Rhizosphere pseudomonas and application thereof

Abstract

This invention relates to the field of microbial biotechnology, specifically to a strain of *Pseudomonas rhizoplanae* and its applications. Through extensive screening, this invention discovered a new strain of *Pseudomonas rhizoplanae*, BB438 (accession number GDMCC No: 68248). Biochemical identification and genomic analysis confirmed that this strain possesses nitrogen-fixing, potassium-solubilizing, and amylase, cellulase, β-1,3-glucanase, ferrophosphate, and indoleacetic acid production capabilities. This bacterium can promote seed and plant growth in crops, and its application can help reduce the use of chemical fertilizers and pesticides, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial biotechnology, and in particular to a rhizosphere Pseudomonas rhizoplanae strain and its applications. Background Technology

[0002] Chemical fertilizers and pesticides were once considered key to maintaining intensive farming yields. However, these inputs have adverse effects on the environment, natural resources, and the food chain, such as environmental pollution and pesticide residues. Therefore, finding environmentally friendly and safe alternatives has become a key research direction for sustainable agricultural development. The rhizosphere is an ecological niche with highly interactive interactions between plant roots and microorganisms. Plant growth-promoting rhizobacteria (PGPRs) are a class of microorganisms that colonize the plant rhizosphere ecosystem and exert beneficial effects on plants through various mechanisms. They can promote plant growth, induce plant resistance, promote beneficial plant-microbe symbiosis, and inhibit plant pathogens. In the context of sustainable agricultural development, PGPRs have become an important biological resource for replacing or reducing chemical fertilizers and pesticides due to their environmentally friendly characteristics and multifunctionality. Practice has proven that the application of microbial inoculants in agriculture not only benefits crop yield and quality but also improves the soil environment and addresses the drawbacks of long-term chemical fertilizer application. Currently, a large number of plant rhizosphere proliferators (PGPRs) with significant growth-promoting and yield-enhancing functions, including Bacillus, Azotobacter, Pseudomonas, Acinetobacter, and Burkholderia, have been developed into agricultural microbial inoculants and have emerged in the market. These microorganisms can directly promote plant growth through functions such as phosphorus solubilization and nitrogen fixation, while simultaneously secreting antibacterial extracellular enzymes such as chitinase, cellulase, and protease to degrade the cell walls of pathogenic fungi, thereby effectively inhibiting diseases and indirectly improving plant physiological indicators and agronomic traits. Therefore, based on the diversity of PGPR functions, screening for highly efficient PGPR strains and developing corresponding microbial inoculants can help improve agricultural productivity and promote ecological sustainability. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a microorganism that can reduce the use of chemical pesticides and promote plant growth. Through long-term and extensive screening, this invention has discovered a special rhizosphere Pseudomonas bacterium, thus completing this invention.

[0004] The technical solution of this invention is: In one aspect, the present invention discloses a rhizosphere Pseudomonas strain, wherein the rhizosphere Pseudomonas is Rhizosphere Pseudomonas BB438 ( Pseudomonas rhizoplanae BB438 was deposited on May 11, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province, with accession number GDMCC No:68248.

[0005] In one aspect, the present invention discloses a microbial agent containing *Pseudomonas rhizogenes* BB438.

[0006] In some embodiments, the dosage form of the microbial agent is selected from liquid formulations, lyophilized powders, wettable powders, and solid granules. The liquid formulations further include suspensions, emulsifiable concentrates, aqueous solutions, and microemulsions.

[0007] In one aspect, the present invention discloses a fermentation product prepared by fermentation of Pseudomonas rhizosphereus BB438.

[0008] In one aspect, the present invention discloses the application of rhizosphere Pseudomonas BB438 in promoting crop seed growth.

[0009] In one aspect, the present invention discloses the application of rhizosphere Pseudomonas BB438 in promoting crop growth.

[0010] In this invention, the crop is an agricultural crop; in some embodiments, the crop is selected from one or more of the following: gourd, loofah, cucumber, eggplant, tomato, chili pepper, and sweet potato.

[0011] Preferably, the seeds are soaked in bacterial solution using the seed soaking method; and the rhizosphere Pseudomonas BB438 is applied to the crop using the root irrigation inoculation method.

[0012] In one embodiment, the present invention provides the application of rhizosphere Pseudomonas BB438 strain in promoting the growth of seeds of gourd, loofah, cucumber, eggplant, tomato and pepper.

[0013] In one embodiment, the present invention provides the application of the rhizosphere Pseudomonas BB438 strain in promoting tomato plant growth.

[0014] Preferably, the growth-promoting ability manifests as an increase in the fresh weight of the entire tomato plant, plant height, root length, root fresh weight, dry weight of the entire plant, and dry weight of the roots. More preferably, the inoculation method is root irrigation inoculation with *Pseudomonas rhizogenes* strain BB438, and the final concentration of the colony content in the soil after root irrigation is 1×10⁻⁶. 8 cfu / mL.

[0015] In one embodiment, the present invention provides the application of rhizosphere Pseudomonas BB438 strain in promoting the growth of sweet potato plants.

[0016] Preferably, the growth-promoting ability manifests as an increase in sweet potato root length, root fresh weight, root dry weight, leaf number, leaf fresh weight, and leaf dry weight. More preferably, the inoculation method is root irrigation inoculation with rhizosphere Pseudomonas BB438, and the final concentration of the colony content in the soil after root irrigation is 1×10⁻⁶. 8 cfu / mL.

[0017] In this invention, the amount of *Pseudomonas rhizogenes* BB438 in the aforementioned bacterial agent and fermentation product is not less than 1 × 10⁻⁶. 8 cfu / unit, wherein the unit is preferably g or mL.

[0018] This invention isolated and screened strain BB438 from the rhizosphere soil of pine trees in Meizhou City, Guangdong Province. Through morphological, physiological, and biochemical characteristics and genomic analysis, it was identified as *Pseudomonas rhizoplanae*, and named *Pseudomonas rhizoplanae* BB438. Tests revealed that strain BB438 possesses nitrogen-fixing, potassium-solubilizing, and amylase, cellulase, β-1,3-glucanase, heptaphilin, and indoleacetic acid production capabilities; however, it lacks phosphorus-solubilizing and protease production capabilities. The *Pseudomonas rhizoplanae* strain BB438 promotes the growth of crop seeds and plants, specifically increasing plant height, root length, root fresh weight, root dry weight, leaf number, leaf fresh weight, and leaf dry weight.

[0019] Those skilled in the art will understand that, in this invention, "Rhizosphere Pseudomonas BB438", "strain BB438", and "[other terms]" are used interchangeably. Pseudomonas rhizoplanae "BB438" refers to the strain with accession number GDMCC No:68248. Beneficial effects

[0020] This invention provides a rhizosphere Pseudomonas BB438 and its applications. Through biochemical identification and genomic analysis, it is a novel strain with the ability to fix nitrogen, solubilize potassium, and produce amylase, cellulase, β-1,3-glucanase, ferrophosphate, and indoleacetic acid. This bacterium can promote the growth of crop seeds and plants, and its application can help reduce the use of chemical fertilizers and pesticides, showing good application prospects. Attached Figure Description

[0021] Figure 1 Colony morphology and physiological and biochemical results of strain BB438: A: Colony morphology; B: Gram staining; C: Morphology under scanning electron microscopy.

[0022] Figure 2The genome diagram of strain BB438 shows the following circles from the inside out: First circle, scale; Second circle, GC bias; Third circle, GC content; Fourth and seventh circles, COG associated with each CDS; Fifth and sixth circles, the location of CDS, tRNA, and rRNA on the genome.

[0023] Figure 3 Results of nucleotide identity analysis of the whole genome sequence of strain BB438; Figure 4 Assay for the growth-promoting functions of strain BB438: A: Phosphorus solubilization; B: Nitrogen fixation; C: Protease; D: Potassium solubilization; E: Siderophoresis; F: Amylase; G: Cellulase; H: β-1,3-glucan hydrolase; I: Indoleacetic acid (IAA).

[0024] Figure 5 The growth-promoting effect of strain BB438 on six types of seeds: A: Pumpkin control group; B: Pumpkin treatment group; C: Loofah control group; D: Loofah treatment group; E: Cucumber control group; F: Cucumber treatment group; G: Eggplant control group; H: Eggplant treatment group; I: Tomato control group; J: Tomato treatment group; K: Chili pepper control group; L: Chili pepper treatment group. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0026] Example 1: Isolation and screening of Pseudomonas rhizosphereus BB438 1.1 Materials and Culture Media In 2021, pine rhizosphere soil was collected from Meizhou City, Guangdong Province. A five-point sampling method was used, and soil samples from the same plot were combined to count as one soil sample. Sterile self-sealing bags were used to collect soil samples, and soil collection tools were disinfected with 75% alcohol.

[0027] LB medium is used for the isolation and preservation of Bacillus; Ashbee medium, phosphate-solubilizing medium, potassium-solubilizing medium, protease, amylase, cellulase and β-1,3-glucanase detection medium, siderophore detection medium, and indoleacetic acid detection medium are used to detect whether the strains fix nitrogen, solubilize phosphorus, solubilize potassium, and produce protease, amylase, cellulase, β-1,3-glucanase, ferrophilic acid and indoleacetic acid, etc.

[0028] 1.2. Isolation and Preservation of Rhizosphere Pseudomonas Soil and water were diluted 1:10. The soil suspension was incubated with shaking in a 30°C incubator for 30 mins. The suspension was plated using the dilution method. Different single colonies were picked, the strains were purified, and stored in a -80°C freezer.

[0029] II. Example 2: Identification of Rhizosphere Pseudomonas BB438 The selected strains were identified through morphological, physiological and biochemical characteristics, and whole genome sequencing. A new strain, BB438, was ultimately identified. Specifically: (1) Morphological observation After culturing strain BB438 on LB solid medium at 30 °C for 24 h, the morphology of the colonies was observed, and Gram staining was performed using a Gram staining kit. The morphology of the bacteria was observed using transmission electron microscopy, and their size was measured.

[0030] (2) Physiological and biochemical characteristics Physiological and biochemical characteristics of the strains were determined with reference to the "Handbook of Systematic Classification and Identification of Common Bacteria and Archaea" and "Experimental Guide for Identification of Plant Pathogenic Bacteria (Third Edition)", including: fluorescence observation, accumulation of poly-β-hydroxybutyrate, carbon source utilization test, production of fructan from sucrose, esterase (Tween 80), denitrification, gelatin liquefaction, oxidase, arginine dihydrolase, starch hydrolysis, and growth tests at 4℃ and 41℃.

[0031] (3) Whole genome sequencing analysis Single colonies of strain BB438, activated on LB agar plates, were inoculated into 150 mL of LB liquid medium and cultured at 30 °C and 180 r / min for 18 h. The cells were then collected by centrifugation at 4 °C and 7000 r / min for 15 min. The samples were sent to Shanghai Paisenno Biotechnology Co., Ltd. for sequencing analysis. Sequencing was performed using a combination of Illumina NovaSeq and Nanopore technologies. Total nucleotide sequence similarity among the genomes of different strains was calculated using JSpeciesWS.

[0032] Results: After 24 h of culture on LB medium, strain BB438 formed milky white colonies. Figure 1 (A) The surface is moist and smooth with a slightly raised center; the colony diameter is 2-3 mm. Gram staining is red (…). Figure 1 (B); the bacteria are rod-shaped with rounded ends, measuring (1.38–1.84) μm × (0.60–0.77) μm. Figure 1 (C)

[0033] Strain BB438 can utilize glucose and fructose, and can grow at both 4 ℃ and 41 ℃. It can utilize sucrose to produce fructan, has starch hydrolysis ability, is positive for gelatin liquefaction, esterase (Tween 80) and arginine dihydrolase, and is negative for denitrification and oxidase. It cannot accumulate poly-β-hydroxybutyrate.

[0034] The BB438 genome is 6,600,159 bp in size with a GC content of 65.02%. This genome is predicted to contain 5,960 protein-coding genes, 16 rRNA genes, 69 tRNA genes, and 64 ncRNA genes. Annotated using the COG database, 99.35% (5,921) of the protein-coding genes have defined functions, distributed across 23 COG subclasses. The two most abundant subclasses are "function unknown" and "amino acid transport and metabolism," containing 1,213 and 581 genes respectively, accounting for 20.35% and 9.75% of the total protein-coding genes. Figure 2 The results of whole-genome nucleotide identity alignment analysis showed that BB438 had the highest ANI value of 92.31% compared with existing Pseudomonas strains. Figure 3 According to the classification criteria that an ANI value greater than 96% indicates the same species, and less than 95% indicates different species, BB438 is a different species from existing Pseudomonas strains. Therefore, based on morphological, physiological, and biochemical characteristics, as well as whole-genome ANI analysis, strain BB438 was identified as a new species of Pseudomonas and named [name missing]. Pseudomonas rhizoplanae Rhizobium, strain BB438, is deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province, with accession number GDMCC No:68248 and deposit date May 11, 2026.

[0035] III. Example 3: Identification of growth-promoting factors in strain BB438 The BB438 strain was inoculated onto Assumption nitrogen-free medium using the streak method, and the strain was subcultured three times consecutively. If growth was observed in all three subcultures, the strain possessed nitrogen-fixing ability. The BB438 strain was inoculated onto phosphate-solubilizing medium and CAS detection medium using the single-spot method, and onto potassium-solubilizing medium and protease medium using the three-spot method; a clear zone around the inoculation point indicated a positive result. The BB438 strain was inoculated onto amylase medium using the three-spot method, and then the amylase detection medium was submerged with Gram's iodine solution; a clear zone indicated a positive result. The BB438 strain was inoculated onto cellulase medium and β-1,3-glucanase medium using the three-spot method. The cellulase medium was stained with 0.1% Congo red for 20 min, washed with 1 mol / L NaCl for 5 min, and then rinsed with sterile water. The β-1,3-glucanase medium was stained with 0.01% Congo red for 30 min and then rinsed with sterile water; a clear zone around the colony indicated a positive enzyme-producing strain. The ability of the strain to produce indoleacetic acid (IAA) was tested according to previous methods, with 5% Congo red added to each test tube. mL of indoleacetic acid was added to the liquid culture medium for detection, followed by the addition of 100 μL of OD. 600 For strain BB438 (value = 1), 100 μL of sterile water was added as a blank control. After incubation at room temperature in the dark for 20 min, the color change was observed; a red color in the test tube indicated a positive result. All culture plates were incubated at 30 ℃ for 2-3 days after inoculation.

[0036] Analysis showed that strain BB438 could be continuously subcultured three times on Assumption nitrogen-free medium, indicating its nitrogen-fixing ability. After culturing for 2 days on potassium-solubilizing, amylase, cellulase, and β-1,3-glucanase media, a clear zone appeared around the colony, indicating its ability to solubilize potassium and produce amylase, cellulase, and β-1,3-glucanase. After culturing for 3 days on CAS detection medium, a brown clear zone appeared around the colony, indicating its ability to produce heptaphilin. The indoleacetic acid (IAA) test showed a red color, indicating its ability to produce IAA. However, after culturing for 2 days on phosphate-solubilizing and protease-producing media, no clear zone appeared around the colony, indicating that it does not have the ability to solubilize phosphate or produce protease. Figure 4 ).

[0037] IV. Example 4: Growth-promoting effect of strain BB438 on crop seeds Seeds of bottle gourd, loofah, cucumber, eggplant, tomato, and chili pepper were soaked in 0.5% sodium hypochlorite for 10 minutes, followed by soaking in a solution of 1×10⁻⁶ sodium hypochlorite. 8 Seeds were soaked in a CFU / mL bacterial suspension of strain BB438 for 30 min, placed in seed bags, and cultured for 10 days in an incubator with 8 h light and 16 h dark. Root length and plant height were recorded afterward. The control group was soaked in sterile water. Each treatment used 10 seeds, and the results were repeated three times. The results are shown in Table 1.

[0038] The results showed that strain BB438 had a growth-promoting effect on various plants including bottle gourd, loofah, cucumber, tomato, eggplant, and pepper. Figure 5 ).

[0039] V. Example 5: Growth-promoting ability of strain BB438 on crops 5.1 Growth-promoting ability of tomatoes Five tomatoes (Xinxing 101) with uniform leaf age and growth were selected, and the prepared bacterial solution (strain concentration of 1×10⁻⁶) was added. 8 Slowly pour 50 ml of water (cfu / mL) around the tomato roots and stems, using the same volume of water as a control group. Each group consisted of 5 plants, with 3 replicates. The roots were irrigated once every 7 days for 2 consecutive treatments. On day 15 after the second irrigation, the tomato plant height, root length, root fresh weight, root dry weight, and whole plant fresh and dry weight were measured.

[0040] 5.2 Growth-promoting ability of sweet potatoes After removing the leaves from sweet potato (Jishu 33) cuttings, the cuttings were planted. The prepared bacterial solution (strain concentration 1×10⁸ CFU / mL) was slowly poured around the sweet potato rootstock (50 ml per plant). A control group of 10 plants was used, with 3 replicates per group. The roots were irrigated once every 7 days for two consecutive treatments. On day 10 after the second irrigation, the root length, root weight, root dry weight, leaf fresh weight, dry weight, and number of leaves were measured.

[0041] Results: As shown in Tables 2 and 3:

[0042]

[0043] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A rhizosphere Pseudomonas strain, characterized in that, The rhizosphere pseudomonas is rhizosphere pseudomonas BB438 ( Pseudomonas rhizoplanae BB438), which was preserved in Guangdong Microbial Culture Collection Center on May 11, 2026, the preservation address is No. 59 Building, 5th Floor, Guangdong Microbial Culture Collection Center, 100, Martyrs Road, Guangzhou, and the preservation number is GDMCC No:68248.

2. A microbial agent, characterized in that, The bacterial agent contains the rhizosphere pseudomonas BB438 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The dosage form of the microbial agent is selected from liquid preparations, lyophilized powders, wettable powders, and solid granules.

4. The microbial agent according to claim 3, characterized in that, The liquid formulation is selected from suspensions, emulsifiable concentrates, aqueous solutions, and microemulsions.

5. A fermentation product, characterized in that, The fermentation product was prepared by fermentation of the rhizosphere pseudomonas BB438 as described in claim 1.

6. The application of a rhizosphere Pseudomonas BB438 in promoting crop seed growth, characterized in that, The preservation number of the rhizosphere pseudomonad BB438 is GDMCC No:68248.

7. The application of a rhizosphere Pseudomonas BB438 in promoting crop growth, characterized in that, The preservation number of the rhizosphere pseudomonad BB438 is GDMCC No:68248.

8. The application according to claim 6 or claim 7, characterized in that, The crop is selected from one or more of the following: gourd, loofah, cucumber, eggplant, tomato, chili pepper, and sweet potato.

9. The application according to claim 6 or claim 7, characterized in that, Seeds are soaked in bacterial solution using the seed soaking method; rhizosphere Pseudomonas BB438 is applied to crops using the root irrigation inoculation method.

10. The microbial agent according to claim 2, and the fermentation product according to claim 5, characterized in that, The amount of rhizosphere Pseudomonas BB438 in the bacterial agent, the ferment is not less than 1 x 10 8 cfu / unit, the unit is preferably g or mL.

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