Plant growth-promoting rhizobacterium nfb10 and application thereof

Abstract

The present application relates to the field of microbial technology, and specifically provides a plant growth promoting rhizobacteria NFB10 and its application. The strain is isolated from the rhizosphere soil of rubber tree in Hainan province, and is a potential new species of Pseudomonas. The similarity of 16S rDNA sequence of the strain to similar strains is higher than 99.8%, but the average nucleotide identity of whole genome between species is lower than the inter-species threshold of 95%. The strain has the abilities of phosphorus solubilization, potassium solubilization and nitrogen fixation, and can produce indole acetic acid, cellulase, amylase and ammonia and other growth promoting substances. The strain grows well in the salt concentration of 3% or less and the pH range of 5.0-9.0. Experiments show that the strain can significantly improve the germination rate, root length and sprout length of wheat seeds, and has obvious promoting effects on the plant height, ground diameter, biomass and chlorophyll content of the leaves of the seedlings of Hevea brasiliensis. The strain can be used for preparing plant growth promoting bacteria agents, and is suitable for green cultivation and yield improvement of rubber trees and other crops.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a plant rhizosphere growth-promoting bacterium NFB10 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 applicable to 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. Against this backdrop, there is an urgent need to find microbial solutions that can ensure crop nutrient supply and promote healthy crop growth through multiple biological pathways even with lower chemical fertilizer inputs. 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 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 fertilizer application, this invention provides a strain of Pseudomonas (…). PseudomonasThe potential new species NFB10 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 Pseudomonas ( ). Pseudomonas NFB10 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. Telephone: 020-37656629. It is classified and named as follows: Pseudomonas sp., with accession number GDMCC NO: 67523, accession date December 24, 2025, 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 24 hours on LB solid medium, single colonies are translucent to white, round and raised, with a smooth and moist surface, and a diameter of 1.0~2.0 mm; the bacteria are rod-shaped, about 0.4~0.7×0.8~1.6 μm in size, have flagella, and are Gram-negative.

[0009] 2. Physiological characteristics: It can utilize glucose, maltose, xylose, fructose, and citrate, but cannot utilize rhamnose, mannitol, and other carbon sources; it is positive for oxidase, catalase, ornithine utilization, lysine utilization, dynamic puncture, gelatin liquefaction, and nitrate reduction reactions, and negative for urea utilization and VP reaction; it has the ability to produce amylase and cellulase.

[0010] 3. Molecular biological characteristics: Whole genome sequencing was performed using Illumina and PacBio platforms. The genome consists of a single circular chromosomal DNA strand, 5,599,696 bp in size, with a GC content of 62.18%. It contains 5,019 coding sequences (CDS), 77 tRNAs, and 22 rRNAs (including 7 16S rDNAs). The full-length 16S rDNA sequence is 1,532 bp (as shown in SEQ ID NO:1). The average nucleotide identity (ANI) with closely related strains is below the 95% threshold for interspecies division, indicating that it is a potential new species of Pseudomonas.

[0011] 4. Environmental tolerance: It can grow well in the range of NaCl concentration of 1%~3% and pH 4.0~9.0, and is suitable for most farmland soil environments.

[0012] (III) Growth-promoting function of strain NFB10 Phosphorus solubilization function: It can hydrolyze insoluble phosphorus in the soil and convert it into soluble phosphorus for plant absorption; Potassium-degrading function: It can decompose insoluble potassium compounds (such as potassium feldspar) and release soluble potassium ions; Nitrogen fixation function: It can convert nitrogen in the air into ammonia nitrogen that can be used by plants; Plant hormone production: Indoleacetic acid (IAA) can be produced in L-tryptophan-containing medium, with a yield of 19.64±0.64 μg / mL; Ammonia production function: The fermentation supernatant can react with Nessler's reagent to form a yellowish-brown precipitate, which indicates that it has the ability to produce ammonia; Produces hydrolytic enzymes: It can produce amylase and cellulase, which decompose soil organic residues and improve soil structure.

[0013] (iv) Application of strain NFB10 1. Enhance wheat seed germination: After disinfecting the wheat seeds, use a solution with a concentration of 1.0 × 10⁻⁶. 8 Soaking seeds in a CFU / mL NFB10 bacterial suspension for 1 hour followed by constant temperature incubation at 30℃ can increase seed germination rate by 13.72%, root length by 78.57%, and shoot length by 31.73%.

[0014] 2. To promote the growth of rubber tree seedlings: Rubber tissue culture seedlings were treated with root irrigation, with 200 mL of 1.0×10⁻⁶ solution applied at the 1st, 3rd, and 9th weeks after transplanting. 8 After 150 days of continuous use of CFU / mL bacterial suspension, the seedling height growth value increased by 69.41%, the ground diameter growth value increased by 28.43%, the whole plant fresh weight increased by 13.26%, and the leaf chlorophyll content increased by 7.31%.

[0015] 3. Soil improvement: As the core microbial strain of soil conditioner, it enhances soil fertility by activating insoluble nutrients in the soil, decomposing organic matter, and regulating the rhizosphere microenvironment, making it suitable for agricultural production under conditions of low fertility or reduced application of chemical fertilizers.

[0016] 4. Fertilizer preparation: As an active ingredient in bio-phosphate fertilizer, bio-potassium fertilizer, and bio-nitrogen fertilizer, it directly provides nutrients to plants, solves the problem of "nitrogen deficiency, phosphorus deficiency, and potassium deficiency" and rapidly increases yield and replenishes nutrients.

[0017] The beneficial effects of this invention are: (1) The Pseudomonas NFB10 provided by this invention is a potential new species of Pseudomonas, which fills the gap in the resources of native growth-promoting bacteria for rubber trees and enriches the germplasm bank of probiotic microorganisms for tropical crops. (2) The strain has comprehensive growth-promoting functions, integrating functions such as phosphorus solubilization, potassium solubilization, nitrogen fixation, hormone production, and hydrolytic enzyme production. It can enhance crop growth potential in multiple dimensions and has a significant growth-promoting effect. (3) The strain has strong environmental adaptability, a wide range of salt and acid tolerance, high colonization efficiency, stable functional expression, and is suitable for different farmland soil conditions. (4) The application method is simple (seed soaking, root irrigation), which can reduce the amount of chemical fertilizer used, reduce agricultural production costs, reduce environmental pollution, promote the green and sustainable development of rubber tree, wheat and other crop planting industries, and has broad application prospects. Attached Figure Description

[0018] Figure 1 The colony morphology of NFB10 on nitrogen-free medium.

[0019] Figure 2 The colony morphology of strain NFB10 on LB medium.

[0020] Figure 3 The cell morphology of strain NFB10 under a transmission electron microscope.

[0021] Figure 4 This is a circumscribed map of the chromosomes of strain NFB10.

[0022] Figure 5 Phylogenetic tree of NFB10 16S rDNA.

[0023] Figure 6 A heatmap showing the calculated average nucleotide identity (ANI) value for the entire genome sequence.

[0024] Figure 7 The results show the salt (NaCl) concentration tolerance of strain NFB10.

[0025] Figure 8 The results show the pH tolerance test results for strain NFB10.

[0026] Figure 9 The phosphorus solubilization effect of strain NFB10 on phosphorus-solubilizing medium.

[0027] Figure 10 The colony morphology of NFB10 on potassium-solubilizing medium.

[0028] Figure 11 The results show the cellulase production of strain NFB10.

[0029] Figure 12 The results show the amylase production of strain NFB10.

[0030] Figure 13 The results of IAA (indoleacetic acid) production by strain NFB10 are shown on the left. The left side represents the control treatment, and the right side represents the NFB10 bacterial culture treatment.

[0031] Figure 14The results show the ammonia production capacity of strain NFB10. The left side represents the control treatment, and the right side represents the NFB10 bacterial solution treatment.

[0032] Figure 15 The effect of NFB10 bacterial suspension on improving the germination rate of wheat seeds and promoting root and shoot growth is shown on the left, with the control treatment on the left and the NFB10 bacterial suspension treatment on the right.

[0033] Figure 16 The NFB10 strain has a growth-promoting effect on rubber seedlings. The left side is the control, and the right side is the NFB10 treatment. Detailed Implementation

[0034] 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.

[0035] Example 1: Isolation and screening of Pseudomonas NFB10 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 nitrogen-free medium (Assab medium) plates, with three replicates per gradient. After incubation upside down at 30°C for 3–5 days, single colonies were selected and streaked onto nitrogen-free medium plates. This process was repeated three times to verify the stability of nitrogen-fixing capacity.

[0036] Results: NFB10 was able to be stably passaged and grown on nitrogen-free medium, and it had nitrogen-fixing ability. Figure 1 ).

[0037] 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.

[0038] Example 2 Identification of NFB10 Based on morphological, physiological, and molecular biological identification results, strain NFB10 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 24 hours, then observing the colony morphology.

[0039] NFB10 colony morphology as follows Figure 2 As shown, its single colonies are translucent to white, round and raised, with a smooth and moist surface, and a diameter of 1.0 to 2.0 mm.

[0040] (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.

[0041] Under a transmission electron microscope, NFB10 cells are rod-shaped, measuring approximately 0.4–0.7 × 0.8–1.6 μm, and possess flagella. Cell morphology is shown in [reference needed]. Figure 3 As shown.

[0042] 2.2 Physiological identification The physiological characteristics of NFB10 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 NFB10

[0043] Note: + indicates a positive reaction, - indicates a negative reaction.

[0044] Table 1 shows that Pseudomonas NFB10 is Gram-negative; it can utilize glucose, maltose, xylose, fructose, and citrate, but cannot utilize rhamnose, mannitol, inositol, sorbitol, melibiose, raffinose, calendula alcohol, arabinose, sucrose, inulin, lactose, and galactose. It is positive for oxidase, catalase, ornithine utilization, lysine utilization, motility puncture, gelatin liquefaction, and nitrate reduction reactions, but negative for urea utilization and VP reaction.

[0045] 2.3 Molecular biological identification The whole genome of NFB10 was sequenced using both the second-generation sequencing platform Illumina and the third-generation sequencing platform PacBio. Sequencing results showed that the strain contains one circular chromosomal DNA strand, 5,599,696 bp in size, with a GC content of 62.18%.

[0046] 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 NFB10 contains 5,019 coding sequences (CDS), 77 tRNAs, and 22 rRNAs, including 7 16S rDNAs, 7 23S rDNAs, and 8 5S rDNAs. The chromosome circle diagram of the NFB10 strain genome is shown below. Figure 4 As shown.

[0047] The 16S rDNA sequence of NFB10 was extracted and compared with the NCBI database (https: / / www.ncbi.nlm.nih.gov) and EZBioCloud data (www.ezbiocloud.ne) using BLAST. The results showed that NFB10 belongs to the genus *Pseudomonas*. Pseudomonas Bacteria, and their strains Pseudomonas juntendi BML3 (NCBI Registry Number: NR180457.1) and Pseudomonas machongensis The sequence similarity of MH2 (NCBI accession number: NR199517.1) was the highest, at 99.93% and 99.93% respectively, with strain [missing information]. Pseudomonas taiwanensis DSM21245 (NCBI Registry Number: NR116172.1), Pseudomonas plecoglossicidaThe sequence similarity of FPC951 (NCBI accession number: NR024662.1) was high, at 99.80% and 99.80%, respectively.

[0048] The full-length 16S rDNA sequence of NFB10 is 1532 bp, as shown in SEQ ID NO:1.

[0049] After selecting and downloading 16S rDNA sequences with high homology to the tested strain sequences 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 NFB10 is shown below. Figure 5 As shown. Phylogenetic tree analysis indicates that NFB10 is related to strain... Pseudomonas asiatica RYU5 and Pseudomonas taiwanensis DSM21245 is the most closely related, and they cluster together.

[0050] Download from NCBI database Pseudomonas juntendi BML3, Pseudomonas machongensis MH2 Pseudomonas taiwanensis DSM21245 and Pseudomonas plecoglossicida The full genome sequences of strains with high 16S rDNA sequence similarity to FPC951 and NFB10 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 EvolMicrobiol. 66: 1100-1103.). The calculation results showed that NFB10 and... Pseudomonas plecoglossicida FPC951, Pseudomonas asiatica RYU5 Pseudomonas juntendi BML3, Pseudomonas machongensis MH2 and Pseudomonas taiwanensis The average nucleotide similarities of DSM21245 were 91.89%, 86.64%, 85.20%, 84.29%, and 85.26%, respectively. The average nucleotide similarity with other reference strains was also below the 95% interspecies threshold. Based on the strain's morphological and physiological characteristics, 16S rDNA sequence similarity, and phylogenetic analysis, NFB10 was preliminarily identified as belonging to the genus *Pseudomonas*. Pseudomonas Potential new species of bacteria.

[0051] The calculated average nucleotide identity (ANI) value of the whole genome sequence of strains with high similarity to the NFB10 16S rDNA sequence is as follows: Figure 6 As shown.

[0052] Example 3: Salt concentration and pH tolerance test of Pseudomonas NFB10 (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), with three replicates for each treatment. 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.

[0053] Result: As Figure 7 As shown, strain NFB10 can grow well when the NaCl concentration is in the range of 1% to 3%.

[0054] 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.

[0055] (2) pH tolerance test Test method: The pH of the culture medium was adjusted using 0.1 mol / L sodium hydroxide or 0.1 mol / L hydrochloric acid to prepare LB medium with a pH of 2.0 to 10.0. The medium was filtered through a 0.22 μm sterile filter membrane to avoid pH changes caused by high temperature. The activated bacterial solution was inoculated into the above-mentioned pH gradient LB medium at an inoculation rate of 1% (v / v), with 3 replicates for each gradient. After incubation at 30℃ and 200 rpm for 24 h, the absorbance value was measured by OD600 to detect the growth status of the strain.

[0056] Conclusion: Figure 8 As shown, strain NFB10 can grow well in the pH range of 4.0 to 9.0.

[0057] 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.

[0058] Example 4: Growth-promoting ability test of Pseudomonas NFB10 4.1 Identification of the phosphate-solubilizing ability of Pseudomonas NFB10 Test method: Pick a single colony from the preservation plate and inoculate it into a test tube containing 3 mL of LB liquid medium. Incubate at 30°C and 220 rpm for 24 hours to activate the strain. Subculture the activated strain three times on a phosphate-solubilizing medium plate. If the strain can be stably subcultured and grow and form a phosphate-solubilizing hydrolysis zone, it proves that the strain has phosphate-solubilizing ability.

[0059] Conclusion: Figure 9 As shown, NFB10 has phosphorus-solubilizing ability, can grow stably on inorganic phosphorus solid culture medium, and hydrolyzes solid Ca3(PO4)2 to form hydrolysis zones.

[0060] 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.

[0061] 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.

[0062] 4.2 Identification of potassium-solubilizing ability of Pseudomonas NFB10 Test method: Pick a single colony from the preservation plate and inoculate it into a test tube containing 3 mL of LB liquid medium. Incubate at 30°C and 220 rpm for 24 hours to activate the strain. Then, streak the activated strain on potassium-solubilizing medium (with insoluble potassium feldspar powder as the sole potassium source) three times. Stable subculturing proves that the strain has potassium-solubilizing ability.

[0063] Results: NFB10 was able to be stably passaged and grown on potassium-solubilizing medium, demonstrating potassium-solubilizing ability. Figure 10 ).

[0064] 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. Soak the potassium feldspar powder in deionized water overnight to remove free potassium, then dry before use.

[0065] 4.3 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.

[0066] Conclusion: Figure 11 As shown, a hydrolysis zone is visible at the original strain's growth location (center of the plate), indicating that NFB10 has the ability to produce cellulase.

[0067] 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.

[0068] 4.4 Amylase Production Capacity Determination Test method: After transferring the strain to the amylase-producing selection medium plate, incubate at 30°C for 48 h, scrape off the colonies, add Lugol's iodine solution to cover the entire medium, let stand for a while, and observe whether hydrolysis zones appear.

[0069] Conclusion: Figure 12 As shown, a hydrolysis zone is visible at the original strain's growth location (center of the plate), indicating that NFB10 has the ability to produce amylase.

[0070] The amylase-producing screening medium is formulated as follows: 5 g yeast extract, 10 g peptone, 5 g NaCl, 10 g soluble starch, 20 g agar powder, 1000 ml deionized water, pH 7.2~7.4. Sterilize at 121℃ for 15 min.

[0071] 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 an uninoculated solution. The IAA concentration in each test tube was calculated based on the standard curve.

[0072] Conclusion: Figure 13 As shown, the NFB10 culture supernatant can undergo a colorimetric reaction with the IAA detection reagent. The IAA content in the NFB10 culture medium under the above conditions was determined to be 19.64 ± 0.64 μg / mL.

[0073] 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.

[0074] 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, sterilized at 121 ℃ for 20 min.

[0075] 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.

[0076] 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.

[0077] 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 of a yellowish-brown precipitate was observed.

[0078] Conclusion: Figure 14 As shown, the fermentation supernatant of NFB10 can form a yellowish-brown precipitate with Nessler's reagent, while no precipitate is formed in the control, indicating that NFB10 has the ability to produce ammonia.

[0079] 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.

[0080] 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.

[0081] Example 5: Determination of the ability of Pseudomonas NFB10 to promote wheat seed germination 5.1 Test method: Wheat seeds were soaked in 75% ethanol for 3 min, soaked in 5% sodium hypochlorite (NaClO) for 5 min, washed with sterile water 3 times, and then washed with sterile water for 2 hours, during which the water was changed at least 3 times to remove sodium hypochlorite.

[0082] 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 bacterial suspension used was approximately CFU / mL. Wheat seeds were soaked in the bacterial suspension for 1 hour, then transferred to 15 cm sterile Petri dishes lined with filter paper, 30 seeds per dish, and moistened with 5 mL of deionized water. The control group consisted of seeds treated with sterile water. Each group had 5 replicates, with 30 seeds per replicate. After incubation at 30℃ for 5 days, the germination rate was calculated, and the root and shoot lengths of the germinated seeds were measured. The germination rate was calculated as follows: Germination rate (%) = (Number of germinated seeds / Number of tested seeds) × 100%. Experimental data were analyzed using Excel and SPSS 25.0 statistical software for ANOVA and multiple comparisons.

[0083] 5.2 Test Results: As shown in Table 2, NFB10 bacterial suspension significantly improved wheat seed germination rate and promoted root and shoot growth. The germination rate, root length, and shoot length of the NFB10-treated group were increased by 13.72%, 78.57%, and 31.73%, respectively, compared to the control group. Figure 15 ).

[0084] Table 2 Effects of NFB10 strain on wheat seed germination

[0085] Note: Different letters in the same column indicate significant differences between treatments (p < 0.05).

[0086] Example 6: Determination of the growth-promoting ability of Pseudomonas NFB10 on rubber tissue culture seedlings 6.1 Rubber seedling pot experiment Test Method: The rubber seedlings used were tissue-cultured 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 were filled with approximately 5 L of soil. Healthy, uniformly growing rubber tissue-cultured seedlings were selected and planted into the pots. Each seedling was supplemented with 1.23 g of urea, 0.58 g of superphosphate, and 0.46 g of potassium chloride. Each treatment was replicated in four groups, with eight seedlings per replicate.

[0087] 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.

[0088] 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.

[0089] 6.2 Methods for determining growth indicators of rubber seedlings 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 value / initial diameter at ground level × 100%.

[0090] 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 entire rubber seedling was dug up, washed, and brought back to the laboratory to be weighed. The seedlings were then cut at the rhizome, 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.

[0091] 6.3 Results of the test on the growth-promoting ability of rubber tissue culture seedlings NFB10 promotes the growth and increases the biomass of rubber tissue culture seedlings. Figure 16 ).

[0092] As shown in Table 3, NFB10 treatment promoted the growth of rubber seedlings. The diameter growth of rubber tissue culture seedlings treated with NFB10 was 28.43% higher than that of the control, and the plant height growth was significantly higher by 69.41%.

[0093] Table 3 Effects of bacterial strains on the growth of rubber tissue culture seedlings

[0094] Note: Different letters in the same column indicate significant differences between treatments (p < 0.05).

[0095] As shown in Table 4, the NFB10 treatment increased the biomass of rubber seedlings. The total fresh weight, aboveground fresh weight, and aboveground dry weight of the NFB10-treated rubber tissue culture seedlings were significantly increased by 13.26%, 14.99%, and 22.06% compared with the control (CK), respectively, while the underground fresh weight and underground dry weight were increased by 10.49% and 8.39% compared with the control, respectively.

[0096] In addition, NFB10 can increase the chlorophyll content of plant leaves and enhance plant photosynthesis. The chlorophyll content of the whole leaves of rubber tissue culture seedlings treated with NFB10 increased by 7.31% compared with the control.

[0097] Table 4. Effects of bacterial strains on biomass and leaf chlorophyll content of rubber tissue culture seedlings

[0098] Note: Different letters in the same column indicate significant differences between treatments (p < 0.05).

Claims

1. A strain of Pseudomonas ( Pseudomonas strain NFB10, characterized in that, The strain NFB10 was classified and named Pseudomonas sp., accession number: GDMCC NO: 67523, was deposited at Guangdong Provincial Center for Microbial Culture Collection on December 24, 2025; the 16S rDNA sequence of strain NFB10 is shown in SEQ ID NO:

1.

2. Fermentation broth or bacterial suspension containing the strain NFB10 described in claim 1.

3. The application of the strain NFB10 as described in claim 1 or the bacterial suspension as described in claim 2, characterized in that, The application is any one of the following: A1) Application in improving the germination rate of wheat seeds; A2) Application in promoting root growth in wheat seeds; A3) Application in promoting the growth of wheat seed buds; A4) Application in promoting the growth of rubber tree seedling height; A5) Application in promoting the growth of rubber tree seedling diameter at ground level; A6) Application in promoting the increase of biomass in rubber tree seedlings; A7) Application in promoting the increase of chlorophyll content in the leaves of rubber tree seedlings.

4. A bacterial agent comprising the strain NFB10 of claim 1.

5. A bio-phosphate fertilizer comprising the strain NFB10 of claim 1.

6. A bio-potassium fertilizer comprising the strain NFB10 of claim 1.

7. A bio-nitrogen fertilizer comprising the strain NFB10 of claim 1.

8. A soil conditioner comprising the strain NFB10 of claim 1.

9. A method for improving the germination rate of wheat seeds, characterized in that, The strain NFB10 described in claim 1 is prepared into a bacterial suspension, and wheat seeds are soaked in the bacterial suspension. The preparation method of the bacterial suspension is as follows: Inoculate strain NFB10 into LB liquid medium at a 1% v / v inoculation rate, incubate at 200 rpm and 30℃ for 48 h, centrifuge the fermentation broth at 5000 rpm for 5 min, discard the supernatant, wash the bacterial precipitate twice with sterile water, resuspend, and dilute with sterile water to a bacterial concentration of 1.0 × 10⁻⁶. 8 cfu / mL is the bacterial suspension used.

10. A method for promoting the growth of rubber tree seedlings, characterized in that, The strain NFB10 described in claim 1 was prepared into a bacterial suspension and applied to rubber tree seedlings by root irrigation. The promotion of rubber tree seedling growth includes promoting the growth of rubber tree seedling height, promoting the growth of rubber tree seedling diameter, promoting the increase of rubber tree seedling biomass, and promoting the increase of chlorophyll content in rubber tree seedling leaves. The preparation method of the bacterial suspension is as follows: Inoculate strain NFB10 into LB liquid medium at a 1% v / v inoculation rate, incubate at 200 rpm and 30℃ for 48 h, centrifuge the fermentation broth at 5000 rpm for 5 min, discard the supernatant, wash the bacterial precipitate twice with sterile water, resuspend, and dilute with sterile water to a bacterial concentration of 1.0 × 10⁻⁶. 8 cfu / mL is the bacterial suspension used.

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