Pseudomonas palleroniana bacterial microorganism and application thereof

Abstract

This application relates to the field of microbial technology, disclosing a Pseudomonas palleroniana bacterium and its applications, including strain AQY14, whose taxonomic name is Pseudomonas palleroniana, strain number 16S rRNA, and which was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 23, 2026, with accession number GDMCC No:67636. Furthermore, this invention also provides a bacterial agent containing live bacteria of the aforementioned strain AQY14 and its culture. The bacterial culture includes fermentation broth, fermentation filtrate, bacterial suspension, and purified bacterial solution obtained by culturing strain AQY14 in a culture medium. The bacterial agent is a liquid preparation. The culture medium includes Ashby medium, R2A medium, and PDA medium. The provided strain AQY14 possesses multiple growth-promoting and biocontrol functions, exhibiting significant synergistic applications. This strain simultaneously possesses multiple plant growth-promoting characteristics such as nitrogen fixation, dissolution of organic and inorganic phosphorus, potassium solubilization, siderophore production, and IAA production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Pseudomonas palleroniana bacterium and its applications. Background Technology

[0002] Rice is one of the most important food crops in my country and even globally. During its growth, it is frequently infected by various pathogens, among which sheath blight (mainly caused by Rhizoctonia solani), rice blast, and bacterial blight are the major diseases causing yield losses. Currently, the control of these diseases mainly relies on the frequent use of chemical pesticides. However, long-term and excessive application of chemical pesticides not only leads to pesticide resistance in pathogens but also causes a series of problems such as pesticide residues, environmental pollution, and soil microecological imbalance. Therefore, developing safe, efficient, and environmentally friendly biological control strategies has become an important direction in the field of plant disease control.

[0003] Utilizing beneficial microorganisms and their metabolites for disease control is a hot topic in biopesticide research and development. Currently, some antagonistic strains have been reported for application in rice disease control. However, existing biocontrol bacteria generally suffer from limited functionality; most strains only inhibit a single or a few pathogens, or only have growth-promoting effects without broad-spectrum antagonistic capabilities, making it difficult to simultaneously meet the dual needs of crop growth promotion and integrated disease control. Furthermore, the control efficacy of existing strains against soil-borne diseases such as sheath blight is still not ideal, and their stability and adaptability in field application need improvement. Therefore, screening for a novel microbial strain that possesses multiple growth-promoting properties, broad-spectrum antagonistic effects against various rice pathogens, and exhibits stability in practical applications is of great significance for developing new microbial agents, reducing dependence on chemical pesticides, and promoting the sustainable development of green agriculture. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a Pseudomonas palleroniana bacterium and its application, which solves the problem that existing biocontrol bacteria have limited functions and cannot simultaneously promote crop growth and effectively control multiple rice diseases.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: a Pseudomonaspalleroniana bacterium, comprising strain AQY14, whose taxonomic name is Pseudomonaspalleroniana, strain number 16S rRNA, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 23, 2026, with accession number GDMCC No: 67636.

[0006] In addition, the present invention provides a microbial agent containing live bacteria of the strain AQY14 and a culture of the strain.

[0007] Preferably, the bacterial culture is a fermentation broth, fermentation filtrate, bacterial suspension, or purified bacterial solution obtained by culturing the bacterial strain AQY14 in a culture medium, and the bacterial agent is a liquid preparation.

[0008] Preferably, the culture medium includes Ashby medium, R2A medium, and PDA medium.

[0009] Furthermore, the present invention also provides an application of the Pseudomonas palleroniana bacterium microbial strain, such as the application of the Pseudomonas palleroniana bacterium microbial strain or the application of the microbial agent as described, in the preparation of biopharmaceuticals.

[0010] Preferably, the biological drug is used for any one or more of the following purposes: (1) Used to antagonize rice sheath blight; (2) Used to antagonize rice blast; (3) Used to antagonize rice bacterial blight; (4) Used to antagonize Fusarium graminearum in rice; (5) Used to antagonize *Corydalis oryzae* in rice; (6) Soluble phosphorus, dissolve potassium, produce IAA, produce iron carrier, fix nitrogen.

[0011] This invention provides a Pseudomonas palleroniana bacterial microorganism and its application. It has the following beneficial effects: 1. The strain AQY14 provided by this invention possesses multiple growth-promoting and biocontrol functions, exhibiting significant synergistic effects. This strain simultaneously possesses various plant growth-promoting characteristics, including nitrogen fixation, dissolution of organic and inorganic phosphorus, potassium solubilization, iron carrier production, and IAA production. It can also secrete biocontrol-related enzymes such as proteases and chitinases, and exhibits a negative hemolytic reaction, demonstrating good safety. This integration of multiple functions lays a solid foundation for its application as a multifunctional microbial agent in agricultural production.

[0012] 2. The strain AQY14 provided by this invention exhibits broad-spectrum and highly effective antagonistic effects against a variety of rice pathogens, with unexpectedly strong antibacterial effects. This strain not only shows significant inhibitory effects against Rhizoctonia solani (71.48% inhibition rate on plate confrontation and 58.64% inhibition rate on sclerotium germination), but also causes abnormal swelling of the pathogenic mycelium. Simultaneously, it achieves inhibition rates of 69.98%, 69.40%, and 70.13% against rice blast, Fusarium graminearum, and Cephalosporium graminearum, respectively, and also shows significant inhibitory effects against bacterial blight. Its broad-spectrum inhibitory ability against different groups of pathogens far exceeds that of conventional antagonistic strains.

[0013] 3. After treatment with the fermentation broth of strain AQY14, the length of rice sheath blight lesions significantly decreased from 2.81 cm to 0.38 cm, and the disease index decreased from 58.85% to 40.00%, with a relative control effect of 32.03%. This result confirms the practical application value of this strain in the control of rice sheath blight and provides excellent strain resources for the development of new biological pesticides. Attached Figure Description

[0014] Figure 1 This is a single colony morphology diagram of strain AQY14 in this invention; Figure 2 This is a schematic diagram of the phylogenetic tree of strain AQY14 in this invention; Figure 3 This is a diagram illustrating the inhibitory effect of strain AQY14 on Rhizoctonia solani in this invention; Figure 4 This is a schematic diagram illustrating the inhibitory effect of strain AQY14 on Rhizoctonia solani in this invention; Figure 5 This is a diagram illustrating the effect of strain AQY14 on the hyphae of Rhizoctonia solani in this invention; Figure 6 This is a schematic diagram illustrating the broad-spectrum antibacterial activity of strain AQY14 in this invention; Figure 7 This is a schematic diagram illustrating the potted plant control effect of strain AQY14 in this invention. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Please see the appendix Figure 1 - Appendix Figure 7This invention provides a Pseudomonas palleroniana bacterial microbial strain, including strain AQY14, whose taxonomic name is Pseudomonas palleroniana, strain number is 16S rRNA, and was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 23, 2026, with accession number GDMCC No:67636.

[0017] The following examples provide a detailed description. Unless otherwise specified, the experimental methods used in these examples are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0018] The culture medium used in the examples: Ashby medium: 10g mannitol, 0.2g NaCl, 5g calcium carbonate, 0.2g K2HPO4, 0.1g CaSO4·2H2O, 18g agar, 1L H2O, pH=7.2±0.2, 121℃, sterilize for 20min; R2A medium: 0.5g yeast extract, 0.5g peptone, 0.5g acid-hydrolyzed casein, 0.5g glucose, 0.5g soluble starch, 0.3g sodium pyruvate, 0.024g MgSO4·7H2O, 0.3g K2HPO4, adjust pH to 7.0±0.2, sterilize at 121℃ for 20min; PDA medium: 3g potato extract powder, 20g glucose, adjust pH to 7.0±0.2, sterilize at 121℃ for 15min.

[0019] Example 1: Isolation and Identification of Strains AQY14 The soil sample of strain AQY14 involved in this invention was obtained from rice soil in Yunnan Province, and the specific isolation method is as follows: Take 10g of soil sample and add it to an Erlenmeyer flask containing 90mL of sterile water. Incubate at 30℃ and 160r / min for 20min to prepare 10 -1 Soil suspension: Take 1 mL of soil suspension and add it to 9 mL of sterile water. Mix thoroughly and record as 10. -2 And so on, up to 10 -5 Select 10 -3 10 -4 10 -5Three gradient dilutions were used for dilution and plating. 200 µL of each dilution was evenly spread onto Ashby solid plates and incubated upside down in a 30°C constant temperature incubator. After 3-5 days, as many strains with different colony morphologies as possible were streaked onto Ashby solid medium for isolation and purification, and finally single colonies were obtained.

[0020] (1) Morphological characteristics of the bacterial cells: Please see the appendix Figure 1 On R2A medium, single colonies are round, opaque, with a raised center and a smooth surface. They are Gram-positive and non-motile. The strain grows in a temperature range of 4-35℃ (optimal 30℃); tolerates a pH range of 5-8 (optimal pH=7.0); and tolerates a salinity range of 0-5% (optimal 0%).

[0021] (2) Physiological and biochemical characteristics of AQY14 Please refer to Table 1. The physiological and biochemical reactions of AQY14 were identified. The results showed that strain AQY14 was positive for eight indicators: nitrogen fixation, organic phosphorus, inorganic phosphorus, siderophoresis, potassium solubilization, IAA production, siderophoresis, protease, and chitinase. It was negative for two indicators: hemolysis and β-glucanase.

[0022] Table 1: Physiological and biochemical characteristics of strain AQY14

[0023] In this context, "+" indicates a positive result, and "-" indicates a negative result.

[0024] (3) Molecular biological identification Please see the appendix Figure 2 The 16S rRNA gene sequence of strain AQY14 was uploaded to the NCBI GenBank database, obtaining the accession number PZ153170. The 16S rRNA gene sequence of strain AQY14 was aligned using BLAST, and the 15 most similar strains' 16S rRNA gene sequences were downloaded. A phylogenetic tree of strain AQY14 was constructed using the NJ method in MEGAX software. From the phylogenetic tree (… Figure 2As can be seen, strain AQY14 is most closely related to *Pseudomonas palleroniana* CFBP 4389T in evolution, with a 99.79% similarity in their 16S rRNA gene sequences. Genome alignment of strain AQY14 with *Pseudomonas palleroniana*, calculating their dDNA-DNA hybridization values ​​and average nucleotide identity (ANI) values ​​based on genome sequences, revealed that the dDDH and ANI values ​​between AQY14 and *Pseudomonas palleroniana* were 93.50% and 98.71%, respectively, both higher than the critical values ​​(dDDH < 70%, ANI < 95-96%). Based on the morphological characteristics, phylogenetic tree, and genome comparison results, strain AQY14 was identified as belonging to the genus *Pseudomonas*. In systematic taxonomy, this strain belongs to the kingdom Bacteria, phylum Proteobacteria, class Gamma-Proteobacteria, order Pseudomonales, family Pseudomonaceae, and genus *Pseudomonas*.

[0025] Example 3: Analysis of the broad-spectrum antibacterial activity of strain AQY14 (1) Determination of the antagonistic effect of AQY14 on Rhizoctonia solani Laboratory-preserved *Rhizoctonia solani* was activated on PDA medium and used for later use. A single colony of AQY14 was inoculated into R2A liquid medium and incubated at 30℃ and 160 rpm until the logarithmic growth phase (18-24 h). Using the plate confrontation method, *Rhizoctonia solani* mycelial discs with a diameter of 8 mm were placed in the center of the PDA medium using a punch, and 5 µL of purified bacterial solution was inoculated 3 cm away from the discs. After incubation at 30℃, the diameter of the inhibition zone was measured (cross-cross method), and the inhibition rate was calculated. Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - mycelial cake diameter) × 100%.

[0026] Select sclerotia of *Rhizoctonia solani* of similar size and soak them in sterile fermentation filtrate for 30 min. Place the treated sclerotia on PDA plates, using sclerotia soaked in sterile water as a control. Four sclerotia per plate, with each treatment repeated three times. Incubate at 28℃ in the dark. After 48 h, observe the colony diameter of sclerotia germination and calculate the inhibition rate. Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter) × 100.

[0027] The results of the plate confrontation method and the sclerotium germination inhibition method showed that strain AQY14 had a significant inhibitory effect on Rhizoctonia solani and sclerotia, with inhibition rates of 71.48% and 58.64%, respectively.

[0028] Please see the appendix Figure 3 Appendix Figure 4and appendix Figure 5 Rhizoctonia solani near the antagonistic bacteria was picked and placed on a glass slide, with a plate inoculated only with Rhizoctonia solani as a control. Changes in hyphal morphology were observed under a microscope. The hyphae in the control group grew well and branched at right angles; the hyphae in the AQY14 treatment group showed deformities and swelling.

[0029] The results are shown in the table below (Table 2): Table 2: Inhibitory effect of strain AQY14 on Rhizoctonia solani

[0030] (2) Antagonistic effect of strain AQY14 against other rice pathogens Please see the appendix Figure 6 Laboratory-preserved rice blast fungus, *Corydalis graminearum*, and *Fusarium graminearum* were activated on PDA medium, while bacterial blight pathogens were activated on TSA medium. After activation, they were ready for use. Single colonies of AQY14 were picked and inoculated into R2A liquid medium and cultured at 30℃ and 160rpm until the logarithmic growth phase (18-24h). Using the plate confrontation method, a 1cm diameter pathogenic fungal disc was placed in the center of the PDA medium, and 5µL of purified bacterial solution was inoculated 3cm away from the disc. After incubation at 30℃, the diameter of the inhibition zone was measured (cross-cross method), and the inhibition rate was calculated. Select bacterial blight pathogens and inoculate them into TSA liquid medium. Incubate at 30℃ and 160rpm until the logarithmic phase (30-36h). Take 500µL of bacterial blight pathogen solution and spread it evenly on TSA medium. Inoculate 5µL of purified AQY14 bacterial solution 3cm away from the center. After incubating at 30℃ for 24h, measure the diameter of the inhibition zone.

[0031] The results are shown in the table below (Table 3): Table 3: Broad-spectrum antibacterial activity of strain AQY14

[0032] Example 5: Control effect of strain AQY14 on sheath blight Please see the appendix Figure 7 A pot experiment was conducted using bacterial suspensions of the strain Chujing 28 to verify the results, with sterile water as a control. Five replicates were set up for each treatment. Single colonies were picked and inoculated into R2A liquid medium, and single colonies of AQY14 were picked and inoculated into R2A liquid medium. The cultures were incubated at 30℃ and 160 rpm for 24 h. The fermentation broth was then centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the culture was resuspended in sterile water to OD0.05. 600=0.1, when rice seedlings reached the three-leaf-one-heart stage, the antagonistic bacteria were evenly sprayed onto the leaf surface. After 24 hours of treatment, Rhizoctonia solani cake was inoculated onto the rice leaf sheaths. Twenty days after inoculation, the incidence of rice sheath blight was investigated, and the disease index and control effect were calculated. The results showed that the fermentation broth of the strain could reduce the incidence of rice sheath blight.

[0033] The results are shown in the table below (Table 4): Table 4: Effects of AQY14 fermentation broth on disease incidence in Chujing 28 rice

[0034] Summarize: The strain AQY14 provided in this application exhibits surprisingly broad-spectrum inhibitory capabilities: it not only shows significant antagonistic effects against Rhizoctonia solani (inhibition rate of 71.48% on plate confrontation and 58.64% inhibition rate of sclerotium germination), but also causes abnormal swelling of the pathogenic mycelium; even more unexpectedly, it achieves inhibition rates of 69.98%, 69.40%, and 70.13% against rice blast, Fusarium graminearum, and Cephalosporium graminearum, respectively, and also shows a clear inhibition zone against bacterial blight; in pot experiments, after treatment with this strain, the length of rice sheath blight lesions decreased sharply from 2.81 cm to 0.38 cm, and the disease index decreased from 58.85% to 40.00%, with a relative control effect of 32.03%, a result far exceeding conventional expectations.

[0035] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A Pseudomonas palleroniana bacterium, comprising strain AQY14, characterized in that, Its taxonomic name is Pseudomonas palleroniana, strain number 16S rRNA, and it was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 23, 2026, with accession number GDMCC No:67636.

2. A microbial agent, characterized in that, The bacterial agent contains live bacteria of strain AQY14 as described in claim 1, as well as strain culture.

3. The bacterial agent according to claim 2, characterized in that, The bacterial culture is the fermentation broth, fermentation filtrate, bacterial suspension, and purified bacterial solution obtained by culturing the bacterial strain AQY14 in a culture medium, and the bacterial agent is a liquid preparation.

4. The bacterial agent according to claim 2, characterized in that, The culture media include Ashby medium, R2A medium and PDA medium.

5. The application of a Pseudomonas palleroniana bacterium microbial strain, characterized in that, The use of the Pseudomonas palleroniana bacterium microbial strain as described in claim 1 or the bacterial agent as described in any one of claims 2-4 in the preparation of biopharmaceuticals.

6. The application of the Pseudomonas palleroniana bacterium microbial strain according to claim 5, characterized in that, The biological drug is intended for use in one or more of the following applications: (1) Used to antagonize rice sheath blight; (2) Used to antagonize rice blast; (3) Used to antagonize rice bacterial blight; (4) Used to antagonize Fusarium graminearum in rice; (5) Used to antagonize rice graminearum; (6) Soluble phosphorus, dissolve potassium, produce IAA, produce iron carrier, fix nitrogen.

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