Pseudomonas Jersei LB4 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to Pseudomonas Jergeri LB4 and application thereof.The Pseudomonas Jergeri LB4 is obtained by screening and separating pear tree rhizosphere of calcareous soil by the applicant and has the capacity of producing IAA, solubilizing phosphorus, fixing nitrogen and producing siderophores, pot experiment shows that the LB4 can remarkably improve the root development form of pyrus betulaefolia seedlings, increase the seedling biomass and improve the yield of pyrus betulaefolia seedlings, and the yield of pyrus betulaefolia seedlings is increased. A good growth promoting effect is achieved on plants; the strain and iron oxide powder are mixed and cultured to prepare microbial iron-soluble fermentation liquor, and a foliage spraying method is adopted to find that the microbial iron-soluble fermentation liquor prepared by combining LB4 and exogenous iron can effectively promote peanut growth, increase the biomass of plants and remarkably improve the content of active iron and total iron in peanut leaves and root systems; and the feed has good growth promoting and iron supplementing functions.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Pseudomonas japonicus LB4 and its applications. Background Technology

[0002] Iron is one of the essential micronutrients for plant growth and development, participating in various physiological and metabolic processes throughout the plant life cycle. Although soil contains abundant iron resources, it exists mostly in oxidized forms that are difficult for plants to absorb and utilize. Especially in calcareous soils with high pH and high bicarbonate content, the bioavailability of iron is extremely low, leading to varying degrees of iron deficiency chlorosis in plants in such soils, which seriously affects the yield and quality of local fruit trees and crops.

[0003] Traditional fertilization methods, due to the excessive use of chemical fertilizers, have had a negative impact on the environment. For the sustainable development of green agriculture, utilizing beneficial microorganisms to maintain healthy plant development, improve nutrient absorption rates, and increase crop yields is an effective alternative. *Pseudomonas gypseus*, a common rhizosphere growth-promoting bacterium, promotes plant growth through direct mechanisms such as IAA production, phosphorus solubilization, potassium solubilization, and nitrogen fixation, or indirect mechanisms such as competing for ecological niches with pathogens and degrading plant toxins. Furthermore, *Pseudomonas gypseus* can activate insoluble iron in the soil environment through non-specific pathways such as secreting protons, phenolic compounds, and producing organic acids, as well as through a specific pathway of synthesizing and secreting microbial siderophores. Therefore, *Pseudomonas* is considered a core microbial group closely related to improving plant iron nutrition.

[0004] In actual agricultural production, foliar spraying of iron is the most common measure to correct crop chlorosis. However, most foliar fertilizers on the market are mainly ferrous sulfate, which is easily oxidized and lost, or expensive chelated iron fertilizers. At present, iron-producing microorganisms are widely used for plant root irrigation because they can directly utilize the abundant insoluble iron sources in the soil. However, there are few reports on the application of chelates between microorganisms and exogenous iron in foliar spraying. Summary of the Invention

[0005] In view of the above, it is necessary to address the technical shortcomings of commercially available foliar fertilizers, which are mostly based on easily oxidized and lost ferrous sulfate or expensive chelated iron fertilizers. This would involve utilizing microorganisms to produce chelates containing exogenous iron, conducting experiments on these chelates, developing corresponding foliar fertilizers to promote plant growth, increase plant iron content, prevent chlorosis, and prepare a new type of iron-containing bio-fertilizer.

[0006] To achieve the above objectives, this invention has screened out a new strain: *Pseudomonas jejuni*. Pseudomonas jessenii Strain LB4, its classification name is: Pseudomonas jesseniiLB4, Chinese classification name: Pseudomonas jejuni LB4, accession number CCTCC NO: M20241258; this strain is deposited at the China Center for Type Culture Collection, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on June 17, 2024.

[0007] The present invention also includes *Pseudomonas japonicus*. Pseudomonas jessenii Inoculum of strain LB4.

[0008] The present invention also includes the aforementioned *Pseudomonas jejuni*. Pseudomonas jessenii Application of strain LB4 or the aforementioned microbial agent in the preparation of bio-organic fertilizer and / or foliar fertilizer.

[0009] The present invention also includes the aforementioned *Pseudomonas jejuni*. Pseudomonas jessenii Application of strain LB4 or the aforementioned inoculant in promoting the growth of pears and / or peanuts.

[0010] The present invention also includes the aforementioned *Pseudomonas japonicus*. Pseudomonas jessenii The use of strain LB4 or the bacterial agent in the production of IAA, phosphorus solubilization, phosphorus solubilization, nitrogen fixation and / or siderophore production.

[0011] The present invention also includes the aforementioned *Pseudomonas japonicus*. Pseudomonas jessenii A method for planting plants using strain LB4 or the aforementioned microbial agent, wherein the method comprises adding a microbial suspension containing strain LB4, bio-organic fertilizer, and / or foliar fertilizer during the seedling stage of the plant.

[0012] Furthermore, the plant is a pear and / or a peanut.

[0013] The present invention also includes a method for preparing the foliar fertilizer, the method comprising: using activated Pseudomonas gypseum... Pseudomonas jessenii The bacterial suspension of strain LB4 was inoculated into 10% MSA medium containing excess iron oxide powder, and the bacterial iron dissolution fermentation broth was obtained. The supernatant recovered after centrifugation was the foliar fertilizer.

[0014] The present invention has the following beneficial effects: The strain LB4 of the present invention was isolated by the applicant from the rhizosphere of pear trees in calcareous soil. It has the ability to produce IAA, solubilize phosphorus, fix nitrogen and produce iron carriers. Pot experiments showed that LB4 can significantly improve the root development morphology of pear seedlings, increase seedling biomass and has a good growth-promoting effect on plants. The microbial iron-solubilizing fermentation liquid made by combining this strain with exogenous iron can effectively promote peanut growth, significantly increase peanut biomass, and significantly increase the active iron and total iron content in peanut leaves and roots by foliar spraying. It also has a good growth-promoting and iron-supplementing function. Attached Figure Description

[0015] Figure 1 This is a colony morphology diagram of strain LB4.

[0016] Figure 2 Phylogenetic tree diagram of strain LB4 constructed based on the 16S rRNA sequence.

[0017] Figure 3 Figure showing the results of a potted experiment on pear trees inoculated with strain LB4.

[0018] Figure 4 This is a diagram showing the experimental results of strain LB4 on the aboveground dry weight of pear seedlings.

[0019] Figure 5 The figure shows the experimental results of strain LB4 on the root dry weight of pear seedlings.

[0020] Figure 6 The figure shows the experimental results of strain LB4 on the total dry weight of pear seedlings.

[0021] Figure 7 This is a diagram showing the experimental results of strain LB4 on the root growth of pear seedlings.

[0022] Figure 8 This is a diagram showing the experimental results of strain LB4 on the root growth of pear seedlings.

[0023] Figure 9 The figure shows the experimental results of strain LB4 on the root surface area of ​​pear seedlings.

[0024] Figure 10 The figure shows the experimental results of strain LB4 on the number of root tips in pear seedlings.

[0025] Figure 11 Figure 1 shows the results of a pot experiment on peanut seedlings treated with iron soluble fermentation broth from strain LB4.

[0026] Figure 12 The figure shows the experimental results of the iron dissolving fermentation broth of strain LB4 on the aboveground dry weight of peanut seedlings.

[0027] Figure 13 The figure shows the experimental results of the iron dissolution fermentation broth of strain LB4 on the dry weight of peanut seedling roots.

[0028] Figure 14 The figure shows the experimental results of the iron dissolution fermentation broth of strain LB4 on the total dry weight of peanut seedlings.

[0029] Figure 15 The figure shows the experimental results of the iron dissolution fermentation broth of strain LB4 on the total iron content of peanut seedling leaves.

[0030] Figure 16 The figure shows the experimental results of the iron soluble fermentation broth of strain LB4 on the total iron content of peanut seedling roots.

[0031] Figure 17 The figure shows the experimental results of the total iron accumulation in peanut seedlings by the iron dissolution fermentation broth of strain LB4. Information on the preservation of biological materials

[0032] The strain information deposited in this application is: *Pseudomonas gypseum*. Pseudomonas jessenii Strain LB4, its classification name is: Pseudomonas jessenii LB4, Chinese classification name: Pseudomonas jejuni LB4, accession number CCTCC NO: M20241258; this strain is deposited at the China Center for Type Culture Collection, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on June 17, 2024. Detailed Implementation

[0033] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0034] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features. Example 1

[0035] This example demonstrates the isolation and identification of Pseudomonas jejuni LB4.

[0036] 1. Isolation and purification of strain LB4: 1g of rhizosphere soil from pear orchards in calcareous soil of Xinxiang, Henan Province was collected and added to a 50ml Erlenmeyer flask containing glass beads and 9ml of SM buffer. The soil suspension was obtained by shaking at 170°C for 30min at 30°C. The suspension was then gradually diluted with sterile water to a concentration of 10⁻⁵ to 10⁻⁷. The suspension was spread on TSA medium and incubated at 30°C for 48h. Single bacteria were picked by streak plate method to purify the strain.

[0037] 2. Identification of strain LB4: ① Morphological identification: The specific procedure is as follows: Inoculate strain LB4 onto TSB medium and incubate in a constant temperature incubator. After 24 hours of incubation, observe until round colonies appear on the petri dish. Figure 1 As shown, Figure 1 In the middle, the colonies are milky white, opaque, with neat and smooth edges, and a smooth and moist surface. The colonies are regularly round, relatively uniform in size, with no wrinkles or protrusions on the surface, and no diffuse or flocculent edges are observed, which is consistent with the morphological characteristics of Pseudomonas jejuni LB4; ② After extracting and sequencing the DNA of the strain, molecular identification was performed, and a phylogenetic tree was constructed. Combined with the phylogenetic tree ( Figure 2 Based on morphological identification, the strain was classified and named. Pseudomonas jessenii . Example 2

[0038] This example demonstrates the growth-promoting function of strain LB4.

[0039] 1. Qualitative determination of IAA function of strain LB4: LB4 strain was added to TSB medium containing L-tryptophan at an inoculum of 1%, and incubated at 30℃ and 170 r·min. -1 After shaking for 48 hours, the supernatant was obtained by centrifugation at 10,000 rpm for 5 minutes. 100 μL of the supernatant was mixed with an equal volume of Salkowski colorimetric solution. 100 μL of uninoculated culture medium was mixed with an equal volume of Salkowski colorimetric solution as a blank control. The mixture was reacted for 30 minutes in the dark. The solution turning red indicates that the strain has the ability to produce IAA.

[0040] 2. Determination of nitrogen fixation and phosphorus solubility of the strain: 10 μL of the activated LB4 bacterial suspension was added to Ashube nitrogen fixation medium, Monkina organic phosphorus medium and PKO inorganic phosphorus medium respectively, with three replicates for each. The culture was carried out in an incubator at 28℃ for 5 days. The presence of halos or clear zones on the medium was observed. The ability of the strain to fix nitrogen, solubilize organic phosphorus and inorganic phosphorus was determined by the ratio of the halo or clear zone (D) to the colony diameter (d).

[0041] 3. Determination of siderophore production capacity: Following the method described by Schwyn and Neilands, the bacterial suspension cultured in MSA medium under iron-limited conditions for 48 h was centrifuged at 3500 rpm for 10 min through a perforated membrane filter. The supernatant was mixed with CAS detection solution at a 1:1 ratio, allowed to stand for 2 h, and the OD was measured. 630 The absorbance (A) at nm was measured; the control was sterile MSA medium, which was also mixed with the CAS test solution in the same proportion, and the absorbance (Ar) was measured. The relative content of siderophores was calculated according to the formula [(Ar-As) / Ar]×100%.

[0042] The results of the above growth-promoting characteristics are shown in Table 1.

[0043]

[0044] As shown in Table 1, strain LB4 has the ability to produce IAA, fix nitrogen, solubilize inorganic phosphorus, solubilize organic phosphorus, and produce siderophores. Example 3

[0045] Study on the growth-promoting effect of strain LB4 on pear seedlings.

[0046] (1) Pear seedling cultivation: Pear seeds are soaked in sterile water for 24 hours, then soaked in 75% alcohol for 30 seconds, rinsed 3-5 times with sterile water, then soaked in 2% sodium hypochlorite for 20 minutes, and finally rinsed clean with sterile water. The sterilized seeds are spread evenly on a culture medium with sterile water-soaked filter paper, and placed in a 28℃ constant temperature incubator to germinate for 3-5 days. Germinated seeds are selected and sown into sterilized substrate for seedling cultivation. When the pear seedlings in the substrate have 2-3 true leaves, healthy and uniform seedlings are selected and transplanted into sterilized substrate flower pots.

[0047] (2) Inoculation: The prepared LB4 bacterial suspension (final concentration 10) 7 CFU / g soil was inoculated into the rhizosphere of *Pyrus pyrifolia* seedlings, with no inoculation as a control. Each treatment was replicated 12 times. After 30 days of culture, samples were collected to determine the phenotypic traits of *Pyrus pyrifolia*.

[0048] The test results are as follows Figure 3 It is evident that the growth of pear seedlings inoculated with strain LB4 was significantly better than that of uninoculated pear seedlings, which clearly demonstrates that inoculation with strain LB4 promotes the growth of pear seedlings.

[0049] Figures 4-6 To compare the dry weight of pear seedlings, from Figures 4-6 As can be seen, compared with the control, the aboveground dry weight, root dry weight and total dry weight of the pear seedlings in the LB4 strain experimental group were significantly increased by 65.63%, 77.11% and 67.35%, respectively.

[0050] Figure 7 The results of the comparison of root morphology of *Pyrus pyrifolia* seedlings are as follows: Figure 7 It is evident that the root system of pear seedlings inoculated with LB4 strain was more developed, and the root morphology was significantly improved. Figures 8-10 This shows a comparison of root length, root surface area, and root tip. Figures 8-10 It can be seen that, compared with the control group, the root length, root surface area and root tip number of the pear seedlings in the LB4 strain experimental group were significantly increased by 33.69%, 29.40% and 29.40%, respectively. Example 4

[0051] Study on the effects of fermentation broth of strain LB4 on peanut growth and iron accumulation.

[0052] (1) Preparation of LB4 iron dissolving fermentation broth: The activated LB4 bacterial suspension was inoculated into 10% MSA medium containing excess iron oxide powder (0.5 g) at an inoculation rate of 1%, and incubated at 30°C and 170 r·min. -1 The bacterial iron-dissolving fermentation broth was obtained by shaking the flask for 24 hours. After centrifugation, the supernatant was recovered and used for foliar spraying.

[0053] (2) Seedling raising: Peanut seeds are soaked in sterile water for 12 hours, then disinfected with 1% hydrogen peroxide for 20 minutes, and finally rinsed with sterile water. The disinfected seeds are spread evenly on a culture medium with sterile water-soaked filter paper, and placed in a 28℃ constant temperature incubator for 3-5 days to germinate. Peanut seeds with sprouted white leaves are selected and sown in sterilized substrate for seedling raising. When the peanut seedlings have 2-3 true leaves, healthy and uniform seedlings are selected and transplanted into sterilized mixed soil with a calcareous soil:quartz sand ratio of 3:2.

[0054] (3) Spraying treatment: Add surfactant (0.1% Tween 20) before spraying to ensure better spraying effect. When spraying, pay attention to spraying both the front and back of the leaves until they are wet. Spray once every 7 days. Spray a total of 3 times in the whole experiment. After 21 days of treatment, collect samples and measure the indicators.

[0055] Experimental results: ① The experimental results on the growth of peanut seedlings are as follows: Figure 11 visible, Figure 11 The growth chart of peanut seedlings shows that the LB4 experimental group sprayed with LB4 iron-containing fermentation liquid and the experimental group sprayed with EDTA-Fe solution have similar growth, which is significantly better than the control group that was not sprayed with any iron-containing solution. Figures 12-14 The figure shows the dry weight experimental results for each experimental group. As can be seen from the figure, compared with the control, the aboveground dry weight, root dry weight, and total dry weight of peanut seedlings treated with LB4 soluble iron fermentation broth increased significantly by 40.62%, 43.20%, and 40.91%, respectively. The aboveground dry weight and total dry weight were comparable to those treated with EDTA-Fe, while the root dry weight significantly exceeded that of the EDTA-Fe treatment. ② The experimental results of peanut iron nutrition are as follows: Figures 15-16 As shown in the figure, compared with the control, the total iron content in peanut leaves and roots treated with LB4 iron-soluble fermentation broth was significantly increased, by 112.28% and 50.89% respectively, with the total iron content in leaves significantly exceeding that of the EDTA-Fe treatment. Compared with the control, the total iron accumulation in peanuts treated with LB4 iron-soluble fermentation broth was significantly increased by 128.33%, comparable to the effect of the EDTA-Fe treatment (e.g., ...). Figure 17 (As shown).

[0056] In summary, the *Pseudomonas jejuni* LB4 strain screened by the applicant of this invention has the ability to produce IAA, solubilize phosphorus, fix nitrogen, and produce iron carriers. Pot experiments showed that LB4 can significantly promote the growth of pear and peanut seedlings and effectively improve root development morphology, thus having a good growth-promoting effect on plants. In addition, foliar spraying of a microbial iron-solubilizing fermentation broth made by combining LB4 with exogenous iron can significantly increase peanut biomass, significantly increase the active iron and total iron content in peanut leaves and roots, and has a good growth-promoting and iron-supplementing function.

[0057] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Pseudomonas japonicus Pseudomonas jessenii The strain LB4 has a deposit number of CCTCC NO:M20241258.

2. Comprising the Pseudomonas japonicus according to claim 1 Pseudomonas jessenii Inoculum of strain LB4.

3. Pseudomonas japonicus according to claim 1 Pseudomonas jessenii Use of the strain LB4 or the bacterial agent as claimed in claim 2 in the preparation of biological organic fertilizer and / or foliar fertilizer.

4. Pseudomonas japonicus according to claim 1 Pseudomonas jessenii Use of strain LB4 or the bacterial agent as claimed in claim 2 in promoting the growth of pears and / or peanuts.

5. Pseudomonas japonicus according to claim 1 Pseudomonas jessenii Use of strain LB4 or the bacterial agent as claimed in claim 2 in producing IAA, phosphate solubilization, phosphate dissolution, nitrogen fixation and / or siderophore production.

6. Use of the Pseudomonas japonicus as claimed in claim 1 Pseudomonas jessenii The method for planting plants using the strain LB4 or the bacterial agent as claimed in claim 2, characterized in that: The method comprises: adding a bacterial suspension containing strain LB4, biological organic fertilizer and / or foliar fertilizer to plants at the seedling stage; and the plants are pears and / or peanuts.

7. A method for preparing the foliar fertilizer as claimed in claim 3, characterized in that: The method comprises: Pseudomonas jessenii The bacterial suspension of strain LB4 is inoculated into a 10% MSA medium containing excess iron oxide powder, and the bacterial iron-dissolving fermentation liquid is obtained by culture. The supernatant recovered after centrifugation is the foliar fertilizer.

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