] Figure 16 The results of IAA (indoleacetic acid) production by SPN5 strain are shown on the left, with the control treatment on the left and the SPN5 bacterial solution treatment on the right.

[0035] Figure 17 The results show the ammonia production capacity of the SPN5 strain. The left side represents the control treatment, and the right side represents the SPN5 bacterial solution treatment.

[0036] Figure 18 The SPN5 strain promotes the growth of lettuce. The left side shows the control treatment, and the right side shows the SPN5 bacterial solution treatment.

[0037] Figure 19 The growth-promoting effect of SPN5 strain on rubber seedlings is shown from left to right as follows: control (fertilizer applied), SPN5 bacterial solution treatment + fertilizer applied, SPN5 bacterial solution treatment + fertilizer reduced by 20%, and SPN5 bacterial solution treatment + fertilizer reduced by 20%. Detailed Implementation

[0038] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0039] Experimental Example 1: Isolation and Screening of Pantotheca SPN5 The rhizosphere soil samples used were collected from rubber tree rhizosphere soil at Guangba Farm, Dongfang City, Hainan Province. The rhizosphere soil was obtained using the shaking method. 1.0 g of rubber tree rhizosphere soil was weighed and placed in an Erlenmeyer flask containing glass beads and 99 mL of sterile water. The flask was then shaken at 30℃ and 200 rpm for 30 min to disperse the soil, yielding 10... -2 Gradient soil suspensions were prepared by sequentially diluting them to prepare 10... -3 10 -4 10 -5 10 -6 10 -7 Gradient soil suspensions were prepared. 0.1 mL of each gradient suspension was spread onto phosphate-solubilizing medium plates, with three replicates per gradient. After incubation at 30°C for 2–3 days, colonies exhibiting phosphate-solubilizing zones were selected and streaked onto LB agar plates. This process was repeated three times to obtain pure cultures. The obtained pure cultures were then inoculated onto phosphate-solubilizing medium plates, and the presence of phosphate-solubilizing zones was observed. This process was repeated three times to verify the stability of phosphate-solubilizing ability.

[0040] Conclusion: Figure 1 As shown, SPN5 has phosphorus-solubilizing ability, can grow stably on inorganic phosphorus solid culture medium, and hydrolyzes solid Ca3(PO4)2 to form hydrolysis zones.

[0041] The phosphorus-solubilizing medium is formulated as follows: Ca3(PO4)2 5.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.003 g, yeast extract 0.5 g, glucose 10 g, agar powder 20 g, distilled water 1000 mL, pH 7.0. Sterilize at 115 ℃ for 20 min.

[0042] The LB solid medium formulation is as follows: 10 g peptone, 5 g yeast extract, 10 g NaCl, 20 g agar powder, 1000 mL distilled water, pH 7.0. The liquid medium does not contain agar. Sterilize at 121℃ for 15 min.

[0043] Experimental Example 2: Identification of Pantotheca SPN5 Based on morphological, physiological, and molecular biological identification results, strain SPN5 was identified as... Pantoea It belongs to the category of microorganisms, and the specific identification steps are as follows: 2.1 Morphological identification (1) Colony morphology characteristics Streaking the bacterial culture onto LB solid medium plates and incubating at 30°C for 48 hours, then observing the colony morphology.

[0044] SPN5 colony morphology as follows Figure 2As shown, its single colonies are pale yellow, round and raised, with a smooth and moist surface, and a diameter of 1.5~3.0 mm.

[0045] (2) Identification of bacterial cell morphology Single colonies were collected from the pipette tip and placed in 30 μL of sterile deionized water. They were allowed to disperse naturally and then fixed by adsorption using a 200-mesh copper mesh. The colonies were stained with 1% sodium phosphotungstenate for 2 min. After blotting the staining solution with filter paper at the edge of the copper mesh, the morphology of the bacterial cells was observed using a Hitachi HT7700 transmission electron microscope.

[0046] Under a transmission electron microscope, SPN5 cells appear as short, rod-shaped organisms, approximately 0.8–1.2 × 1.2–2.5 μm in size, and possess flagella. Cell morphology is shown in [reference needed]. Figure 3 As shown.

[0047] 2.2 Physiological identification The physiological characteristics of SPN5 were identified using commercially available biochemical identification tubes and kits. Biochemical identification tubes for glucose, maltose, rhamnose, xylose, mannitol, inositol, sorbitol, melibiose, raffinose, citrate, calendula alcohol, arabinose, sucrose, fructose, inulin, lactose, galactose, oxidase, catalase, ornithine, lysine, urea, dynamic puncture, VP, nitrate, and gelatin liquefaction were purchased from Qingdao Haibo Biotechnology Co., Ltd., and Gram staining reagent kits were purchased from Beijing Solarbio Technology Co., Ltd. The procedures were performed according to the instructions. The identification results are shown in Table 1. Table 1. Some physiological characteristics of SPN5

[0048] Note: + indicates a positive reaction, - indicates a negative reaction.

[0049] Table 1 shows that Pantotheca SPN5 is Gram-negative; it can utilize glucose, maltose, rhamnose, mannitol, arabinose, fructose, and galactose, but cannot utilize xylose, inositol, sorbitol, melibiose, raffinose, citrate, calendula alcohol, sucrose, inulin, and lactose. It is positive for catalase and nitrate reduction reactions, but negative for oxidase, motility puncture, gelatin liquefaction, ornithine utilization, lysine utilization, urea utilization, VP reaction, and nitrate reduction reaction.

[0050] 2.3 Molecular biological identification The whole genome of SPN5 was sequenced using both the Illumina second-generation sequencing platform and the PacBio third-generation sequencing platform. Sequencing results showed that this strain contains two circular chromosomal DNA strands and two plasmid DNA strands, totaling 5,915,721 bp, with an average GC content of 52.95%. Specifically, chromosome A was 4,324,144 bp with a GC content of 53.42%; chromosome B was 1,078,765 bp with a GC content of 51.96%; plasmid A was 299,435 bp with a GC content of 50.37%; and plasmid B was 213,377 bp with a GC content of 52.01%.

[0051] Using software such as Glimmer (http: / / ccb.jhu.edu / software / glimmer / index.shtml), tRNAscan-SE v2.0 (http: / / trna.ucsc.edu / software / ), and Barrnap (https: / / github.com / tseemann / barrnap), the types, locations, and sequence information of coding sequences (CDS), tRNAs, and rRNAs in the genome were predicted. The prediction results showed that SPN5 contained 5,450 coding sequences (CDS), 80 tRNAs, and 22 rRNAs, including 7 16S rDNAs, 7 23S rDNAs, and 8 5S rDNAs.

[0052] The genomes of SPN5 chromosome A, chromosome B, plasmid A, and plasmid B are as follows: Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown in the diagram, the outermost ring indicates the genome size; the second and third rings represent the CDS on the positive and negative strands, with different colors indicating the functional classification of different COGs in the CDS; the fourth ring represents rRNA and tRNA; the fifth ring represents GC content, with the red portion indicating a region with a GC content higher than the genome-wide average, and the blue portion indicating a region with a GC content lower than the genome-wide average, with higher peak values ​​indicating a larger difference from the average GC content; the innermost ring represents the GC-Skew value, specifically calculated as GC / G+C.

[0053] The 16S rDNA sequence of SPN5 was extracted and compared with the NCBI database (https: / / www.ncbi.nlm.nih.gov) using BLAST. The results showed that SPN5 belongs to the genus *Ureaplasma*. Pantoea ) bacteria, and their relationship with the type strain Pantoea rodasiiLMG26273 (NCBI Registry Number: MLFP01000054) Enterobacter cancerogenus ATCC33241 (NCBI Registry Number: FYBA01000020) Pantoea leporis R (NCBI Registry Number: GCA_028048155.1_15) Pantoea rwandensis The sequence similarity of LMG26275 (NCBI accession number: MLFR01000061) was high, at 99.25%, 98.77%, 98.70%, and 98.50%, respectively.

[0054] The full-length 16S rDNA sequence of SPN5 is 1539 bp, as shown in SEQ ID NO:1.

[0055] After selecting and downloading 16S rDNA sequences with high sequence similarity to the tested strain from the NCBI database, a phylogenetic tree was constructed using the Neighbor-Joining method in Mega 5.0 software, with the bootstrap value set to 1000. The 16S rDNA phylogenetic tree of SPN5 is shown below. Figure 8 As shown. Phylogenetic tree analysis revealed that SPN5 is related to the model strain. Pantoea rodasii LMG26273 and Pantoea rwandensis LMG26275 are the most closely related and cluster together.

[0056] Download pattern strains from the NCBI database Pantoea rwandensis LMG26275 Pantoea rodasii DSM26611 and Enterobacter cancerogenus The full genome sequences of strains with high 16S rDNA sequence similarity to SPN5, such as ATCC33241, were compared using OAT software (version 0.93.1) according to the method of Lee et al. (Lee, I., Kim, YO, Park, SC, & Chun, J.(2015). OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 66: 1100-1103.). The calculation results showed that SPN5 and... Pantoea rwandensis LMG26275 Pantoea rodasii DSM26611 and Enterobacter cancerogenus The average nucleotide similarity of ATCC33241 was 93.10%, 85.08%, and 73.05%, respectively. Its average nucleotide similarity with other reference strains was also below the 95% interspecies threshold. Based on strain morphology, physiological and biochemical characteristics, 16S rDNA sequence similarity, and phylogenetic analysis, SPN5 was preliminarily identified as belonging to the genus *Panthera*. Pantoea Potential new species of bacteria within.

[0057] The calculated average nucleotide identity (ANI) value of the whole genome sequence of strains with high similarity to SPN5 16S rDNA sequence is as follows: Figure 9 As shown.

[0058] Experimental Example 3: Salt Concentration, pH, and Temperature Tolerance Tests of Pantothenia glutinosa SPN5 (1) Salt concentration tolerance test Test method: LB medium was prepared, and the NaCl concentration was adjusted to final concentrations (w / v) of 1%, 3%, 5%, 7%, and 9%. Bacterial suspensions were inoculated into each of the above concentration gradients at an inoculum rate of 1% (v / v). Each treatment was replicated in triplicate. After incubation at 30℃ and 220 rpm for 24 h with shaking, OD was measured. 600 The absorbance value was measured to detect the growth status of the strain.

[0059] Result: As Figure 10 As shown, the SPN5 strain can grow well when the NaCl concentration is in the range of 1% to 3%.

[0060] The basic formulation of the LB medium is as follows: 10 g peptone, 5 g yeast extract, NaCl concentration adjusted according to the requirements of the test experiment, 1000 mL distilled water, pH 7.0. Sterilize at 121℃ for 15 min.

[0061] (2) pH tolerance test Test method: Adjust the pH of the culture medium using 0.1 mol / L sodium hydroxide or 0.1 mol / L hydrochloric acid to prepare LB medium with a pH of 2.0–10.0. Filter the medium through a 0.22 μm sterile membrane to avoid pH changes caused by high temperature. Inoculate the activated bacterial solution at a rate of 1% (v / v) into each of the above pH gradients of LB medium, with three replicates per gradient. Incubate at 30°C with constant temperature shaking at 200 rpm for 24 h. OD 600 The absorbance value was measured to detect the growth status of the strain.

[0062] Conclusion: Figure 11 As shown, the SPN5 strain can grow well in the pH range of 5.0 to 9.0.

[0063] The basic formulation of the LB medium is as follows: 10 g peptone, 5 g yeast extract, 10 g NaCl, and 1000 mL distilled water. The pH value was adjusted according to the requirements of the test experiment, and sterilization was performed by filtration.

[0064] Experiment Example 4: Growth-promoting ability test of Pantotheca SPN5 4.1 Identification of potassium-solubilizing ability of Pantothenia glutinosa SPN5 Test method: Pick a single colony from the preservation plate and inoculate it into a test tube containing 3 mL LB liquid medium. Incubate at 30°C and 220 rpm for 24 hours to activate the strain. Streak the activated strain onto a potassium-solubilizing medium (using insoluble potassium feldspar powder as the sole potassium source) and subculture three times. Stable subculturing proves that the strain has potassium-solubilizing ability. Figure 12 ).

[0065] Results: SPN5 was able to be stably passaged and grown on potassium-solubilizing medium, demonstrating its potassium-solubilizing ability.

[0066] The potassium-solubilizing medium is formulated as follows: Na₂HPO₄ 2.0 g, MgSO₄•7H₂O 0.5 g, FeCl₃ 0.005 g, CaCO₃ 0.1 g, sucrose 5.0 g, potassium feldspar powder 1.0 g, agar powder 20 g, pH 7.0, distilled water 1000 mL, pH 7.0. Sterilize at 121℃ for 15 min. The potassium feldspar powder is soaked overnight in deionized water to remove free potassium, then dried for later use.

[0067] 4.2 Identification of nitrogen-fixing capacity of Pantotheca acuminata SPN5 Test method: Single colonies were picked from the preservation plate and inoculated into test tubes containing 3 mL LB liquid medium. The colonies were activated by incubation at 30°C and 220 rpm for 24 hours. The activated strains were then streaked onto nitrogen-free medium (Assab medium) plates for three consecutive subcultures. Stable subculturing indicates that the strain has nitrogen-fixing ability. Figure 13 ).

[0068] Results: SPN5 was able to be stably passaged and grown on nitrogen-free medium, and it has nitrogen-fixing ability.

[0069] The nitrogen-free culture medium (Assab medium) has the following formula: K₂HPO₄ 0.2 g, NaCl 0.2 g, CaCO₃ 2.0 g, mannitol 10 g, CaSO₄ 0.1 g, MgSO₄ 0.2 g, agar powder 20 g, distilled water 1000 mL, pH 7.0. Sterilize at 115℃ for 20 min.

[0070] 4.3 Determination of Iron Production Capacity Test method: Pick a single colony from the preservation plate and inoculate it into a test tube containing 3 mL LB liquid medium. Incubate at 30 ℃ and 220 rpm for 24 hours to activate the colony. Take 0.5 μL of the activated bacterial solution and inoculate it onto solid CAS detection medium. After incubation at 30 ℃ for 48 h, observe whether the blue color around the colony fades and forms a halo. Figure 14 ).

[0071] Conclusion: Figure 14 As shown, strain SPN5 can competitively bind to Fe in the CAS detection plate complex. 3+ A faded halo forms around the colony.

[0072] The formulation and preparation steps of the CAS detection culture medium are as follows: Prepare the following two solutions respectively: ① 10 g peptone, 5 g yeast extract, 10 g NaCl, 24.27 g Na2HPO4·12H2O, 5.91 g NaH2PO4·2H2O, 0.75 g KH2PO4, 40 g agar, 1000 ml deionized water, pH 6.8. Sterilize at 121℃ for 20 min.

[0073] ② Solution A: Dissolve 60.5 mg CAS in 50 mL of deionized water, and add 10 mL of FeCl3 solution (containing 1 mM FeCl3 and 10 mM HCl); Solution B: Dissolve 72.9 mg HDTMA (hexadecyltrimethylammonium bromide) in 40 mL of deionized water; Slowly pour Solution A along the beaker wall into Solution B to obtain the CAS blue detection solution. Sterilize at 121℃ for 20 min.

[0074] When solutions ① and ② are heated and cooled to approximately 60°C, they are mixed in equal volumes and shaken thoroughly to obtain the CAS detection culture medium.

[0075] 4.4 Determination of Cellulase Production Capacity Test method: After transferring the strain to cellulase-producing screening medium plates, incubate at 30℃ for 48 h, scrape off the colonies, add 0.25% Congo red aqueous solution to cover the entire medium, stain for 30 min, and then decolorize with 1 M NaCl aqueous solution for 10 min. Observe whether hydrolysis zones appear.

[0076] Conclusion: Figure 15 As shown, a hydrolysis zone is visible at the original growth location (center of the plate), indicating that SPN5 has the ability to produce cellulase.

[0077] The cellulase screening medium formula is as follows: 5 g yeast extract, 10 g peptone, 5 g NaCl, 15 g sodium carboxymethyl cellulose (CMC-Na), 20 g agar powder, 1000 ml deionized water, pH 7.2~7.4. Sterilize at 121℃ for 15 min.

[0078] 4.5 Determination of IAA (Indoleacetic Acid) Production Capacity Test method: 0, 5, 10, 15, 20, and 25 μg / mL standard solutions were prepared using analytical grade IAA (indoleacetic acid). Each gradient standard solution was thoroughly mixed with an equal volume of Salkowski reagent and allowed to stand in the dark for 20 min. Distilled water was used as a blank control. The absorbance at 530 nm was measured to plot a standard curve. After bacterial activation, the strain was inoculated at a 1% (v / v) inoculum into liquid medium containing a final concentration of 0.5 g / L L-tryptophan NA. The culture was incubated at 30℃ and 200 rpm for 24 h. The fermentation broth was centrifuged at 5000 rpm for 3 min. The supernatant was collected, and an equal volume of Salkowski reagent was added. The mixture was allowed to stand in the dark for 20 min, and the absorbance at 530 nm was measured using a spectrophotometer. The blank control was the uninoculated solution. The IAA concentration in each test tube was calculated based on the standard curve. Figure 16 ).

[0079] Conclusion: Figure 10 As shown, the SPN5 culture supernatant can undergo a colorimetric reaction with the IAA detection reagent. The IAA content in the SPN5 culture medium under the above conditions was determined to be 41.33 ± 1.91 μg / mL.

[0080] The Salkowski reagent formula is as follows: FeCl3·6H2O 1.015 g, ddH2O 250 mL, H2SO4 150 mL. After preparation, the reagent should be stored away from light.

[0081] The formulation and preparation steps of the liquid culture medium with a final concentration of 0.5 g / L L-tryptophan NA are as follows: ①NA liquid culture medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1000 mL distilled water, pH 7.0, 121 ℃, sterilize for 20 min.

[0082] ②L-Tryptophan solution (4g / L): Weigh 0.2 g L-Tryptophan, dissolve it in 50 mL of deionized water, filter it through a 0.22 μm filter membrane for sterilization and storage.

[0083] ③ Add the solution obtained in ② to the culture medium obtained in ① until the final concentration of L-tryptophan solution is 0.5 g / L, which is the required culture medium.

[0084] 4.6 Ammonia production capacity determination Test method: The activated strain was inoculated into LB liquid medium at a 1% (v / v) inoculum and cultured at 30℃ and 200 rpm for 2 days. After centrifuging the fermentation broth at 5000 rpm for 3 min, 0.9 mL of the supernatant was taken, and 0.1 mL of Nessler's reagent was added. The presence or absence of a yellowish-brown precipitate was observed.

[0085] Conclusion: Figure 17 As shown, the fermentation supernatant of SPN5 can form a yellowish-brown precipitate with Nessler's reagent, while no precipitate is formed in the control, indicating that SPN5 has the ability to produce ammonia.

[0086] The LB liquid culture medium formula is as follows: 10 g peptone, 5 g yeast extract, 10 g NaCl, 1000 mL distilled water, pH 7.0. Sterilize at 121℃ for 15 min.

[0087] The Nessler's reagent is formulated as follows: 16.0 g of sodium hydroxide is dissolved in 50 ml of water and cooled to room temperature. 7.0 g of potassium iodide and 10.0 g of mercuric iodide are weighed, dissolved in water, and slowly added to the above 50 ml sodium hydroxide solution under stirring. The solution is then diluted with water to 100 mL.

[0088] Example 6: Determination of the growth-promoting ability of Pantotheca SPN5 on lettuce Inoculate 1% of the culture medium into LB liquid medium and incubate at 30°C and 200 rpm for 1-2 days. Centrifuge at 5000 rpm for 5 min, discard the supernatant, wash the bacterial pellet twice with sterile water, and dilute to 1.0 × 10⁻⁶. 8 Approximately CFU / mL.

[0089] The lettuce variety used was Italian lettuce, and the potting soil was horticultural potting mix. Lettuce seeds were placed on a 9cm plate lined with moistened filter paper and left to germinate for 2 days in the dark at room temperature. Germinated seeds were then transferred to pots filled with horticultural potting mix and cultured at 22℃ for 7 days under a 16h / 8h light cycle before root drenching. Each plant received 5 mL of the nasal solution, while the control group received an equal volume of sterile water for root drenching.

[0090] After culturing at 22℃ for 25 days with a 16 h / 8 h photoperiod, the number of cotyledons was counted, and the chlorophyll content of the leaves was measured using a SPAD-502Plus chlorophyll meter (Konica Minolta, Inc. Japan). The soluble sugar content of the leaves was determined by the anthrone colorimetric method. The fresh weight of the whole plant was measured by gravimetric method, and the fresh weight of the aboveground and underground parts of the plant was measured separately after the plant was cut at the rhizome. The aboveground and underground tissues of the plant were dried in an oven at 75℃ to constant weight and then measured by dry weight. Figure 18 ).

[0091] The effects of SPN5 strain on lettuce biomass and growth are shown in Table 2.

[0092] SPN5 promoted plant growth, with plant height, stem diameter, and cotyledon number increasing by 12.52%, 31.33%, and 9.93%, respectively, compared to the control.

[0093] SPN5 increased plant biomass, with the whole plant fresh weight, aboveground fresh weight, and underground fresh weight of the treated plants increasing by 32.73%, 34.05%, 23.13%, 45.45%, and 33.33%, respectively, compared to the control.

[0094] SPN5 also improves the quality of lettuce, with its soluble sugar content significantly increasing by 22.61% compared to the control.

[0095] SPN5 promotes photosynthesis in lettuce, and the chlorophyll content of lettuce leaves treated with it increased by 5.58% compared with the control.

[0096] Table 2 Effects of SPN5 strain on lettuce biomass and growth

[0097] Note: Different letters in the same column indicate significant differences between treatments (p < 0.05).

[0098] Experiment Example 7: Determination of the growth-promoting ability of Pantotheca SPN5 on rubber tissue culture seedlings 6.1 Rubber seedling pot experiment Test method: The rubber seedlings used were tissue culture seedlings of the Thermo-Research 7-33-97 variety, provided by the Hainan Natural Rubber New Planting Material Innovation Base. The potting soil was local red soil from Danzhou. The pots had a base diameter, top diameter, and height of 12 cm, 18 cm, and 32 cm, respectively, and contained approximately 5 L of soil. Healthy, uniformly growing rubber tissue culture seedlings were selected and planted into the pots.

[0099] Four treatment groups were set up: CK control (fertilizer applied), T1 (bacterial solution treatment + fertilizer applied), T2 (bacterial solution treatment + fertilizer reduced by 20%), and T3 (bacterial solution treatment + fertilizer reduced by 20%). The amount of fertilizer per plant (pot) for each treatment is shown in Table 3. Each treatment was replicated in four replicates, with eight seedlings per replicate.

[0100] Table 3 Experimental setup and fertilizer application rate per seedling

[0101] The strain was inoculated into LB liquid medium at a 1% (v / v) inoculum and cultured at 200 rpm and 30°C for 48 h. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were washed and resuspended with sterile water. The bacterial suspension was then diluted with sterile water to a concentration of 1.0 × 10⁻⁶. 8 The concentration of the bacterial suspension is approximately CFU / mL. Root irrigation was performed on rubber seedlings at weeks 1, 3, and 9 after transplanting. 200 mL of the prepared bacterial suspension was applied to the soil around the roots of each seedling. The control group received an equal volume of sterile water. The pot experiment lasted 150 days.

[0102] The LB liquid culture medium formula is as follows: 10 g peptone, 5 g yeast extract, 10 g NaCl, 1000 mL distilled water, pH approximately 7.0. Sterilize at 121℃ for 15 min.

[0103] 6.2 Measurement of Rubber Seedling Growth Indicators Test method: Before and after the experiment, the height and diameter at ground level of the rubber seedlings were measured, and the growth values ​​of plant height and diameter at ground level were calculated. Plant height was the height from the soil surface to the main stem growth point, and the growth value was calculated as: final plant height - initial plant height. Diameter at ground level was the diameter of the seedling stem approximately 2.0 cm from the soil surface, and the growth value was calculated as: final diameter at ground level - initial diameter at ground level. The growth rate of diameter at ground level was calculated as: (diameter at ground level growth / initial diameter at ground level) × 100%.

[0104] Before the experiment ended, the chlorophyll content of the leaves was measured using a SPAD-502Plus chlorophyll meter (Konica Minolta, Inc. Japan). After the experiment, the rubber seedlings were dug up, washed, and brought back to the laboratory to be weighed. The seedlings were then cut at the root collar, and the fresh weight of the above-ground and underground parts was measured separately. The seedlings were dried at 85℃ to constant weight, and the dry weight of the above-ground and underground parts was measured.

[0105] 6.3 Results of the test on the growth-promoting ability of rubber tissue culture seedlings As shown in Tables 4 and 5, SPN5 promoted the growth and biomass of rubber tissue culture seedlings, and even with reductions in fertilizer application of 20% and 40%, it still increased seedling height and total plant biomass. SPN5 is particularly suitable for promoting rubber growth and biomass under low fertility conditions. With a 40% reduction in fertilizer application, the SPN5 treatment increased seedling height by 79.22%, total plant fresh weight by 23.40%, aboveground fresh weight by 30.68%, and underground fresh weight by 10.29% compared to the control (Tables 4 and 5). Figure 19 ).

[0106] As shown in Table 4, SPN5 promoted the growth of rubber tissue culture seedlings. The plant height growth values ​​under the conditions of applying chemical fertilizer, reducing chemical fertilizer by 20%, and reducing chemical fertilizer by 40% were 33.20%, 13.07%, and 79.22% higher than the control, respectively.

[0107] Table 4 Effects of bacterial strains on the growth of rubber tissue culture seedlings

[0108] Note: CK: Control, fertilizer applied; T1: SPN5 bacterial solution treatment, fertilizer applied; T2: SPN5 bacterial solution treatment, fertilizer application reduced by 20%; T3: SPN5 bacterial solution treatment, fertilizer application reduced by 40%; Different letters in the same column represent significant differences between treatments (p < 0.05).

[0109] As shown in Table 5, SPN5 increased the biomass of rubber tissue culture seedlings, primarily by increasing the aboveground biomass. Under conditions of combined application of chemical fertilizer, 20% reduction in chemical fertilizer application, and 40% reduction in chemical fertilizer application, the fresh weight of the aboveground parts of the plants increased by 20.61%, 12.65%, and 30.68% compared to the control, respectively, while the fresh weight of the whole plant increased by 6.94%, 5.29%, and 23.40% compared to the control, respectively. Under the condition of 40% reduction in chemical fertilizer application, the fresh weight of the underground parts of the rubber seedlings treated with SPN5 increased by 10.29% compared to the control. In addition, SPN5 increased the chlorophyll content of the leaves of rubber tissue culture seedlings. Under conditions of combined application of chemical fertilizer, 20% reduction in chemical fertilizer application, and 40% reduction in chemical fertilizer application, the chlorophyll content of the leaves increased by 6.34%, 4.56%, and 5.22% compared to the control, respectively.

[0110] Table 5 Effects of bacterial strains on the biomass of rubber tissue culture seedlings

[0111] Note: CK: Control, fertilizer applied; T1: SPN5 bacterial solution treatment, fertilizer applied; T2: SPN5 bacterial solution treatment, fertilizer application reduced by 20%; T3: SPN5 bacterial solution treatment, fertilizer application reduced by 40%; Different letters in the same column represent significant differences between treatments (p < 0.05).

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Pantoea ( Pantoea strain SPN5, characterized in that, The strain SPN5 was classified and named Pantoea sp., accession number: GDMCC NO: 66466, was deposited at Guangdong Provincial Center for Microbial Culture Collection on June 4, 2024.

2. The strain SPN5 according to claim 1, characterized in that, The 16S rDNA sequence of strain SPN5 is shown in SEQ ID NO:

1.

3. Fermentation broth or bacterial suspension containing the strain SPN5 described in claim 1.

4. The application of the strain SPN5 as described in claim 1 or the bacterial suspension as described in claim 3, characterized in that, The application is any one of the following: A1) Its application in promoting plant growth; A2) Application in the preparation of products that promote plant growth; The plants mentioned include lettuce and rubber tree seedlings.

5. A bacterial agent comprising the strain SPN5 of claim 1.

6. A bio-phosphate fertilizer comprising the strain SPN5 of claim 1.

7. A bio-potassium fertilizer comprising the strain SPN5 of claim 1.

8. A bio-nitrogen fertilizer comprising the strain SPN5 of claim 1.

9. A soil conditioner comprising the strain SPN5 of claim 1.

10. A method for promoting the growth of lettuce or rubber tree seedlings, characterized in that, The strain SPN5 described in claim 1 was prepared into a bacterial suspension and applied to lettuce or rubber tree seedlings by root irrigation. The preparation method of the bacterial suspension is as follows: SPN5 strain was inoculated into LB liquid medium at a 1% inoculum size, cultured at 200 rpm and 30℃ for 48 h, then the fermentation broth was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, the bacterial precipitate was washed twice with sterile water, resuspended, and diluted with sterile water to a bacterial concentration of 1.0 × 10⁻⁶. 8 cfu / mL is the bacterial suspension used.

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