Application of pseudomonas

Abstract

The invention discloses application of pseudomonas, and belongs to the field of microorganism application. The bacterial strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 28, 2025, the preservation number is CGMCC NO.36820, the bacterial strain belongs to Pseudomonas sp., and the name of the bacterial strain is Pseudomonas T9. The invention also discloses a preparation method of the bacterial strain. The strain has the capabilities of fixing nitrogen, producing ammonia and dissolving phosphorus, can secrete siderophores, indoleacetic acid (IAA) and protease, and has the function of promoting growth of various plants.

Description

Technical Field

[0001] This invention belongs to the field of microbial applications, and more specifically, relates to the application of a Pseudomonas bacterium. Background Technology

[0002] Plant growth-promoting rhizobacteria (PGPRs) are a class of beneficial microorganisms that live freely in the soil or attach to plant roots. They promote plant growth, enhance the absorption and utilization of mineral nutrients, and effectively suppress harmful organisms. Several PGPR strains have been successfully isolated and identified, among which *Pseudomonas* is widely studied and applied due to its significant growth-promoting and stress-resistance capabilities. Other common PGPRs include *Rowstoneella*, *Burkholderia*, *Methylobacterium*, *Sphingomyelin*, *Bacteroides*, *Enterobacter*, and *Massezia*. PGPRs exert their growth-promoting effects through various mechanisms, such as releasing volatile organic compounds, dissolving phosphorus, fixing nitrogen, synthesizing ironophiles, and secreting plant hormones (such as IAA).

[0003] In recent years, the potential of plant growth promoters (PGPRs) in promoting plant growth and enhancing disease resistance has attracted widespread attention. For example, the growth-promoting bacterium DM11 isolated from the rhizosphere of Codonopsis pilosula can produce auxin and heparin, and possesses properties such as phosphorus solubilization, nitrogen fixation, and salt tolerance. Its volatile organic compounds have a significant promoting effect on the growth of cucumber and tomato. Bacillus subtilis isolated from potato can promote growth rate, dry matter yield, and leaf area, and enhance drought resistance. Pseudomonas fluorescens can stimulate seed germination and seedling growth under drought stress. The strain S258 isolated from cotton has also been shown to promote the growth of Arabidopsis thaliana. Therefore, conducting research on the identification and screening of PGPRs is of great significance for improving plant growth potential and disease resistance, and provides a new technical pathway for promoting sustainable agriculture.

[0004] The hardy kiwifruit variety exhibits vigorous growth and profuse growth, with wild vines reaching up to 30 meters in length and stems up to 15 centimeters in diameter. However, significant differences in growth performance exist within the same orchard for the same variety, with some plants being stunted while others are exceptionally robust. Besides inherent differences in the seedlings themselves, this variation in growth may be closely related to the abundance of growth-promoting bacteria in the rhizosphere soil. Summary of the Invention

[0005] In view of the above-mentioned problems in the existing technology, the purpose of this invention is to provide a rhizosphere growth-promoting bacterium T9 for jujube kiwifruit and its application.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows: This study extracted a strain from the rhizosphere soil samples of robust kiwifruit plants in a kiwifruit orchard in Yangmuchuan Town, Kuandian Manchu Autonomous County, Dandong City, Liaoning Province, China (40°N, 124°E). This strain possesses nitrogen-fixing, ammonia-producing, and phosphorus-solubilizing abilities, and can secrete siderophores, indoleacetic acid (IAA), and proteases, demonstrating its potential as a microbial fertilizer.

[0007] A strain of Pseudomonas, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.36820, is named Pseudomonas T9. This strain is used to promote plant growth.

[0008] Furthermore, the plant mentioned includes the hardy kiwifruit.

[0009] Furthermore, the plant mentioned includes corn.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: Pseudomonas T9 possesses nitrogen-fixing, ammonia-producing, and phosphorus-solubilizing abilities, and can secrete siderophores, indoleacetic acid (IAA), and proteases, which can significantly promote plant growth. It can be used as a plant rhizosphere growth-promoting bacterium and has the potential to be used as a microbial fertilizer. Attached Figure Description

[0011] Figure 1 The colony morphology of Pseudomonas T9 on nutrient agar plates after being cultured at 28°C for 48 hours. Figure 2 A magnified image of a single colony of Pseudomonas T9; Figure 3 This is a scanning image of Pseudomonas T9 under an electron microscope. Figure 4 Phylogenetic analysis diagram of Pseudomonas T9 strain and related strains; Figure 5 This diagram illustrates the assay for functional substances in Pseudomonas T9. A represents the cellulase activity assay; B represents the siderophore secretion assay; C represents the phosphate dissolution assay; D represents the nitrogen fixation assay; E represents the protease activity assay; F represents the ammonia generation assay; and G represents the IAA generation assay using Solkov's reagent. a is the positive control (IAA standard), b is the negative control (no IAA), and c is the result of the Pseudomonas T9 culture supernatant. Figure 6 This diagram illustrates the growth-promoting effect of Pseudomonas T9 on maize. In the diagram, A represents the T9 experimental group, and B represents the control group. Figure 7This image shows the growth-promoting effect of Pseudomonas T9 on tissue culture seedlings of Actinidia arguta. In the image, A represents the T9 experimental group, and B represents the control group. Detailed Implementation

[0012] The present invention will be further described below with reference to specific embodiments.

[0013] The strains provided in this protocol were extracted from rhizosphere soil samples of robust jujube kiwifruit plants in Yangmuchuan Town, Kuandian Manchu Autonomous County, Dandong City, Liaoning Province, China (40°N, 124°E). Classic microbial isolation and culture methods were used to isolate the strains from the rhizosphere soil samples.

[0014] Molecular biological identification confirmed that the strain belongs to the genus Pseudomonas sp., and it was named Pseudomonas T9. It was deposited on November 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36820, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] Pseudomonas T9 has any one of the following nucleotide sequences in its 16S rDNA: I. Possesses the nucleotide sequence shown in SEQ ID NO:1; II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 1; III. A sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO:1; IV. Complementary sequences to sequences shown in I, II, or III.

[0016] The morphological identification process of the strain is as follows: First, morphological observation was performed. After culturing the strain on NA agar at 28°C for 48 hours, it exhibited typical colony morphology: colonies were round, moist, raised, and glossy; the color was pale yellow to light black, with neat edges, and a relatively fast growth rate. Figure 1 and Figure 2 As shown.

[0017] Further observation using a scanning electron microscope (SEM) revealed that its morphology was as follows: Figure 3 As shown, the bacterial cells of the strain are rod-shaped (bacilli), with regular morphology and relatively smooth surface, with a length of approximately 5.35–7.41 µm and a width of approximately 0.43–0.54 µm.

[0018] The molecular identification process of the strain is as follows: The bacterial 16S rDNA universal primers 16F27 / 1492R and rpoA gene primers were used for PCR amplification and sequencing of this strain. The obtained 16S rDNA and rpoA gene sequences were compared for homology with sequences in GenBank. Representative strains were selected, and a phylogenetic tree was constructed using MEGA 6.0 software. The phylogenetic tree is shown below. Figure 4 As shown.

[0019] Based on the morphological identification results, the strain was finally identified as belonging to the genus Pseudomonas, and was closely related to P. allokribbensis and P. kribbensis.

[0020] Fresh bacterial culture was sent to Meiji Biotechnology for whole-genome sequencing. A second-generation + third-generation sequencing strategy combining Illumina and PacBio was adopted to ensure that each sample simultaneously obtained PacBio sequencing data with a coverage of no less than 100× and Illumina sequencing data with a coverage of 100×, so as to guarantee the integrity and accuracy of genome assembly.

[0021] Based on sequencing data, statistical analysis of base distribution and quality fluctuations was performed for each sequencing cycle of all reads. The base quality, error rate, and distribution of each sample were evaluated to reflect the overall library construction quality and sequencing quality.

[0022] The clean data after quality control was assembled using the Unicycler assembly software, followed by three generations of sequence assembly. Pilon software was used for sequence correction during the process. If there was a significant overlap at both ends of the final assembled sequence, the sequence was circularized and one end of the overlapping sequence was removed, ultimately yielding complete chromosome and plasmid sequences.

[0023] Based on the GTDB online service platform (GTDB - Genome Taxonomy Database), the average nucleotide identity (ANI) value among 23 genome sequences, including this strain and its closest Pseudomonas genus type strain, was calculated.

[0024] ANI analysis based on the complete genome of the strain showed that it had the highest ANI value of 93.53% with P. kribbensis, which was lower than the 95% threshold commonly used for bacterial species identification. The specific data of the analysis are shown in Table 1.

[0025] Table 1. Analysis of average nucleotide identity (ANI) between strains and the closest type of strains in the genus Pseudomonas: sample name ANI(%) ZB1P45 86.74 IT-P374 88.83 IzPS23 93.00 inb918 91.72 2180 88.59 PgBE89 88.8 IzPS32d 91.52 YJY915 91.71 BM06 88.74 iranensisSWRI54 87.93 FIT81 91.66 46-2 93.53 FP1962 86.72 PMCC200367 86.58 B21-045 87.95 OTU6ESPEB1 87.86 BS3782 86.74 YK-310 91.63 FIT28 88.07 PGSB8459 88.45 CTB12 88.04 BIM-B-582 88.13

[0026] The above results indicate that this strain is significantly differentiated from known Pseudomonas species at the genomic level, and it has been preliminarily identified as a new species. The strain has been named Pseudomonas T9.

[0027] The physiological and biochemical identification process of the strain is as follows: Pseudomonas T9 was inoculated into NA solid medium and cultured at 28°C for 48 h. The following physiological and biochemical properties of Pseudomonas T9 were determined: Voges–Proskauer (VP) test, indole formation, gelatin liquefaction, oxidase activity, growth at pH 5.7, hydrogen sulfide production, nitrate reduction, urease activity, glucose utilization, and starch hydrolysis.

[0028] The test results are shown in Table 2, where “+” indicates a positive reaction and “-” indicates a negative reaction.

[0029] Table 2. Physiological and biochemical characteristics of Pseudomonas T9: phenotypic characteristics Reaction characteristics phenotypic characteristics Reaction characteristics VP test - Hydrogen sulfide production - Indole formation - Nitrate reduction - Gelatin liquefaction + Urease activity - Oxidase activity + glucose utilization + Growth at pH 5.7 + Starch hydrolysis -

[0030] The functional substances of the strain were determined as follows: Cellulase production: The activated strain was inoculated onto Congo red cellulose medium with cellulose as the sole carbon source and cultured at 25°C for 7 days. The presence of transparent hydrolysis zones around the colonies was observed to determine its cellulase production capacity.

[0031] Siderophore generation: The activated strain was inoculated on chromium azurite (CAS) agar medium and cultured at 25°C for 7 days. The color change around the colony was observed to determine the generation of siderophores.

[0032] Phosphate solubilization capacity: The activated strain was inoculated into the National Botanical Garden of the United States phosphate (NBRIP) medium and cultured at 25°C for 7 days. The presence of a transparent dissolution zone was observed to determine its phosphate solubilization capacity.

[0033] Nitrogen fixation capacity: The activated strain was passaged twice on Assumption nitrogen-free solid medium, then inoculated into the same medium and cultured at 25°C for 7 days. The growth of the strain was observed to determine its nitrogen fixation capacity.

[0034] Protease production: The activated strain was inoculated into skim milk medium and cultured at 25°C for 7 days. The presence or absence of a transparent hydrolysis zone around the colony was observed to determine the production of protease.

[0035] Ammonia production capacity: The activated strain was inoculated into peptone ammoniated medium and cultured at 25°C for 7 days. Using uninoculated medium as a blank control, 3–5 drops of Nessler's reagent were added to the plate. The appearance of a yellow or brownish-red precipitate indicated that the strain had ammonia production capacity.

[0036] Indoleacetic acid (IAA) production capacity: The activated strain was inoculated into LB medium containing 5 mmol / L L-tryptophan and cultured at 25°C for 7 days. The bacterial culture filtrate was collected, and 1.5 mL of Salkawski chromogenic reagent was added. The mixture was allowed to stand at room temperature for 30 min. Using sterile medium as a control, if the filtrate turned pink, it indicated that the strain could produce IAA.

[0037] Specific test results are as follows: Figure 5 As shown, no transparent hydrolysis zone appeared on the Congo red cellulose medium, indicating that this bacterium does not produce cellulase. Figure 5 A); The appearance of a yellow halo on CAS medium indicates the potential for siderophore production ( Figure 5 B); the formation of a transparent dissolution zone around the colony on NBRIP medium indicates phosphate solubility (B). Figure 5 C); it can grow on Assumption nitrogen-free medium, indicating that it has nitrogen-fixing ability ( Figure 5 D); the formation of a clear hydrolysis zone in skim milk culture medium indicates the production of a protease ( Figure 5 E); the appearance of a yellow / brownish-red precipitate after the addition of Nessler's reagent indicates the ability to produce ammonia (E). Figure 5 F); The fermentation broth turned pink after adding Salkawski color developer (F); Figure 5 c in G), and Figure 5 Comparing a and b in G shows that IAA ( Figure 5 In G, a is the positive control, b is the negative control, and c is the supernatant of the fermentation broth of Pseudomonas T9 medium.

[0038] The above results indicate that Pseudomonas T9 possesses nitrogen-fixing, ammonia-producing, and phosphorus-solubilizing abilities, and can secrete siderophores, indoleacetic acid (IAA), and proteases, but cellulase activity was not detected. Pseudomonas T9 has multiple growth-promoting functions and has the potential to be used as a microbial fertilizer.

[0039] To verify the growth-promoting effect of Pseudomonas T9, a pot experiment was conducted on maize. The specific method is as follows: Corn seeds were surface-sterilized with 75% alcohol for 1–2 minutes, rinsed with sterile water 3–5 times, placed in a petri dish lined with moist sterile filter paper, and germinated in the dark at 25–28°C for 2 days.

[0040] Thirty seeds with the same germination status were selected and soaked in bacterial suspension (OD600≈0.8) for 2 hours. The control group was soaked in sterile water for 2 hours. Then, they were sown in flower pots containing 0.5 kg of soil.

[0041] After sowing, water regularly, and on the 7th and 14th days, add bacterial solution by root irrigation. Add 10 mL of bacterial suspension to each plant. OD600≈0.8. In the control group, an equal amount of sterile water was used for irrigation.

[0042] Throughout the experiment, regular watering and weeding were carried out to ensure normal corn growth. The plants were harvested 30 days after sowing, and relevant biomass indicators were measured.

[0043] Its plant growth status is as follows Figure 6 As shown in Table 3, the growth results are as follows: T9 is the experimental group irrigated with Pseudomonas T9 bacterial suspension, and CK is the control group that does not use Pseudomonas T9 bacterial suspension.

[0044] Table 3. Maize growth parameters: deal with Plant height (cm) Stem diameter (mm) Number of leaves (pieces) Fresh weight of above-ground parts (g) Fresh weight of underground part (g) Dry weight of aboveground parts (g) Dry weight of underground part (g) CK 16.77 7.71 5.30 10.26 4.89 0.92 0.44 T9 17.76 8.54 5.77 12.53 6.09 1.12 0.53 Corn treated with Pseudomonas T9 suspension showed significantly higher morphological indicators and biomass than the control group, specifically: plant height increased by 5.88%, stem diameter by 10.72%, leaf number by 8.81%, aboveground fresh weight by 22.01%, underground fresh weight by 24.33%, aboveground fresh weight by 21.46%, and underground fresh weight by 18.95%.

[0045] The above results indicate that Pseudomonas T9 can significantly promote the growth of maize plants.

[0046] To verify the growth-promoting effect of Pseudomonas T9 on Actinidia arguta, the following tissue culture seedling experiment was conducted: Pseudomonas T9 was inoculated into LB liquid medium and cultured at 28°C with shaking for 2 days until the logarithmic growth phase. The cells were collected by centrifugation at 966×g, the supernatant was discarded, the precipitate was washed twice with sterile water, and finally resuspended with an appropriate amount of sterile water to prepare a T9 bacterial suspension.

[0047] Tissue culture seedlings of *Actinidia arguta* with uniform growth were selected and pre-cultured on rooting medium for 7 days until the roots reached approximately 5 mm in length. These seedlings were then used for the experiment. In a sterile laminar flow hood, the roots of the rooted seedlings were immersed in a T9 bacterial suspension for 2 minutes; the control (CK) group was immersed in an equal volume of sterile water for 2 minutes. Each treatment was replicated with 30 seedlings. After treatment, all tissue culture seedlings were transferred to fresh rooting medium for further culture. After 6 weeks of culture, the fresh weight, number of leaves, and plant length were recorded.

[0048] Its plant growth status is as follows Figure 7 As shown in the figure, A represents the T9 experimental group, and B represents the control group. The growth results are shown in Table 4, where T9 is the experimental group treated with Pseudomonas T9 bacterial suspension, and CK is the control group that did not use bacterial suspension.

[0049] Table 4. Growth parameters of tissue culture seedlings of Actinidia arguta: deal with Plant height (cm) Number of leaves (pieces) Fresh weight of the plant (g) CK 5.37 7.83 0.42 T9 5.43 9.33 0.51 Compared with the uninoculated control, inoculation with Pseudomonas T9 strain significantly promoted the growth of Actinidia arguta, with a 22.09% increase in plant fresh weight, a 19.15% increase in leaf number, and a slight increase in plant height (1.2%), but the increase was not significant.

[0050] The above results indicate that Pseudomonas T9 can significantly promote the growth of tissue culture seedlings of Actinidia arguta.

Claims

1. Use of a Pseudomonas bacterium, characterized in that, The strain is preserved in China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC NO. 36820, belongs to Pseudomonas sp., is named as Pseudomonas T9, and is used for promoting plant growth.

2. Use of a Pseudomonas according to claim 1, characterized in that The plant includes Actinidia deliciosa.

3. The use of a Pseudomonas according to claim 1, characterized in that The plant includes Zea mays.

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