Pseudomonas rhizophila strain and application thereof

Abstract

The invention relates to the technical field of microbiology and agricultural biology, in particular to a Pseudomonas rhizophila strain 19C11, which has the functions of dissolving inorganic phosphorus and mineralizing organic phosphorus, can improve the content of available phosphorus in a culture system and soil, and can synergistically enhance the phosphorus absorption of plants with arbuscular mycorrhizal fungi, so that the phosphorus absorption rate of plants is increased, and the phosphorus absorption rate of plants is increased. The compound can be used for promoting plant growth, improving quality, preparing bio-fertilizer and the like.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and agricultural biotechnology, and in particular to a strain of Pseudomonas rhizophila and its applications. Background Technology

[0002] Phosphorus is an essential macronutrient for plant growth and development, playing a crucial role in energy metabolism, photosynthesis, cell division, and nucleic acid synthesis. However, the amount of available phosphorus in soil that can be directly absorbed and utilized by plants is usually low. Most phosphorus exists in the form of insoluble inorganic phosphate salts (such as tricalcium phosphate, aluminum phosphate, and ferric phosphate) or organic phosphorus compounds, resulting in generally low utilization rates of phosphate fertilizers by crops. Long-term, excessive application of chemical phosphate fertilizers not only wastes resources but also easily leads to problems such as soil structure degradation, eutrophication of water bodies, and ecosystem imbalance.

[0003] Phosphate-soliciting microorganisms (PSMs) can convert insoluble phosphorus into soluble phosphorus through biological processes, improving the efficiency of phosphorus absorption and utilization by plants, and have significant application potential in the fields of sustainable agricultural development and ecological restoration. Reported PSMs mainly include Pseudomonas, Bacillus, and Flavobacterium. These microorganisms typically release fixed phosphorus by secreting metabolic products such as organic acids, phosphatases, and polyphosphatases, thereby increasing the available phosphorus content in the soil. However, the phosphorus-soliciting effects and growth-promoting properties of existing PSMs vary in different ecological environments, and their activity and functional stability are greatly affected by environmental conditions, limiting their application in different crops and soil types.

[0004] With the development of molecular biology and genomics technologies, researchers have gradually utilized whole-genome sequencing and functional annotation to reveal the metabolic pathways and functional gene characteristics of phosphate-solubilizing microorganisms, elucidating their phosphate-solubilizing mechanisms and ecological adaptability at the gene level. This type of research provides a theoretical basis for the screening and precise application of highly efficient functional strains. However, current phylogenetic information on phosphate-solubilizing bacteria of the genus *Pseudomonas* remains relatively limited, especially regarding their functional gene composition and phosphate-solubilizing potential in specific plant rhizosphere environments, where in-depth analysis is still lacking.

[0005] Some plants are highly sensitive to soil phosphorus supply, forming unique rhizosphere microbial communities around their roots. Licorice (Glycyrrhiza uralensis Fisch.) is an important medicinal plant. Studies have shown that the rational use of functional bacteria with phosphorus-solubilizing capabilities can improve phosphorus nutrition and growth performance of licorice. However, there is currently a lack of highly efficient phosphorus-solubilizing strains specifically screened for the rhizosphere ecological environment of licorice.

[0006] Furthermore, arbuscular mycorrhizal fungi (AMF) are mycorrhizal fungi that are ubiquitous in the rhizosphere of plants, expanding the root absorption range and participating in plant carbon and phosphorus exchange. Previous studies have shown that some phosphate-solubilizing bacteria can interact with mycorrhizal fungi to promote plant phosphorus absorption and growth; however, research on the synergistic mechanisms within the rhizosphere system of medicinal plants is still limited, and stable and efficient strains for combined application are lacking.

[0007] Therefore, how to screen and analyze phosphorus-solubilizing strains with clear origins, stable functions, good growth-promoting potential, and synergistic effects with mycorrhizal fungi has become a key scientific and technological issue for improving the efficiency of phosphorus nutrient utilization in plants and reducing dependence on chemical phosphate fertilizers. Summary of the Invention

[0008] This invention aims to overcome the problems of unstable activity, poor adaptability, and limited growth-promoting effects of existing phosphorus-solubilizing microorganisms. It provides *Pseudomonas rhizophila* strain 19C11, which has a clear origin, stable function, and highly efficient phosphorus-solubilizing and growth-promoting properties. This strain can effectively dissolve insoluble inorganic phosphorus and mineralize organic phosphorus compounds, increasing the available phosphorus content in the soil and promoting plant root development and growth. Simultaneously, it can form a good symbiotic relationship with plant rhizosphere microorganisms, making it suitable for improving phosphorus nutrition in plants such as licorice (*Glycyrrhiza uralensis*).

[0009] This invention also provides the application of Pseudomonas rhizophila strain 19C11 in agricultural production, particularly including compound inoculants combined with arbuscular mycorrhizal fungi, providing a new technical approach for promoting plant growth and developing green agriculture in low-phosphorus environments.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] On the one hand, the present invention provides Pseudomonas rhizophila strain 19C11, which has the accession number CGMCC No.33985.

[0012] On the other hand, the present invention provides a plant growth promoter composition comprising the aforementioned Pseudomonas rhizophila strain 19C11.

[0013] In some embodiments, the composition further comprises a matrix and / or nutrient components.

[0014] In some embodiments, the composition is a liquid bacterial agent, a powdered bacterial agent, or a granular bacterial agent, preferably a suspension, bacterial powder, or a freeze-dried bacterial agent.

[0015] On the other hand, the present invention provides a plant growth promoter combination comprising the aforementioned Pseudomonas rhizophila strain 19C11 and arbuscular mycorrhizal fungi, preferably, the arbuscular mycorrhizal fungi being Rhizophagus irregularis AH01.

[0016] On the other hand, the present invention provides the use of the aforementioned Pseudomonas rhizophila strain 19C11, the above-described composition, or the aforementioned plant growth promoter combination in promoting plant growth, plant phosphorus uptake, and / or soil phosphorus activation.

[0017] In some implementation schemes, promoting plant growth includes: promoting an increase in plant biomass and promoting plant root growth.

[0018] In a specific implementation plan, the plant is licorice.

[0019] On the other hand, the present invention provides a method for promoting plant growth, plant phosphorus uptake, and / or soil phosphorus activation, comprising:

[0020] Apply the above-described composition of *Pseudomonas rhizophila* strain 19C11 or the above-described combination of plant growth promoters to the plant, the soil near the plant, or the soil of the plant to be planted.

[0021] In some implementations, the application methods include drip irrigation, irrigation, basal fertilizer application, fertigation, and root irrigation.

[0022] In the specific implementation plan, the application rate of the rhizotrophic Pseudomonas strain 19C11 is 0.5 × 10⁻⁶. 7 Up to 1×10 9 Cells / plant, preferably 9.6 × 10⁶ 8 Cells per plant.

[0023] The application rate of Rhizocystis heterophylla AH01 inoculant is 10-40g (containing approximately 600-2400 spores), preferably 30g (containing approximately 1800 spores).

[0024] In a specific implementation plan, the plant is licorice.

[0025] Compared with existing technologies, the present invention has the following beneficial effects: The *Pseudomonas rhizophilus* strain 19C11 of the present invention has significant phosphorus-solubilizing and mineralizing capabilities, effectively increasing the available phosphorus content in the culture system and soil. Inoculation with this strain can promote plant root development and increase biomass, improving plant phosphorus absorption capacity and soil phosphorus activation levels. Simultaneously, this strain can form a synergistic system with mycorrhizal fungi, further enhancing plant phosphorus absorption and growth performance. Using the formulation containing *Pseudomonas rhizophilus* strain 19C11 of the present invention can improve plant phosphorus nutrient utilization efficiency and reduce the use of chemical fertilizers (especially phosphate fertilizers).

[0026] The *Pseudomonas rhizogenes* strain 19C11 of this invention is functionally stable and suitable for farmland and medicinal herb cultivation systems in low-phosphorus or arid regions. It has good environmental adaptability and broad application prospects, and can provide important technical support for phosphorus reduction fertilization and ecological sustainable production in agriculture. Attached Figure Description

[0027] To more clearly illustrate the technical solution of the present invention, the accompanying drawings further illustrate the embodiments. The accompanying drawings are merely illustrative diagrams and are not intended to limit the scope of protection of the present invention.

[0028] Figure 1 Phylogenetic tree of strain 19C11 and its closely related strains.

[0029] Figure 2 This is a diagram of the genome structure of strain 19C11.

[0030] Figure 3 The in vitro phosphorus solubility of strains ZN6 and 19C11 is shown, where Figure A shows the effective phosphorus concentration measured in tricalcium phosphate medium; and Figure B shows the effective phosphorus concentration measured in calcium magnesium phytate medium.

[0031] Figure 4 The effects of inoculation with Pseudomonas rhizophila strain 19C11 on licorice growth and soil phosphorus content were shown. Figure A shows the aboveground biomass of licorice under different treatments; Figure B shows the root biomass of licorice under different treatments; and Figure C shows the available phosphorus concentration in the soil under different treatments.

[0032] Figure 5 The effect of co-inoculation of *Pseudomonas rhizophila* strain 19C11 with arbuscular mycorrhizal fungi on licorice growth is shown in Figure [Figure number missing].

[0033] Figure A shows the aboveground biomass of licorice under different treatments; Figure B shows the root biomass of licorice under different treatments; Figure C shows the root phosphorus content under different treatments.

[0034] Preservation Instructions

[0035] Taxonomous Pseudomonas rhizophila

[0036] Strain name: 19C11

[0037] Latin name: Pseudomonas rhizophila

[0038] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0039] Collection institution abbreviation: CGMCC

[0040] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0041] Deposit date: March 26, 2025

[0042] CGMCC Registration Number: 33985 Detailed Implementation

[0043] This invention provides a strain of Pseudomonas rhizophila, 19C11, which was deposited on March 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33985.

[0044] The genome of this strain has a single circular chromosome structure, with a total length of approximately 6.06 Mb and a GC content of 60.33%, containing 5380 open reading frames (ORFs) and 190 non-coding RNAs. The functional characteristics of the strain's genome include typical phosphorus-related gene clusters: pyrroloquinoline quinone (PQQ) biosynthesis genes pqqB, pqqC, pqqD, and pqqE; glucose dehydrogenase gene gcd; inorganic pyrophosphatase gene ppa; exonuclease gene ppx; alkaline phosphatase gene phoD; and phosphate transport system gene clusters pstA, pstB, pstC, and pstS, as well as the low-affinity transporter pit. These genes collectively participate in the inorganic phosphorus dissolution and organophosphorus mineralization processes, constructing a complete phosphorus metabolism regulatory system.

[0045] The strain also contains genes related to pyruvate metabolism and citric acid cycle pathways, which can produce active organic acids through carbon metabolism to promote the dissolution of insoluble phosphates; it contains genes related to flagella assembly, motility proteins and biofilm formation, giving it rhizosphere movement and colonization capabilities; and it also possesses a phosphatase secretion system and a polyphosphate degradation pathway, which can effectively release fixed phosphorus resources.

[0046] The *Pseudomonas rhizophila* strain 19C11 of this invention can be used alone in agricultural production to increase the available phosphorus content in the soil through biological action, promoting plant root development and biomass accumulation in both above-ground and below-ground parts of the plant. When applied in licorice cultivation, it can enhance the available phosphorus level in the soil and the efficiency of phosphorus absorption by plants. This strain can be used in combination with mycorrhizal fungi (such as arbuscular mycorrhizal fungi) to form a synergistic promoting system, further enhancing the absorption and utilization of phosphorus by plants.

[0047] The composition or formulation containing Pseudomonas rhizophila strain 19C11 in this invention can be a liquid bacterial agent, a powdered bacterial agent, or a granular bacterial agent; the liquid bacterial agent includes suspension or fermentation broth, and the powdered bacterial agent includes bacterial powder or freeze-dried bacterial agent.

[0048] Liquid microbial agents generally refer to microbial preparations that are suspended or dissolved in water or other solvents, and are commonly used for spraying, irrigation, etc. Liquid microbial agents are suitable for large-scale agricultural applications, especially when applied through drip irrigation, irrigation, spraying, etc. They can spread and disperse rapidly in farmland, making them suitable for large-area crops.

[0049] Powdered microbial agents are formulations in which microorganisms are converted into powder form through a drying process, which is generally convenient for storage and transportation. Powders are suitable for direct application to soil or mixing with other materials, and can provide a longer survival environment for microorganisms, but may require more precise control during application.

[0050] Granular microbial agents generally refer to microorganisms that are fixed into granules by a carrier (such as kaolin, humus, vermiculite, etc.) so that they can better contact the soil or plant roots and play a role in continuous release. They are released relatively slowly in the soil and are suitable for application in combination with soil or plant roots.

[0051] The plant growth promoter combination of Pseudomonas rhizophila strain 19C11 and arbuscular mycorrhizal fungi in this invention can be a liquid inoculant, a powder inoculant, or a granular inoculant; the liquid inoculant includes suspension or fermentation broth, and the powder inoculant includes inoculant powder or freeze-dried inoculant.

[0052] The arbuscular mycorrhizal fungus is preferably *Rhizophagus irregularis* AH01. Preferably, the plant growth promoter combination further includes a substrate and / or nutrient components.

[0053] The substrate can be kaolin, diatomaceous earth, talc, vermiculite, peat, or humus, etc., and the nutrient components can include nitrogen sources, phosphorus sources, potassium sources, trace elements, plant growth regulators, and other organic matter that promotes microbial growth. Specifically, the nitrogen source can be urea, ammonium nitrate, ammonia, etc.; the phosphorus source can be potassium dihydrogen phosphate, superphosphate, etc.; the potassium source can be potassium sulfate, potassium chloride, etc.; trace elements can include mineral elements such as iron, zinc, copper, molybdenum, and boron; and plant growth regulators can be gibberellins, cytokinins, indoleacetic acid, etc. The formulation can be prepared separately and then mixed before use, or the two inoculants can be applied separately to plants or soil as needed to improve plant phosphorus nutrient utilization, improve soil fertility, and promote plant growth.

[0054] Insoluble inorganic phosphorus compounds include tricalcium phosphate, aluminum phosphate, iron phosphate, and phosphorus-containing minerals.

[0055] Organophosphorus compounds include: calcium magnesium phytate, nucleic acids, phospholipids, and phytin, etc.

[0056] The method for promoting plant growth, plant phosphorus absorption and / or soil phosphorus activation in this invention includes applying the aforementioned Pseudomonas rhizophila strain 19C11 or the above-mentioned preparation to the plant, the soil near the plant or the soil of the plant to be planted.

[0057] In some implementation schemes, the application methods include drip irrigation, irrigation, basal fertilizer application, fertigation, root irrigation, etc.

[0058] In the specific implementation plan, the application rate of the rhizotrophic Pseudomonas strain 19C11 is 0.5 × 10⁻⁶. 7 Up to 1×10 9 Cells / plant, preferably 9.6 × 10⁶ 8 Cells per plant.

[0059] The application rate of Rhizocystis heterophylla AH01 inoculant is 10-40g (containing approximately 600-2400 spores), preferably 30g (containing approximately 1800 spores).

[0060] Available phosphorus concentration refers to the concentration of phosphorus that can be absorbed by plant roots in soil or water. Methods for determining available phosphorus concentration mainly include: sodium bicarbonate extraction method and molybdenum-antimony spectrophotometric method.

[0061] Soil conditioners are materials added to soil to improve its physical and / or chemical properties, as well as its biological activity, including microbial preparations that promote plant growth and improve soil environmental quality.

[0062] Example

[0063] The following general methods are used in the following embodiments:

[0064] I. Biomass Measurement

[0065] The dry weight method was used. Specifically, the above-ground parts of the licorice were cut off, the pot was inverted, and the roots were collected. The roots were rinsed with clean water and then dried with paper towels. Two licorice plants from each pot were considered as a biological replicate, and the weights of the above-ground parts and roots were recorded as fresh weights. 0.5 g of the mixed licorice root mixture was frozen in liquid nitrogen and stored at -80°C for RNA extraction. The remaining sample was placed in an oven and dried at 75°C for 72 hours until constant weight was achieved, and the dry weight (DW) was obtained.

[0066] II. Determination of Phosphorus Content in Roots

[0067] The dried root samples were pulverized in a ball mill (GT200) at 1600 rpm for 2 minutes to obtain root powder samples. Approximately 100 mg of root powder sample was accurately weighed and placed at the bottom of a dry digestion tube. 5 ml of nitric acid was added, and digestion was performed using a microwave digester (Mars5, CEM) according to the preset program. The digestion program was as follows: the temperature was increased to 120 °C over 8 minutes and held for 3 minutes; then increased to 160 °C over 11 minutes and held for 7 minutes; then increased to 180 °C over 8 minutes and held for 20 minutes. After digestion, the acid was removed by electrothermal extraction, and the sample was transferred to a 50 ml centrifuge tube. The volume was adjusted to 30 ml with ultrapure water, and the solution was filtered through a 0.45 μm filter membrane. The phosphorus content of the roots was determined using an ICP-OES instrument.

[0068] Example 1: Isolation and Identification of Strain 19C11

[0069] 1. Strains Isolation and Screening

[0070] Soil samples were collected from Minqin County, Gansu Province, specifically from the topsoil (0-25 cm). Uniformly germinating licorice seeds were selected and sown in plastic pots containing 600g of soil. The soil was then cultured in an artificial climate chamber under the following conditions: temperature 18-25℃, humidity 50%-60%, and a 14-hour photoperiod per day. After 30 days of growth, the licorice roots were harvested, homogenized, and diluted. The root tissue was cut into approximately 2 mm pieces, mixed, and 0.02g of root tissue was placed in a 1.5ml sterile centrifuge tube. 30ml of sterile phosphate buffer (pH=7) was added, and the root sample was placed on a plate shaker at 180rpm for 15 minutes at room temperature. This washing process was repeated three times. The roots were then homogenized using a sterile glass grinder; during homogenization, 200μl of sterile 10mM magnesium chloride solution was added to the centrifuge tube. Transfer the homogenate to a 50 ml centrifuge tube (containing 25 ml of 10 mM magnesium chloride solution), mix well, and let stand for 15 minutes. Set up three dilutions (2000x, 6000x, and 18000x), and dilute the root homogenate with 1 / 10 TSB medium. Take 160 μl of the sample dilution and aliquot it into 96-well cell culture plates, separating 45 cell culture plates for each dilution. Seal the culture plates and incubate at room temperature for 15-20 days. Based on the Poisson distribution, select the dilutions in which bacteria grow (approximately 30% or less of the wells show bacterial growth) for subsequent bacterial identification. Take samples from the wells with bacterial growth at the 6000x and 18000x dilutions, dip the samples into the plates, and transfer them to 1 / 2 TSB solid plates using the streak plate method. Incubate for 3-5 days at 28°C. Select single colonies and transfer them to new plates for further incubation. Repeat the purification culture three times. Based on the morphology and characteristics of the bacteria on the culture medium, preliminary classification and removal of duplicates were performed, and the purified bacteria were named 19C11.

[0071] 2. Species identification and phylogenetic tree analysis

[0072] To determine the taxonomic classification and genomic characteristics of strain 19C11, whole-genome sequencing and phylogenetic analysis were performed. Sequencing employed a whole-genome shotgun (WGS) strategy, constructing both Illumina short-read and PacBio long-read libraries. The Illumina library underwent paired-end sequencing (2 × 150 bp) using the NovaSeq 6000 platform; the PacBio library underwent single-molecule real-time sequencing (SMRT) using the Sequel II system. During genome assembly, de novo assembly was performed primarily using long-read data from the PacBio platform to obtain a continuous and complete genome sequence. This was then combined with high-precision short-read data from the Illumina platform, and Pilon software (v1.24) (https: / / github.com / broadinstitute / pilon?tab=readme-ov-file) was used for error correction and quality optimization to ensure the integrity and base accuracy of the assembled genome. Initial assembly was performed using HGAP software (https: / / github.com / marbl / canu) and CANU software (https: / / github.com / PacificBiosciences / pbbioconda) to complete contig assembly. After multiple rounds of correction, the complete genome was obtained.

[0073] Based on the assembled chromosome sequences, 20 closely related species were screened in the RefSeq database (https: / / www.ncbi.nlm.nih.gov / refseq / ?mod=article_inline) using fastANI software (v1.33) (https: / / github.com / ParBLiSS / FastANI / releases), and phylogenetic analysis was performed using the UBCG (Up-to-date Bacterial Core Gene) bacterial core gene set. The UBCG core gene set contains 92 single-copy genes that are conserved in most bacteria. A phylogenetic tree was constructed on the core genes of the samples using ubcg software (https: / / help.ezbiocloud.net / ubcg-users-manual / ). The results showed that strain 19C11 and the Pseudomonas rhizophila (GCF_003033885.1) branch share 91 common core genes (out of a total of 92 in UBCG), indicating that they are most closely related. Further analysis using the fastANI software (https: / / github.com / ParBLiSS / FastANI / releases) revealed an average nucleotide identity (ANI) value of 98.06% between strain 19C11 and *Pseudomonas rhizophila*, exceeding the same-species threshold of 95%. Based on the phylogenetic tree and ANI analysis results, strain 19C11 was confirmed to belong to *Pseudomonas rhizophila*.

[0074] Figure 1 A phylogenetic tree of strain 19C11 and related Pseudomonas species is presented, demonstrating the phylogenetic relationships between *Pseudomonas rhizophilus* 19C11 and related *Pseudomonas* strains. The phylogenetic tree was constructed based on the UBCG core gene set to support the taxonomic position of strain 19C11.

[0075] This strain was deposited on March 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33985 and accession name Pseudomonas rhizophila.

[0076] Example 2: Genome-wide functional annotation and phosphate-solubilizing feature analysis of Pseudomonas rhizophila strain 19C11

[0077] To further elucidate the genetic characteristics and phosphate solubilization mechanism of *Pseudomonas rhizogenes* strain 19C11, whole-genome functional annotation and metabolic pathway analysis were performed on this strain. The sequencing and assembly information of strain 19C11 has been described in Example 1.

[0078] Figure 2 The genome structure diagram of strain 19C11 shows the complete circular chromosome structure, functional region distribution, and location distribution characteristics of major phosphate-solubilizing genes of strain 19C11.

[0079] Functional annotation of the genome of strain 19C11 revealed a genome length of approximately 6.06 Mb, a GC content of 60.33%, and predicted 5380 open reading frames (ORFs) and 190 non-coding RNAs, including 16 rRNAs and 67 tRNAs. The functional annotation results indicate that this strain possesses key genes typically associated with inorganic phosphorus dissolution and organophosphorus mineralization.

[0080] Based on the gene function annotation results, functional genes directly related to phosphorus solubilization metabolism were extracted, as shown in Table 1.

[0081] Table 1. Phosphate-related genes of *Pseudomonas rhizotrophus* strain 19C11

[0082]

[0083] Among the detected genes, the pyrroloquinoline quinone (PQQ) biosynthesis gene clusters pqqB, pqqC, pqqD, and pqqE were detected, responsible for the formation of PQQ coenzymes; the glucose dehydrogenase gene gcd was detected, which catalyzes the oxidation of glucose to gluconic acid, thereby lowering the pH of the culture environment to promote the dissolution of insoluble inorganic phosphorus; the inorganic pyrophosphatase gene ppa and the exonuclease gene ppx were detected, participating in the degradation of polyphosphates; the alkaline phosphatase gene phoD was detected, which catalyzes the hydrolysis of organophosphorus esters; and the phosphate-specific transport system gene clusters pstA, pstB, pstC, and pstS, as well as the low-affinity transport gene pit, were detected, mediating the uptake and transmembrane transport of inorganic phosphorus. These genes collectively constitute the phosphorus-solubilizing metabolic system of strain 19C11, providing the genetic basis for its highly efficient phosphorus activation function.

[0084] KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotation results show that strain 19C11 contains 47 functional genes involved in pyruvate metabolism and 27 genes involved in the citric acid cycle, demonstrating its potential to produce organic acids through carbon metabolism pathways, which contributes to the dissolution and release of inorganic phosphorus. This strain also contains 112 genes encoding motility proteins, 38 genes related to flagella assembly, and 109 genes related to biofilm formation, indicating strong rhizosphere motility and colonization capabilities. COG (Clusters of Orthologous Groups) functional classification results show that the main gene categories of this strain are concentrated in processes such as amino acid metabolism and transport, energy production and transport, carbohydrate metabolism, and secondary metabolite synthesis. CAZy (Carbohydrate-Active enZymes) database annotation results show that this strain contains various glycoside hydrolases (GH) and carbohydrate esterases (CE), which can participate in the degradation and utilization of organophosphorus compounds.

[0085] In summary, Pseudomonas rhizogenes strain 19C11 possesses a complete PQQ synthesis pathway, gluconic acid metabolism pathway, phosphatase secretion system, and phosphate transport system. Their synergistic effect enables the dissolution of inorganic phosphorus and the mineralization of organic phosphorus, providing genetic and functional basis for its application in plant growth promotion and compound microbial agent development.

[0086] Example 3: Verification of the ability of *Pseudomonas rhizophila* strain 19C11 to dissolve inorganic phosphorus and mineralize organic phosphorus.

[0087] To verify the ability of *Pseudomonas rhizogenes* strain 19C11 to dissolve inorganic phosphorus and mineralize organic phosphorus under in vitro conditions, *Variovorax paradoxus* ZN6 (ZN6 for short), a strain known to have typical phosphorus-solubilizing functions, was selected as the reference strain. ZN6 was provided by Professor Zhang Lin of China Agricultural University, and its phosphorus-solubilizing ability can be found in the literature (Jin, ZX, Jiang, FY, Wang, LT, Declerck, S., Feng, G., & Zhang, L. (2024). Arbuscular mycorrhizal fungi and Streptomyces: brothers in arms to shape the structure and function of the hyphosphere microbiome in the early stage of interaction. Microbiome, 12(1). doi:10.1186 / s40168-024-01811-2). The phosphorus solubilization ability of strain 19C11 was verified by plate experiments and liquid culture experiments.

[0088] In the plate experiment, the isolated and purified strain 19C11 was inoculated into 1 / 2 TSB liquid medium (Haibo Biotechnology Co., Ltd., HB4114-19) and cultured at 28℃ with constant temperature shaking at 180 r / min for 24 h. 20 μL of the bacterial suspension was then added dropwise to NBRIP solid medium (Shanghai Yuanye Biotechnology Co., Ltd., R30014) supplemented with 0.5% tricalcium phosphate and incubated upside down in a 30℃ incubator for 5 days. After the incubation period, a clear zone appeared around the colonies, indicating that strain 19C11 has phosphorus-soluble ability.

[0089] In the liquid culture experiment, strain 19C11 and the reference strain ZN6 were inoculated into 1 / 2 TSB liquid medium (culture volume 50 mL, placed in a 150 mL Erlenmeyer flask), respectively. After incubation at 28℃ and 180 rpm for 24 h with shaking, the supernatant was removed by centrifugation. The cells were washed twice with sterile water and then suspended in NBRIP liquid medium supplemented with 0.5% tricalcium phosphate or calcium magnesium phytate. Inoculation was performed at a volume ratio of 1%. The uninoculated medium served as a blank control to eliminate the influence of phosphorus autolysis. All treatments were incubated at 28℃ and 180 rpm for 5 days with shaking. After incubation, the culture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The available phosphorus content in the supernatant was determined using the molybdenum antimony spectrophotometric method. The results are as follows: Figure 3 As shown, the in vitro phosphate-solubilizing ability of Pseudomonas rhizogenes strain 19C11 is demonstrated.

[0090] In a medium containing tricalcium phosphate, the available phosphorus concentration in the supernatant of strain 19C11 was 817.7 mg / L, which was 89.4% higher than that of the reference strain ZN6. Figure 3 A); In a medium containing calcium magnesium phytate, the effective phosphorus concentration in the supernatant of strain 19C11 was 274.9 mg / L, which was 10.2% higher than that of ZN6. Figure 3 B). The results showed that strain 19C11 had strong inorganic phosphorus dissolution and organic phosphorus mineralization capabilities.

[0091] Example 4: Effects of Pseudomonas rhizophila strain 19C11 on licorice growth

[0092] To verify the promoting effect of *Pseudomonas rhizogenes* strain 19C11 on licorice growth and soil phosphorus availability, a pot experiment was conducted. The test plant was licorice (*Glycyrrhiza uralensis* Fisch.), variety “Guo Gan No. 1”. Three treatments were set up: control (CK), treatment with added tricalcium phosphate (P), and treatment with added tricalcium phosphate and inoculated with *P. rhizogenes* strain 19C11 (P+19C11). Each treatment was replicated in four places. The tricalcium phosphate dosage was 70 mg / kg, and the *P. rhizogenes* strain 19C11 treatment was supplemented with OD... 600 The value is 0.02 (approximately 10). 7 A suspension of *Pseudomonas rhizogenes* strain 19C11 (cells / ml) was added to each pot and each plant. The available nitrogen, available phosphorus, and available potassium in the tested soil of each treatment group were 26.0 mg / kg, 1.8 mg / kg, and 59.6 mg / kg, respectively. The soil was sterilized using 25 kGy gamma-ray radiation.

[0093] Strain strain 19C11 was inoculated into 1 / 2 volume tryptone soybean broth (TSB) liquid medium and cultured at 28℃ and 180 r / min for 24 h with constant temperature shaking. The bacterial cells were collected by centrifugation and washed three times with 10 mM magnesium sulfate solution to prepare OD. 600 It is 0.02 (approximately 10 7 A bacterial suspension (cells / mL) was prepared. Inoculation was performed by irrigation, with the bacterial suspension evenly added to the soil before sowing to achieve a soil moisture content of 16%. The control treatment was treated with an equal volume of 10 mM magnesium sulfate solution.

[0094] Licorice seeds were treated with 50% sulfuric acid (H2SO4) for 30 min, then surface-sterilized by soaking in 10% hydrogen peroxide (H2O2) solution for 10 min, and rinsed thoroughly with sterile deionized water. They were then placed in sterile petri dishes lined with double-layered filter paper and germinated at 25℃ in the dark for 2–3 days. Four seeds with uniform germination were sown per pot, and after 5 days of emergence, two uniformly growing plants were thinned out. The experiment was conducted in an artificial climate chamber with day / night temperatures of 25℃ / 18℃, a photoperiod of 16h / 8h, and a light intensity of 1000 μmol / m². 2 / s, relative humidity 70%, soil moisture content maintained at 16%. Plants were harvested after 90 days of growth, and aboveground and root biomass was measured. Soil available phosphorus content was determined using the molybdenum-antimony spectrophotometric method. Results are as follows: Figure 4 As shown.

[0095] The results showed that the addition of tricalcium phosphate and the treatment of tricalcium phosphate + inoculation with Pseudomonas rhizophila strain 19C11 both significantly increased the aboveground and underground biomass of licorice. Figure 4 A); Compared with the treatment with added tricalcium phosphate alone, the aboveground biomass of licorice inoculated with strain 19C11 increased by 30.0%, and the underground biomass increased by 5.5% (A). Figure 4 B). The soil available phosphorus concentration in the tricalcium phosphate + strain 19C11 treatment was 16.6% higher than that of tricalcium phosphate alone. Figure 4 C). The results showed that, under the condition of adding tricalcium phosphate, Pseudomonas rhizogenes strain 19C11 could effectively promote the transformation of insoluble phosphorus in the soil, improve the absorption and utilization of phosphorus by licorice, promote plant growth and development, and increase the available phosphorus content in the soil. It has the potential to improve crop yield and phosphorus utilization efficiency in low phosphorus environments.

[0096] Example 5: Interaction between *Pseudomonas rhizophila* strain 19C11 and mycorrhizal fungi

[0097] The interaction between *Pseudomonas rhizophilus* strain 19C11 and mycorrhizal fungi and its effects on licorice growth and root phosphorus uptake were verified using a pot experiment. The mycorrhizal fungi used were *Rhizophagus irregularis* AH01 (CGMCC No. 12157) (abbreviated as Ri). Experimental setup: control group (CK), treatment group inoculated with *Pseudomonas rhizophilus* strain 19C11 (19C11 group), treatment group inoculated with mycorrhizal fungi (Ri group), and treatment group inoculated with both mycorrhizal fungi and *Pseudomonas rhizophilus* strain 19C11 (Ri+19C11 group), with four replicates per group. The soil used was the same as in Example 4.

[0098] The tested mycorrhizal fungus was *Rhizospora heterophylla* AH01. The *Rhizospora heterophylla* AH01 inoculum was a mixture containing soil, mycorrhizalized root segments, spores, and mycelium. It was used after being co-cultured and propagated with sorghum and clover. Each gram of inoculum contained approximately 67 spores. The AH01 inoculum was inoculated using a stratification method: for the Ri group and the Ri+19C11 group, 400g of soil was first added to a plastic pot (upper diameter 10.5cm, lower diameter 7.0cm, height 11.0cm), followed by 200g of soil mixed with 30g of the AH01 fungal inoculum (containing approximately 1800 spores).

[0099] The culture and inoculation methods for Pseudomonas rhizophila strain 19C11 are the same as in Example 4, and the bacterial suspension concentration is OD. 600 =0.02 (approximately 10 7 (cells / mL), 96 ml of bacterial suspension was added to each plant in each pot of the 19C11 group and the Ri+19C11 group.

[0100] The test plant was the cultivar "Guogan No. 1" of licorice (Glycyrrhiza uralensis Fisch.). Seeds were treated with 50% H₂SO₄ for 30 min, then surface-sterilized by soaking in 10% H₂O₂ solution for 10 min, and finally rinsed thoroughly with sterile deionized water. After germination at 25℃ in the dark for 2–3 days, seeds with uniform germination were selected for the experiment. Four seeds were sown per pot, and after 5 days of emergence, thinning was performed, selecting two plants with uniform growth.

[0101] The experiment was conducted in an artificial climate chamber with day / night temperatures of 25℃ / 18℃, a photoperiod of 16 h / 8 h, and a light intensity of 1000 μmol / m². 2 / s, relative humidity 70%, and soil moisture content maintained at 16% throughout the growing season. Plants were harvested after 90 days, and the aboveground and root biomass was measured, as well as the root phosphorus content.

[0102] The results are shown in Table 2 and Figure 5 As shown.

[0103] Table 2

[0104]

[0105] The results showed that, compared with the control (CK), inoculation with mycorrhizal fungi (Ri) and double inoculation (Ri+19C11) significantly increased the aboveground and root biomass of licorice. The double inoculation treatment increased the aboveground and root biomass by 95.4% compared with inoculation with mycorrhizal fungi alone. Figure 5 A) and 95.6% Figure 5 B). Root phosphorus content analysis results showed that both Ri and Ri+19C11 treatments significantly increased root phosphorus content, with the double inoculation treatment increasing it by 90.2% compared to inoculation with mycorrhizal fungi alone. Figure 5 C). This result indicates that *Pseudomonas rhizophilus* strain 19C11 significantly promotes phosphorus absorption in licorice roots and enhances plant growth, demonstrating a synergistic effect between *Pseudomonas rhizophilus* strain 19C11 and mycorrhizal fungi in promoting plant growth and enhancing root phosphorus absorption. Therefore, *Pseudomonas rhizophilus* strain 19C11 and arbuscular mycorrhizal fungi can be used as effective components in compound inoculants.

[0106] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are 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. Pseudomonas rhizophila strain 19C11, with accession number CGMCC No.33985.

2. A plant growth promoter composition comprising the *Pseudomonas rhizophilus* strain 19C11 as described in claim 1.

3. The composition of claim 2, wherein the composition further comprises a matrix and / or nutrient components.

4. The composition according to claim 2 or 3 is a liquid bacterial agent, a powdered bacterial agent, or a granular bacterial agent, preferably a suspension, bacterial powder, or a freeze-dried bacterial agent.

5. A plant growth promoter combination comprising the rhizophilic pseudomonas strain 19C11 of claim 1 and an arbuscular mycorrhizal fungus, preferably, the arbuscular mycorrhizal fungus being Rhizophagus irregularis AH01.

6. Use of the *Pseudomonas rhizophila* strain 19C11 of claim 1, the composition of any one of claims 2-4, or the plant growth promoter combination of claim 5 in promoting plant growth, plant phosphorus uptake, and / or soil phosphorus activation.

7. The use according to claim 6, wherein promoting plant growth comprises: Promotes increased plant biomass and / or promotes plant root growth.

8. A method for promoting plant growth, plant phosphorus uptake, and / or soil phosphorus activation, comprising: Apply the *Pseudomonas rhizogenes* strain 19C11 of claim 1, the composition of any one of claims 2-4, or the combination of plant growth promoters of claim 5 to the plant, the soil near the plant, or the soil of the plant to be planted.

9. The method of claim 8, wherein the application method includes drip irrigation, irrigation, basal fertilizer application, fertigation, or root irrigation.

10. The use as described in claim 6 or 7, or the method as described in claim 8, wherein the plant is licorice.

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