Pseudomonas burcheri strain ZJL-8 and application thereof
By screening and preserving Pseudomonas brunettii ZJL-8, the problems of fertilizer pollution and lack of microbial agents in the blueberry industry have been solved, achieving the goals of promoting blueberry growth and industrial development, and providing an environmentally friendly solution for large-scale planting.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NORTHEAST FORESTRY UNIV
- Filing Date
- 2025-12-29
- Publication Date
- 2026-07-03
AI Technical Summary
The blueberry industry suffers from severe fertilizer pollution, a lack of specialized microbial agents, and poor performance of conventional microbial agents in acidic soils, which limits blueberry production and industrial development.
A strain of *Pseudomonas bryonicus* ZJL-8 was screened and preserved, possessing potential growth-promoting abilities such as nitrogen fixation, inorganic phosphorus dissolution, siderophore secretion, and indoleacetic acid synthesis. It was inoculated into the rhizosphere of blueberry to promote its growth.
It significantly promotes blueberry growth, improves root efficiency in nutrient absorption, reduces reactive oxygen species accumulation, and enhances plant health, making it suitable for large-scale planting and commercial development.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Pseudomonas brucellosa ZJL-8 and its applications. Background Technology
[0002] Blueberry (Vaccinium vitis-idaea L.) is a small, wild, vine-like berry plant belonging to the genus Vaccinium in the family Ericaceae. Its fruit is highly valued for its nutritional and health benefits, being rich in vitamins, trace elements, and unique polyphenols and anthocyanins. Currently, the market demand for blueberries is increasing year by year, but yields remain low and industrialization is not yet large-scale. Therefore, the selection and application of fertilizers are particularly important: excessive use of chemical fertilizers can pollute soil and water sources, and residual substances may pose health risks; meanwhile, the development of specialized microbial agents to promote its growth is lagging behind, far from meeting production needs. This restricts the large-scale development and yield increase of blueberries, and also affects the sustainable development of agriculture and forestry.
[0003] Applying more organic and microbial fertilizers is the core approach to reducing fertilizer use. However, organic fertilizers are expensive, and the scarcity of microbial fertilizers specifically for blueberries restricts policy implementation. In addition, long-term over-harvesting and destruction of the growing environment have led to a sharp decline in wild blueberry populations. Therefore, developing microbial growth promoters to increase blueberry yield and growth rate is of great significance for solving the problem of chemical fertilizer pollution and promoting industrial development.
[0004] Wild blueberries grow in acidic environments due to soil exudates, and blueberries themselves exhibit good adaptability to acidic soils. This soil characteristic significantly limits the effectiveness of conventional microbial agents, making them ineffective in blueberry cultivation. Therefore, screening for growth-promoting microorganisms derived from the blueberry rhizosphere is crucial. Microorganisms from the blueberry rhizosphere have formed stable mutualistic symbiotic relationships through long-term interaction and ecological selection with the host. Compared to non-blueberry rhizosphere microorganisms, they possess higher host affinity and colonization capacity. When prepared as microbial fertilizers, they more easily form a more stable "near-natural mutualistic symbiotic system" with blueberries under artificial additive conditions. Summary of the Invention
[0005] The purpose of this invention is to provide a bacterial strain that can effectively promote the growth of blueberries, thereby solving the environmental pollution problems caused by the application of chemical fertilizers and the shortage of specialized bacterial agents in production. To achieve this objective, this invention screened a bacterial strain ZJL-8 from the rhizosphere soil of blueberries and deposited it at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20242867, named *Pseudomonas brenneri* ZJL-8.
[0006] This strain possesses potential growth-promoting abilities including nitrogen fixation, inorganic phosphorus dissolution, siderophore secretion, and indoleacetic acid (IAA) synthesis. When inoculated into the rhizosphere of blueberry, it significantly promotes the growth and development of blueberry. Furthermore, this strain is easy to preserve, enabling large-scale production, and offers advantages such as environmental friendliness and low cost, providing strong technical support for the large-scale cultivation and commercial development of blueberry. Attached Figure Description
[0007] Figure 1 Morphological observation of bacterial strain ZJL-8;
[0008] Figure 2 Gram staining results of bacterial strain ZJL-8;
[0009] Figure 3 Phylogenetic tree of bacterial strain ZJL-8;
[0010] Figure 4 Image showing the growth status of bacteria ZJL-8 nitrogen-fixing plate;
[0011] Figure 5 Figure 1. Qualitative results of bacterial ZJL-8 dissolving inorganic phosphorus plate assay.
[0012] Figure 6 Figure showing the results of a quantitative experiment on the dissolution of inorganic phosphorus by bacteria ZJL-8.
[0013] Figure 7 Figure showing the qualitative results of the ZJL-8 bacteria siderophore plate experiment;
[0014] Figure 8 Figure showing the quantitative experimental results of siderogenic carrier production by bacterial ZJL-8;
[0015] Figure 9 Graph showing the IAA production capacity test results of bacteria ZJL-8;
[0016] Figure 10 Image showing the condition of blueberry plants after inoculation with bacterial ZJL-8;
[0017] Figure 11 Figure showing the results of malondialdehyde (MDA) content detection in blueberry leaves 30 days after inoculation with bacterial ZJL-8;
[0018] Figure 12 Figure showing the results of proline content detection in blueberry leaves 30 days after inoculation with bacterial ZJL-8. Detailed Implementation
[0019] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.
[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.
[0021] Example 1: Isolation and Identification of Bilberry Rhizosphere Bacterial Strain ZJL-8
[0022] (1) Isolation and purification of rhizosphere bacteria strain ZJL-8
[0023] Weigh 1 g of soil sample using a balance. Aseptically add the sample to a sterile Erlenmeyer flask containing 9 mL of sterile water and 10 sterile glass beads. Shake at 30°C for 20 min to evenly disperse the soil particles in the sterile water. Let stand for 10 min, then perform serial dilution to 1 / 10 of the stock solution. -3 10 -4 10 -5 Three gradient concentrations were used, with 100 μL of each concentration spread onto TSB solid medium in triplicate. The medium was incubated at 30°C for 2 days. Single colonies of dominant bacteria with a high colony ratio were repeatedly streaked until no other bacteria grew.
[0024] (2) Identification of rhizosphere bacterial strain ZJL-8
[0025] Following the methods described in Bergey's Manual of Bacteriological Identification (8th Edition), morphological observation, Gram staining, and molecular identification analysis were performed on the dominant bacterial strains with a large colony proportion. The specific results are as follows:
[0026] Morphological observation and Gram staining results: Strain ZJL-8 was inoculated onto TSB solid medium and incubated at 30℃ for 48 h. Colony morphology was observed. Morphological observation of rhizosphere bacteria strain ZJL-8 is as follows: Figure 1 As shown, the isolated colonies are round, light yellow, smooth, moist, and viscous, easy to pick up, and show no obvious dry or rough characteristics; the Gram staining results of rhizosphere bacteria strain ZJL-8 are as follows. Figure 2 As shown, the rhizosphere bacterial strain ZJL-8 is Gram-negative and has no endospores.
[0027] 16S rDNA Molecular Identification and Analysis: Genomic DNA was extracted from the rhizosphere bacterial strain ZJL-8, and PCR amplification was performed using universal primers for the 16S rDNA gene. The sequencing results are shown below:
[0028]
[0029] The obtained sequences were compared with data in GenBank using the BLAST tool on NCBI. The results showed that the rhizosphere bacterium ZJL-8 shared 99.86% homology with the 16S rRNA gene sequence of Pseudomonas brenneri strain XJC-6 (accession number MT631985.1), and 99.79% homology with the 16S rRNA gene sequence of Pseudomonas brenneri strain 147 (JX417436.1) and the 16S rRNA gene sequence of Pseudomonas brenneri strain s2-1 (PQ803941.1). A phylogenetic tree of the rhizosphere bacterium strain ZJL-8 was constructed using closely related sequences as shown below. Figure 3 As shown, based on morphological observation, Gram staining results, and 16S rDNA molecular identification analysis, this strain was identified as Pseudomonas brenneri.
[0030] The bacterium ZJL-8 was further deposited at the China Center for Type Culture Collection on December 19, 2024, with accession number CCTCC NO: M 20242867, and named Pseudomonas brenneri ZJL-8.
[0031] Example 2: Detection of the in vitro growth-promoting ability of bacteria ZJL-8
[0032] (1) Detection of nitrogen fixation capacity of bacteria ZJL-8
[0033] A single colony of bacteria ZJL-8 was picked and inoculated into 30 mL of TSB medium. After overnight incubation at 30°C and 180 rpm, it was streaked onto Assumption solid medium and cultured under suitable conditions for a period of time to observe whether the strain grew.
[0034] The growth status of bacteria on ZJL-8 nitrogen-fixing plates is as follows: Figure 4 It is evident that bacteria ZJL-8 can grow in Assumption solid medium, proving that it has a certain nitrogen-fixing capacity.
[0035] (2) Detection of the ability of bacteria ZJL-8 to dissolve inorganic phosphorus
[0036] Qualitative experiment on the phosphorus solubility of bacterial strain: A single colony of bacterial strain ZJL-8 was picked and inoculated into 30 mL of TSB medium. After overnight culture at 30℃ and 180 rpm, 30 μL of the bacterial solution was spot-inoculated onto PKO solid medium and cultured in the dark for 7 days. Photos were taken and recorded at 1 day, 3 days, 5 days and 7 days to observe whether phosphorus-solubilizing transparent zones were produced. The inorganic phosphorus solubility of bacterial strain ZJL-8 was preliminarily tested.
[0037] The results of the qualitative test of bacterial ZJL-8 dissolving inorganic phosphorus plates are as follows: Figure 5 It is evident that a clear zone exists on the PKO solid medium, and the diameter of the phosphate-solubilizing zone increases with longer culture time, demonstrating that the strain has good phosphate-solubilizing ability.
[0038] Quantitative experiment on the inorganic phosphorus solubility of bacterial strain ZJL-8: A single colony of the strain was picked and inoculated into 30 mL of TSB medium. After overnight incubation at 30°C and 180 rpm, the bacterial cells were collected by centrifugation and diluted with sterile water to OD200. 600 =0.5, take 1% inoculum, inoculate the activated bacterial strain ZJL-8 into PKO liquid medium, three replicates per group, and incubate at 30℃, 150-180rpm. Take samples at 3 days, 5 days, and 7 days, centrifuge at 12000rpm for 5 min, take the supernatant and mix it with 4 mL of colorimetric solution, make up to 25 mL with deionized water, react for 30 min, and then measure the OD value at 882 nm.
[0039] The results of the quantitative experiment on the dissolution of inorganic phosphorus by bacteria ZJL-8 are as follows: Figure 6 As shown, compared with CK, the phosphorus concentration in the culture medium containing bacterial strain ZJL-8 was significantly increased, and there was a large amount of soluble phosphorus in the culture medium, indicating that the strain had a good phosphorus solubility.
[0040] (3) Detection of siderophore production capacity of bacterial strain ZJL-8
[0041] Qualitative experiment on siderophore production by bacterial strain ZJL-8: A single colony of the strain was picked and inoculated into 30 mL of TSB medium and cultured overnight at 30°C and 180 rpm. Then, 30 μL of the bacterial solution was inoculated onto CAS solid medium and cultured at 30°C in the dark for 48 h. The presence of an orange siderophore halo around the colony was observed.
[0042] The results of the qualitative test of ZJL-8 siderophore plate of bacteria are as follows: Figure 7 As shown, a distinct orange siderophore halo appears in the CAS solid medium, proving that the strain has a certain siderophore production capacity.
[0043] Quantitative experiment on siderophore production by bacterial strain ZJL-8: The isolated strain was cultured in MKB liquid medium at 30℃ and 180 r / min for 48 h. The culture medium was centrifuged at 10000 r / min for 2-3 min, and 0.5 mL of the supernatant was mixed with CAS detection solution at a volume ratio of 1:1. After reacting at room temperature for 1 h, the OD value (As) was measured at 630 nm to determine the OD value (Ar) of the mixture of MKB medium and CAS detection solution without inoculation of the strain.
[0044] The (Ar-As) / Ar ratio represents the relative content of siderophores in the sample; a higher ratio indicates a stronger siderophore-producing ability of the strain. During the experiment, if the strain produces siderophores, the mixture gradually turns pink; conversely, if it does not produce siderophores, the mixture remains dark blue. In this example, the quantitative experimental results of siderophore production by bacterium ZJL-8 are as follows: Figure 8 As shown, the (Ar-As) / Ar ratio was 0.61 after quantitative detection, and the mixture was pink, proving that the ZJL-8 bacterial strain has good siderophore production ability.
[0045] (4) Detection of IAA production capacity of bacterial strain ZJL-8
[0046] Bacterial strain ZJL-8 was inoculated into TSB medium containing 100, 200, and 500 mg / L L-tryptophan, respectively. The blank control was TSB medium without bacteria. After incubation, the liquid was centrifuged at 10,000 r / min for 3 min, and 1 mL of the supernatant was transferred to a test tube containing 4 mL of Salkowski chromogenic solution. After reacting in the dark for 20 min, the OD value at 530 nm was measured.
[0047] The results of the IAA production capacity test of bacteria ZJL-8 are as follows: Figure 9 As shown, the increase in tryptophan concentration in the culture medium was accompanied by an increase in IAA synthesis by the strain, demonstrating a positive correlation between tryptophan concentration and IAA synthesis. At an L-tryptophan concentration of 500 mg / L, strain ZJL-8 synthesized 43.35 mg / L of IAA, indicating a strong IAA production capacity.
[0048] Combining the above four growth-promoting ability tests, the results all indicate that the bacteria ZJL-8 has a good in vitro growth-promoting effect. Further, it was inoculated into the plant root system to test its in vivo growth-promoting effect.
[0049] Example 3: Plant growth-promoting effect of bacteria ZJL-8
[0050] (1) Preparation of bacterial ZJL-8 inoculum
[0051] Bacterial ZJL-8 was inoculated into 100 mL of TSB medium and cultured in a constant temperature shaking incubator at 30℃ and 180 rpm for 24 h. The bacterial cells were then diluted with sterile water to OD. 600 =1, at which point the viable bacteria concentration is approximately 1×10⁻⁶. 7 CFU / mL, which means the preparation of bacterial ZJL-8 inoculum is complete;
[0052] (2) Effects of bacteria ZJL-8 on blueberry growth
[0053] Two mL of the bacterial suspension ZJL-8 prepared in this example was inoculated into the rhizosphere of sterile blueberry seedlings. Growth and physiological indicators of the blueberries were measured 30 days after inoculation. Bacterial ZJL-8 was inoculated only once during the experiment. To facilitate observation of the growth characteristics of blueberry seedlings inoculated with bacterial ZJL-8 (experimental group), a treatment (control group) of uninoculated blueberry seedlings with bacterial ZJL-8 was established. The plant status after inoculation with bacterial ZJL-8 is shown in the figure below. Figure 10 As shown in the table, the growth morphology of blueberry seedlings inoculated with ZJL-8 was significantly better than that of the control group. Further comparison of the average plant height and fresh weight of blueberry seedlings under different treatments, as shown in Table 1 (root and stem length detection results 30 days after inoculation with ZJL-8) and Table 2 (growth status detection results 30 days after inoculation with ZJL-8), reveals that the experimental group of blueberry seedlings had taller plants, heavier fresh weights, and better leaf development. The roots of the blueberry seedlings inoculated with ZJL-8 were also thicker, improving the efficiency of nutrient absorption. Simultaneously, the leaf MDA content detection results 30 days after inoculation with ZJL-8 showed a significant increase. Figure 11 It can be seen that the MDA content in the experimental group decreased significantly, possibly due to the reduction of reactive oxygen species accumulation through the secretion of antioxidants, thereby lowering the MDA content; the proline content in the leaves of blueberries inoculated with bacterial ZJL-8 for 30 days was measured by... Figure 12 It can be seen that the proline content in the experimental group increased significantly, proving that the application of strain ZJL-8 can induce plants to synthesize more proline.
[0054] Table 1. Results of root and stem length measurements of blueberries 30 days after inoculation with bacterial ZJL-8.
[0055]
[0056] Table 2. Growth status of blueberries inoculated with ZJL-8 bacteria 30 days later.
[0057]
Claims
1. A Pseudomonas burcheri ZJL-8 and its application, characterized in that, The strain is classified as Pseudomonas brenneri ZJL-8 and was deposited on December 19, 2024, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20242867.
2. A microbial inoculant, characterized in that, The microbial agent contains a fermentation culture of Pseudomonas brucellae ZJL-8 as described in claim 1.
3. The method for preparing a microbial inoculant according to claim 2, characterized in that, The specific steps are as follows: Pseudomonas BZJL-8 is inoculated into 100 mL TSB medium, and after being cultured in a constant temperature shaking incubator at 30℃ and 180 rpm for 24 h, the bacterial body is diluted to OD 600 =1 with sterile water, at which time the viable bacterial concentration is about 1×10 7 CFU / mL, that is, Pseudomonas BZJL-8 microbial inoculant is obtained.
4. The application of the microbial inoculant according to claim 2 in the growth of blueberries.
5. The application of the microbial inoculant according to claim 2 in promoting the growth of blueberry stems and increasing the fresh weight of stems and leaves.
6. The application of the microbial agent according to claim 2, which reduces malondialdehyde (MDA) content and increases proline content, thereby improving the stress resistance of blueberry plants.
7. A method for promoting blueberry growth, characterized in that, The microbial agent described in claim 2 was used to inoculate blueberry seedlings.
8. The method of promoting growth of blueberries of claim 7, wherein, The treatment method involves inoculating the rhizosphere soil of blueberries with microbial agents at a depth of 1-2 cm.