Pseudomonas jejuni LB4 and its application

Abstract

The invention relates to the field of microbial technology, in particular to Pseudomonas japonicus LB4 and applications thereof. The strain LB4 of the invention is obtained by screening and separating from the rhizosphere of pear trees in calcareous soil by an applicant, has the ability to produce IAA, dissolve phosphorus, fix nitrogen and produce iron carriers. Through potted plant experiments, it is found that LB4 can significantly improve the root development morphology of pear seedlings, increase the biomass of seedlings, and has a good growth-promoting effect on plants. The strain is mixed and cultured with iron oxide powder to prepare a microbial iron-dissolving fermentation liquid, and a foliar spraying method is adopted. It is found that the microbial iron-dissolving fermentation liquid prepared by combining LB4 with exogenous iron can effectively promote the growth of peanuts, increase the biomass of plants, and significantly increase the active iron and total iron content of peanut leaves and roots, and has good growth-promoting and iron-supplementing functions.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Pseudomonas jejuni LB4 and applications thereof. Background Art

[0002] Iron is one of the trace elements necessary for plant growth and development, and is involved in a variety of physiological metabolic processes during the plant life cycle. Although the soil is rich in iron resources, most of them exist in oxidized forms that are difficult for plants to absorb and utilize. Especially in calcareous soils with high pH and high bicarbonate content, the biological effectiveness of iron is extremely low, resulting in varying degrees of iron deficiency and chlorosis in plants on the soil, which seriously affects the yield and quality of local fruit trees and crops.

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

[0004] In actual agricultural production, spraying iron supplements on leaves is the most common measure to correct crop chlorosis, but foliar fertilizers on the market are mostly ferrous sulfate, which is easily oxidized and lost, or expensive chelated iron fertilizers. Currently, iron-carrier-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 foliar application of chelates of microorganisms and exogenous iron. Summary of the invention

[0005] In view of the above, it is necessary to address the technical defects of foliar fertilizers on the market, which are mainly ferrous sulfate that is easily oxidized and lost, or expensive chelated iron fertilizers, and use microorganisms to produce chelates containing exogenous iron, and test the chelates to develop corresponding foliar fertilizers for promoting plant growth, increasing plant iron content, and preventing chlorosis, thereby preparing a new type of iron-containing biological fertilizer.

[0006] In order to achieve the above purpose, the present invention screened out a new strain: Pseudomonas jejuni Pseudomonas jessenii Strain LB4, whose classification is named: Pseudomonas jesseniiLB4, Chinese classification name: Pseudomonas japonicus LB4, preservation number is CCTCC NO: M20241258; the strain is deposited in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is June 17, 2024.

[0007] The present invention also includes the Pseudomonas jesii Pseudomonas jessenii Inoculum of strain LB4.

[0008] The present invention also includes the Pseudomonas jesii Pseudomonas jessenii Application of strain LB4 or the bacterial agent in the preparation of biological organic fertilizer and / or foliar fertilizer.

[0009] The present invention also includes the Pseudomonas jesii Pseudomonas jessenii Application of strain LB4 or the bacterial agent in promoting the growth of pears and / or peanuts.

[0010] The present invention also includes the Pseudomonas jesii Pseudomonas jessenii Application of strain LB4 or the bacterial agent in producing IAA, phosphate solubilization, phosphate dissolution, nitrogen fixation and / or siderophore production.

[0011] The present invention also includes the Pseudomonas jesii Pseudomonas jessenii The method for planting plants with strain LB4 or the bacterial agent comprises: adding a bacterial suspension containing strain LB4, biological organic fertilizer and / or foliar fertilizer to the plants during the seedling stage.

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

[0013] The present invention also includes a method for preparing the foliar fertilizer, which 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.

[0014] The present invention has the following beneficial effects: the strain LB4 of the present invention is obtained by screening and separating from the rhizosphere of pear trees in calcareous soil by the applicant, and has the ability to produce IAA, dissolve phosphorus, fix nitrogen and produce iron carriers. Through potted experiments, it is found that LB4 can significantly improve the root development morphology of pear seedlings, increase the biomass of seedlings, and has a good growth-promoting effect on plants; the microbial iron-dissolving fermentation liquid prepared by combining the strain with exogenous iron can effectively promote the growth of peanuts, significantly increase the biomass of peanuts, and significantly increase the active iron and total iron content of peanut leaves and roots, and has good growth-promoting and iron-supplementing functions. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0017] Figure 3 This is the result of a potted experiment in which strain LB4 was inoculated into Pyrus betula.

[0018] Figure 4 This is the experimental result of strain LB4 on the aboveground dry weight of Pyrus betulaeformis seedlings.

[0019] Figure 5 This is the experimental result of strain LB4 on the dry weight of roots of Pyrus betulaeformis seedlings.

[0020] Figure 6 This is the experimental result of strain LB4 on the total dry weight of Pyrus betulaeformis seedlings.

[0021] Figure 7 This is the experimental result of the effect of strain LB4 on the root growth of Pyrus betulaeformis seedlings.

[0022] Figure 8 This is the experimental result of the effect of strain LB4 on the root length of Pyrus betulaeformis seedlings.

[0023] Fig. 9 This is the experimental result of strain LB4 on the root surface area of ​​Pyrus betulaeformis seedlings.

[0024] Fig.10 This is the experimental result of the effect of strain LB4 on the number of root tips of Pyrus betulaeformis seedlings.

[0025] Fig.11 This is the result of a potted experiment in which peanut seedlings were sprayed with the iron-dissolving fermentation liquid of strain LB4.

[0026] Fig.12 This is the experimental result of the effect of the iron-dissolving fermentation liquid of strain LB4 on the aboveground dry weight of peanut seedlings.

[0027] Fig.13 This is the experimental result of the effect of iron-dissolving fermentation liquid of strain LB4 on the dry weight of peanut seedling roots.

[0028] Fig.14 This is the experimental result of the effect of iron-dissolving fermentation liquid of strain LB4 on the total dry weight of peanut seedlings.

[0029] Fig.15 This is the experimental result of the effect of iron-dissolving fermentation liquid of strain LB4 on the total iron content of peanut seedling leaves.

[0030] Fig.16 This is the experimental result of the effect of the iron-dissolving fermentation liquid of strain LB4 on the total iron content of peanut seedling roots.

[0031] Fig.17 This is the experimental result of the effect of iron-dissolving fermentation liquid of strain LB4 on total iron accumulation in peanut seedlings.

[0032] Biomaterial Deposit Information

[0033] The strain information deposited in this application is: Pseudomonas jejuni Pseudomonas jessenii Strain LB4, whose classification is named: Pseudomonas jessenii LB4, Chinese classification name: Pseudomonas japonicus LB4, preservation number is CCTCC NO: M20241258; the strain is deposited in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is June 17, 2024. DETAILED DESCRIPTION

[0034] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0035] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features. Example 1

[0036] This example is the separation and identification of Pseudomonas jessenii LB4.

[0037] 1. Isolation and purification of strain LB4: 1 g of rhizosphere soil from pear trees was collected from a pear orchard with calcareous soil in Xinxiang, Henan Province. The soil was added to a 50 ml conical flask containing glass beads and 9 ml of SM buffer and shaken at 30 °C and 170 °C for 30 min to obtain a soil suspension. Sterile water was added to gradually dilute the suspension into a concentration of 10-5~10-7. The suspension was spread on TSA medium and cultured at 30 °C for 48 h. A single bacterium was picked by the plate streak method to purify the strain.

[0038] 2. Identification of strain LB4: ① Morphological identification The specific operation is: inoculate the LB4 strain on TSB medium and culture it in a constant temperature incubator. After 24 hours of culture, observe until round colonies appear on the plate. Figure 1 As shown, Figure 1 The colonies were milky white, opaque, with smooth edges and a smooth and moist surface. The colonies were regular circles with relatively uniform sizes, no wrinkles or protrusions on the surface, and no diffuse or flocculent edges, which were consistent with the morphological characteristics of Pseudomonas jejuni LB4; ② After the strain DNA was extracted and sequenced, molecular identification was performed to construct a phylogenetic tree. Combined with the phylogenetic tree ( Figure 2 ) and morphological identification, the strain was classified and named Pseudomonas jessenii . Example 2

[0039] This example is a growth-promoting function assay of strain LB4.

[0040] 1. Qualitative determination of IAA function of strains: LB4 strain was added to TSB medium containing L-tryptophan at a 1% inoculum, 30°C, 170 r·min -1 After shaking for 48 hours, centrifuge at 10,000 rpm for 5 minutes to obtain the supernatant. Take 100 μL of the supernatant and mix it with an equal volume of Salkowski colorimetric solution. Mix 100 μL of uninoculated culture medium with an equal volume of Salkowski colorimetric solution as a blank control. React in dark conditions for 30 minutes. If the solution turns red, it means that the strain has IAA ability.

[0041] 2. Determination of nitrogen fixation and phosphorus solubilization ability of strains: Add 10 μL of the activated LB4 bacterial suspension to the Axubei nitrogen fixation medium, the Montgina organophosphate medium and the PKO inorganic phosphorus medium, respectively, with three replicates each, and culture in a 28°C incubator for 5 days. Observe whether there are halos or transparent circles on the culture medium, and judge the strain's ability to fix nitrogen and solubilize organic and inorganic phosphorus based on the ratio of the halo or transparent circle (D) to the colony diameter (d).

[0042] 3. Determination of iron carrier production capacity: Referring to Schwyn and Neilands, the bacterial suspension cultured in MSA medium for 48 h under iron-limited conditions was centrifuged at 3500 rpm for 10 min through a perforated filter. The supernatant was mixed with CAS detection solution at a ratio of 1:1, allowed to react for 2 h, and the OD was measured. 630 The absorbance value at nm (A); the control is sterile MSA culture medium, which is also mixed with CAS detection solution in equal proportions and the absorbance value (Ar) is measured. The relative content of iron carriers is calculated according to the formula [(Ar-As) / Ar]×100%.

[0043] The above growth-promoting properties test results are shown in Table 1.

[0044]

[0045] As shown in Table 1, strain LB4 has the ability to produce IAA, fix nitrogen, decompose inorganic phosphorus, decompose organic phosphorus and produce siderophore. Example 3

[0046] Study on the growth-promoting effect of strain LB4 on Pyrus betulaeformis seedlings.

[0047] (1) Seedling cultivation of Pyrus betulus: Soak the seeds in sterile water for 24 hours, then soak them in 75% alcohol for 30 seconds, rinse them with sterile water for 3-5 times, add 2% sodium hypochlorite and soak them for 20 minutes, and finally clean them with sterile water. Spread the sterilized seeds on a culture medium covered with filter paper soaked in sterile water, place them in a 28℃ constant temperature incubator for germination for 3-5 days, select the germinated seeds and sow them in a sterilized substrate for seedling cultivation. When the Pyrus betulus seedlings in the seedling substrate have 2-3 true leaves, select healthy and consistent seedlings and transplant them into sterilized substrate pots.

[0048] (2) Inoculation: Inoculate the prepared LB4 bacterial suspension (final concentration of 10 7 CFU / g soil) was inoculated into the rhizosphere of Pyrus betulaeformis seedlings, and the uninoculated treatment was used as the control. Each treatment was repeated 12 times, and samples were collected after 30 days of culture to determine the phenotypic traits of Pyrus betulaeformis.

[0049] The test results are as follows Figure 3 It can be seen that the growth of P. betula seedlings inoculated with LB4 strain is significantly better than that of P. betula seedlings not inoculated with bacteria. It can be seen intuitively that the inoculation strain LB4 has the effect of promoting the growth of P. betula seedlings.

[0050] Figure 4-Figure 6 The comparison results of dry weight of Pyrus betulata seedlings are shown in Table 1. Figure 4-Figure 6 It can be seen that compared with the control, the aboveground dry weight, root dry weight and total dry weight of the Pyrus betulaeformis seedlings in the experimental group inoculated with LB4 strain increased significantly by 65.63%, 77.11% and 67.35%, respectively.

[0051] Figure 7 The comparison results of root morphology of Pyrus betulata seedlings are shown in Figure 2. Figure 7 It can be seen that the root system of the Pyrus betulae seedlings inoculated with the LB4 strain is more developed and the root morphology is significantly improved. Figure 8-Figure 10 It can be seen that the comparison results of root length, root surface area and root tip are Figure 8-Figure 10 It can be seen that compared with the control group, the root length, root surface area and root tip number of the Pyrus betulaeformis seedlings in the experimental group inoculated with LB4 strain were significantly increased by 33.69%, 29.40% and 29.40%, respectively. Example 4

[0052] Study on the growth-promoting and iron accumulation effects of strain LB4 fermentation broth on peanut.

[0053] (1) Preparation of LB4 iron-dissolving fermentation broth: The activated LB4 bacterial suspension was inoculated at a 1% inoculum into 10% MSA medium containing excess iron oxide powder (0.5 g) at 30°C and 170 r·min. -1 The bacterial iron-dissolving fermentation liquid was obtained by shaking the flask for 24 hours. After the shaking flask liquid was centrifuged, the recovered supernatant was used for foliar spraying.

[0054] (2) Seedling cultivation: Soak the peanut seeds in sterile water for 12 hours, add 1% hydrogen peroxide to disinfect for 20 minutes, and finally wash with sterile water. Spread the sterilized seeds on a culture medium covered with filter paper soaked in sterile water, place in a 28°C constant temperature incubator for germination for 3-5 days, select white peanut seeds and sow them in sterilized substrate for seedling cultivation. When the peanut seedlings have 2-3 true leaves, select healthy and consistent seedlings and transplant them into a sterilized mixed soil with a ratio of 3:2 of calcareous soil: quartz sand.

[0055] (3) Spraying treatment: Add surfactant (0.1% Tween 20) before spraying to ensure better spraying effect. When spraying, pay attention to spraying both sides of the leaves until they are wet. Spray once every 7 days. The whole experiment is sprayed 3 times in total. Samples are collected and indicators are measured 21 days after treatment.

[0056] Test results: ① The test results on the growth of peanut seedlings are as follows: Fig.11 visible, Fig.11 This is a growth chart of peanut seedlings. It can be seen from the figure that the growth of the LB4 experimental group sprayed with LB4 iron-dissolving fermentation liquid and the experimental group sprayed with EDTA-Fe solution are similar, and are significantly better than the control group that is not sprayed with any iron-containing solution; Figure 12-14 The figure shows the dry weight experimental results of each experimental group. It can be seen from the figure that compared with the control, the aboveground dry weight, root dry weight and total dry weight of peanut seedlings treated with LB4 iron-dissolving fermentation liquid increased significantly by 40.62%, 43.20% and 40.91% respectively. Among them, the aboveground dry weight and total dry weight were the same as those of spraying EDTA-Fe, and the root dry weight significantly exceeded the treatment of spraying EDTA-Fe. Figure 15-16 As shown in the figure, compared with the control, the total iron content of peanut leaves and roots treated with LB4 iron-dissolving fermentation liquid was significantly increased by 112.28% and 50.89%, respectively, among which the total iron content of leaves was significantly higher than that of the treatment with EDTA-Fe; compared with the control, the total total iron accumulation of peanuts treated with LB4 iron-dissolving fermentation liquid was significantly increased by 128.33%, which was equivalent to the effect of spraying EDTA-Fe (such as Fig.17 as shown).

[0057] In summary, the Pseudomonas japonicus LB4 screened by the applicant of the present invention has the ability to produce IAA, dissolve phosphorus, fix nitrogen and produce iron carriers. Potted experiments found that LB4 can significantly promote the growth of pear seedlings and peanut seedlings, can effectively improve the root development morphology and has a good growth-promoting effect on plants; in addition, foliar spraying of LB4 combined with exogenous iron to make a microbial iron-dissolving fermentation liquid can significantly increase the biomass of peanuts, significantly increase the active iron and total iron content of peanut leaves and roots, and has good growth-promoting and iron-supplementing functions.

[0058] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached 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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