Freydobacterium gordensis B210 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to Freyella gordonii B210 and application thereof.The Freyella gordonii B210 is obtained by being separated from rhizosphere soil of pears by the applicant and has the good capacity of producing IAA, decomposing organophosphorus, dissolving phosphorus, decomposing potassium and inhibiting growth of Ralstonia solanacearum, and the Freyella gordonii B210 has the advantages that the Freyella gordonii B210 has the good capacity of producing IAA, decomposing organophosphorus, dissolving phosphorus, decomposing potassium and inhibiting growth of Ralstonia solanacearum; the application of bacterial suspension and fermentation liquor of the strain in plant potting experiments finds that volatile organic compounds and metabolites generated by the strain can effectively improve development of arabidopsis thaliana root systems and overground parts, and potting experiments find that B210 can significantly improve development forms of tomato and pyrus betulaefolia seedling root systems, increase the absorption rate of the root systems to K < + >, increase the seedling biomass and increase the yield of seedlings. The plant growth promoting agent has a good plant growth promoting effect, and can replace a chemical potash fertilizer to a certain extent after being inoculated with a B210 strain, so that the investment of chemical fertilizers is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Gordon Friedman's bacterium B210 and an application thereof. Background Art

[0002] Potassium is one of the essential macroelements for plant growth and development. It is also the most abundant cation in plant cells and is involved in a variety of physiological and metabolic processes during the plant life cycle. In plant photosynthesis, potassium is the second most important element after nitrogen and is indispensable for the growth and development of fruits. In natural soils, potassium exists in four different forms, of which soluble potassium is most easily absorbed by plants from the soil and plays a role in various physiological and biochemical processes, but it only accounts for 1% to 2% of the total potassium content in the soil. In agricultural production, the long-term and large-scale absorption of potassium by crops, coupled with limited application of potassium fertilizers, leads to reduced effectiveness of potassium in the soil, and ultimately leads to smaller crop seeds, lower yields, and increased susceptibility to diseases and pests.

[0003] In the traditional agricultural production model, the long-term excessive use of chemical fertilizers has caused many negative impacts on the ecological environment and seriously threatened the sustainable development of agriculture. At present, when green agriculture has become an inevitable trend in the development of the industry, the use of beneficial microorganisms to maintain healthy plant growth, improve nutrient absorption efficiency, and increase crop yields has become an effective means to replace traditional fertilization methods. As an important group of microorganisms, many bacteria in the Bacillaceae family have the function of rhizosphere growth-promoting bacteria. They can provide necessary nutrients for plant growth through a variety of direct mechanisms, such as synthesizing and secreting hormones required for plant growth, dissolving insoluble phosphorus in the soil, decomposing and activating potassium in the soil, and fixing nitrogen in the air; at the same time, they can also promote plant growth and development through indirect mechanisms, such as competing with pathogens for ecological niches, preventing pathogens from invading plants, and inducing plants to produce systemic resistance and enhance the immunity of plants themselves. It is particularly worth mentioning that many bacteria in the Bacillaceae family can convert insoluble potassium in the soil environment into effective potassium that can be absorbed and utilized by plants by secreting organic acids, producing extracellular polysaccharides and specific enzymes. Therefore, this family may be a core microbial group closely related to improving plant potassium nutrition.

[0004] Gordonia is an emerging genus under the Bacillaceae family, which was divided and established from the genus Bacillus by Gupta RS et al. in 2020 based on phylogenetic and molecular evidence. However, there is still a lack of research on the mechanism of Gordonia in potassium release and growth promotion in fruit trees, and in-depth research is urgently needed to reveal its potential value and provide more theoretical support and technical means for the development of green agriculture. Summary of the invention

[0005] In view of the above, it is necessary to develop new strains for plant growth and disease resistance, in order to increase the scope of application of new strains and promote plant growth and disease resistance, and provide new ideas for the effective use of biological fertilizers.

[0006] To achieve the above purpose, the present invention screened out a new strain: Gordon Friedman's bacterium Gottfriedia sp. strain B210, which is classified as: Gottfriedia sp. B210, Chinese classification name: Gordon Friedman B210, preservation number is CCTCC NO: M2025285; the strain is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is February 24, 2025.

[0007] The present invention also includes the Gordon Friedman bacteria Gottfriedia sp. strain B210.

[0008] The present invention also includes the Gordon Friedman bacteria Gottfriedia Application of strain B210 or the bacterial agent in the preparation of biological organic fertilizer.

[0009] The present invention also includes the Gordon Friedman bacteria Gottfriedia sp. strain B210 or the bacterial agent in promoting plant growth and / or inhibiting bacterial wilt ( Ralstonia solanacearum ) applications in growth.

[0010] Furthermore, the plant is Arabidopsis thaliana, tomato and / or Pyrus betulaeformis.

[0011] The present invention also includes the Gordon Friedman bacteria Gottfriedia sp. strain B210 or the bacterial agent in the production of IAA, phosphate solubilization, phosphate dissolution and / or potassium solubilization.

[0012] The present invention also includes the Gordon Friedman bacteria Gottfriedia sp. strain B210 or the bacterial agent for planting plants, characterized in that the method comprises: Gottfriedia sp. strain B210 bacterial suspension and / or fermentation liquid is poured into the rhizosphere of plant seedlings.

[0013] Furthermore, the method for preparing the fermentation broth is as follows: Gottfriedia sp. strain B210 was inoculated in TSB medium, and the supernatant was extracted by centrifugation after 24 hours. The supernatant was rotary evaporated and then diluted with sterile water.

[0014] The present invention has the following beneficial effects: the strain B210 of the present invention is isolated by the applicant from the rhizosphere soil of pears, and has good IAA production, organic phosphorus dissolution, phosphorus dissolution, potassium dissolution and inhibition of bacterial wilt ( Ralstonia solanacearum ) growth ability, and the bacterial suspension and fermentation liquid of the strain were used in plant pot experiments. It was found that the volatile organic compounds and metabolites produced by the strain can effectively improve the root and aboveground development of Arabidopsis thaliana. Pot experiments found that B210 can significantly improve the root development morphology of tomato and pear seedlings, and enhance the root resistance to K + It can increase the absorption rate of seedlings and improve the biomass of seedlings, which has a good growth-promoting effect on plants. After inoculation with B210 strain, it can replace chemical potassium fertilizer to a certain extent and reduce the input of chemical fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of strain B210.

[0016] Figure 2 This is the phylogenetic tree of strain B210.

[0017] Figure 3 This is the result graph of the potassium-solubilizing ability of strain B210.

[0018] Figure 4 This is a graph showing the inhibition of strain B210 on different pathogens.

[0019] Figure 5 This is the morphological result of the effect of strain B210 VOC (volatile organic compounds) on the overall growth of Arabidopsis seedlings.

[0020] Figure 6 This is a graph showing the effect of strain B210 VOC (volatile organic compounds) on the growth of Arabidopsis seedlings; in the graph, A is the result of seedling root dry weight, B is the result of aboveground dry weight, and C is the result of total dry weight.

[0021] Figure 7 This is the morphological result of the effect of strain B210 VOC (volatile organic compounds) on the root growth of Arabidopsis seedlings.

[0022] Figure 8 This is a graph showing the effect of strain B210 VOC (volatile organic compounds) on the root system of Arabidopsis seedlings; in the figure, A is the root length result, B is the root surface area result, and C is the root tip number result.

[0023] Fig. 9 The phenotypic results of different concentrations of metabolites of strain B210 on the growth of Arabidopsis seedlings.

[0024] Fig.10The results of different concentrations of metabolites of strain B210 on the growth of Arabidopsis seedlings; in the figure, A is the root dry weight result, B is the aboveground dry weight result, and C is the total dry weight result.

[0025] Fig.11 The results of different concentrations of metabolites of strain B210 on the root growth of Arabidopsis seedlings; in the figure, A is the root length result, B is the root surface area result, and C is the root tip number result.

[0026] Fig.12 This is a graph showing the phenotypic results of strain B210 on the growth of tomato potted seedlings.

[0027] Fig.13 This is a graph showing the effect of strain B210 on tomato seedling growth; in the graph, A is the seedling root dry weight result, B is the aboveground dry weight result, and C is the total dry weight result.

[0028] Fig.14 This is a diagram showing the phenotypic results of the effect of strain B210 on tomato roots.

[0029] Fig.15 These are the results of the effect of strain B210 on tomato root growth; in the figure, A is the root length result, B is the root surface area result, and C is the root tip number result.

[0030] Fig.16 This is a graph showing the phenotypic results of strain B210 on the growth of Pyrus betulaeformis potted seedlings.

[0031] Fig.17 This is a graph showing the effect of strain B210 on the growth of Pyrus betulaeformis seedlings; in the graph, A is the result of seedling root dry weight, B is the result of aboveground dry weight, and C is the result of total dry weight.

[0032] Fig.18 This is a diagram showing the phenotypic results of the effect of strain B210 on the root system of Pyrus betula.

[0033] Fig.19 The results of strain B210 on the root growth of Pyrus betulaeformis. In the figure, A is the root length result, B is the root surface area result, and C is the root tip number result.

[0034] Fig. 20 The strain B210 has an effect on the root system K + Result graph of absorption rate. Biomaterial Deposit Information

[0035] The strain information deposited in this application is: Gordon Friedman Gottfriedia sp. strain B210, which is classified as: Gottfriediasp. B210, Chinese classification name: Gordon Friedman B210, preservation number is CCTCC NO:M2025285; the strain is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is February 24, 2025. DETAILED DESCRIPTION

[0036] 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.

[0037] 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

[0038] This example is the separation and identification of Gordon Friedman's bacterium B210.

[0039] 1. Isolation and purification of strain B210: 1 g of rhizosphere soil was collected from the rhizosphere of pear trees in Lishui District, Nanjing, which had not been fertilized with potassium fertilizer for a long time. The soil was added into a 50 ml conical flask containing glass beads and 9 ml of SM buffer. The soil suspension was obtained by shaking at 30 °C and 170 °C for 30 min. Sterile water was added to gradually dilute the suspension into 10 -5 ~10 -7 The concentration suspension was spread on TSA medium and cultured at 30°C for 48 hours. Then, a single bacterium was picked out by the plate streak method to purify the strain.

[0040] 2. Identification of strain B210: ① Morphological identification The specific operation is as follows: Figure 1 As shown, the B210 strain was inoculated on TSA medium and placed in a constant temperature incubator for 24 hours. After culturing, round colonies were observed on the culture dish. They were light yellow and moist, with smooth surfaces and regular edges. They were consistent with the morphological characteristics of Gordon Friedman's bacteria. ② After the DNA of the strain was extracted and sequenced, molecular identification was performed, and the DNA sequence of the strain was amplified. The phylogenetic tree of the B210 strain was analyzed and identified based on the DNA sequence (such as Figure 2 ), confirming that the strain Gottfriedia sp. is closely related, as shown in the evolutionary tree. Figure 2 Combined with morphological identification, the strain was classified and named Gottfriedia sp. Example 2

[0041] This example is a determination of the growth-promoting function of strain B210.

[0042] 1. Qualitative determination of IAA production function of strains: Add 1% inoculum of B210 strain to TSB medium containing L-tryptophan at 30℃ and 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.

[0043] 2. Determination of the ability of strains to dissolve phosphorus and potassium: Add 10 uL of the activated B210 bacterial suspension to the Montana organic phosphorus medium, PKO inorganic phosphorus medium, and silicate solid medium with potassium feldspar as the potassium source, respectively, in triplicate, and culture in a 28°C incubator for 5 days. Observe whether there is a halo or transparent ring on the medium, and judge the ability of the strain to dissolve organic phosphorus, inorganic phosphorus, and insoluble potassium based on the ratio of the halo or transparent ring (D) to the colony diameter (d). At the same time, add the B210 strain at a 1% inoculation amount to the silicate liquid medium with potassium feldspar as the potassium source, in triplicate, and culture in a 28°C incubator for 2 days. Take the supernatant and determine the potassium ion content on a flame spectrophotometer.

[0044] 3. Determination of the strain's antagonistic ability to pathogens: Add 10 uL of the activated B210 bacterial suspension to NA and PDA culture media, respectively. In the center of NA culture medium, add Ralstonia solanacearum, and in the center of PDA culture medium, add Alternaria alternata, Fusarium oxysporum cucumber-specific type, Fusarium oxysporum banana-specific type, and rice seedling pathogen. After 5 days of culture, observe the growth of pathogens to determine the strain's antagonistic ability.

[0045] The above growth-promoting properties are shown in Table 1 and Figure 3-4 shown.

[0046]

[0047] As shown in Table 1, strain B210 has the ability to produce IAA, decompose organic phosphorus, dissolve insoluble potassium and antagonize Ralstonia solanacearum.

[0048] like Figure 3 As shown, strain B210 has good potassium-solubilizing ability; Figure 4 As shown, strain B210 had no antibacterial effect on Alternaria alternata, Fusarium oxysporum cucumber-specific type, Fusarium oxysporum banana-specific type and rice seedling pathogen, but had good antibacterial ability against bacterial wilt. Example 3

[0049] This example is a study on the growth-promoting effect of strain B210 on Arabidopsis thaliana.

[0050] (1) Arabidopsis seedling cultivation: Arabidopsis seeds were placed in sterile water and soaked for 24 hours, then soaked in 75% alcohol for 30 seconds, rinsed with sterile water for 3-5 times, then soaked in 2% sodium hypochlorite for 5 minutes, and finally cleaned with sterile water. The sterilized seeds were spread on agar culture medium and placed in a 28°C constant temperature incubator for germination for 3-5 days, and the germinated seeds were selected for transplanting.

[0051] (2) Study on the growth-promoting effect of B210 VOC (volatile organic compounds): The prepared B210 bacterial suspension (final concentration of 10 8 CFU / mL) was inoculated on one side of the partition plate, and three Arabidopsis seedlings were transplanted on the other side. No bacterial suspension was used as a control. Each treatment was repeated 12 times, and samples were collected after 20 days of cultivation to determine the phenotypic traits of Arabidopsis (because the bacterial suspension was not directly injected into the roots of the plants but inoculated on the other side of the partition plate, we believe that the effect of this strain on the plants is mainly caused by the volatile organic compounds (VOCs) produced during the growth of the strain).

[0052] (3) Investigation of the growth-promoting effect of B210 metabolites: B210 was inoculated in TSB medium, and TSB medium without B210 was used as a control. The medium was cultured in a shaker at 28°C and 170 rpm for 24 h, and then the supernatant was extracted by centrifugation at 5000 g. The supernatant was rotary evaporated in units of 1 ml and graded diluted 10 2 , 10 3 and 10 4 In the culture medium, 5 Arabidopsis seedlings were transplanted into the culture medium at the same time, with 12 replicates for each treatment. After culturing for 20 days, samples were collected to determine the phenotypic traits of Arabidopsis.

[0053] Test results: 1. Figure 5 As shown in Figure 2, the VOC (volatile organic compounds) of B210 can significantly promote the growth of Arabidopsis seedlings; Figure 6 As shown, compared with the control, the root dry weight, aboveground dry weight and total dry weight of Arabidopsis seedlings inoculated with B210 were significantly increased by 1036.00%, 234.29% and 293.24%, respectively.

[0054] 2. If Figure 7 As shown, the VOC (volatile organic compounds) of B210 can improve the root morphology of Arabidopsis thaliana; Figure 8 As shown, the root length, root surface area and number of root tips of B210 in the VOC experimental group increased by 353.13%, 439.50% and 163.16%, respectively.

[0055] 3. If Fig. 9 As shown in Figure 2, different concentrations of B210 metabolites can significantly promote the growth and root morphology of Arabidopsis seedlings; Fig.10and Fig.11 As shown in Figure 2, compared with the control, the root dry weight, aboveground dry weight and total dry weight of Arabidopsis seedlings inoculated with B210 metabolites increased significantly, as did the root length, root surface area and number of root tips. However, the concentration of B210 metabolites reached 10 4 Afterwards, the growth-promoting ability of Arabidopsis seedlings' root dry weight, aboveground dry weight and total dry weight decreased significantly. Example 4

[0056] This example is a study on the growth-promoting effect of strain B210 on pear seedlings and tomato seedlings.

[0057] (1) Tomato and pear seedling cultivation: Soak the corresponding plant 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 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 seedlings in the substrate have 2-3 true leaves, select healthy and consistent seedlings and transplant them into sterilized substrate pots.

[0058] (2) Inoculation: Inoculate the prepared B210 bacterial suspension (final concentration of 10 7 CFU / g soil) was inoculated into the rhizosphere of seedlings, and the uninoculated treatment was used as the control. Each treatment was repeated 12 times. After 60 days of cultivation, samples were collected to determine the phenotypic traits of tomato and pear.

[0059] (3) K + Flow rate measurement: Select plants with consistent growth and good condition in the group, use filter paper strips and resin blocks to fix the complete roots at the bottom of the culture dish, exposing the root tip. Add test solution (0.05mM KCl, 0.2mM MES, pH5.8) to the culture dish to immerse the roots. After standing for 30 minutes, add the sample for testing. Find the root site to be tested under a microscope (a point on the root surface 1200μm away from the root tip, which belongs to the elongation zone), and place K + The flow rate microsensor was placed 5 μm away from the site to be tested and did not touch the sample, and the test was started. Data was recorded for 5 minutes for each sample, and 6 biological replicates were tested in each group. K was directly read and output by imFluxes V3.0 software + Flow rate data, flow rate unit is pico mol • cm -2 • s -1 The positive and negative values ​​of the flow rate only represent K + Transport direction, negative value indicates K + Flows from outside the root cells into the root cells; positive value is the opposite.

[0060] Test results: 1. Fig.12As shown in Figure 2, B210 can significantly promote the growth of tomato seedlings; Fig.13 As shown, compared with the control, the root dry weight, aboveground dry weight and total dry weight of tomato seedlings inoculated with B210 increased significantly by 50.00%, 25.75% and 28.08%, respectively.

[0061] like Fig.14 As shown in Figure 2, inoculation with B210 can improve tomato root morphology; Fig.15 As shown, the root surface area and root tip number increased by 35.18% and 23.43%, respectively.

[0062] 3. If Fig.16 As shown in Figure 2, B210 can significantly promote the growth of Pyrus betulaeformis seedlings; Fig.17 As shown, compared with the control, the root dry weight, aboveground dry weight and total dry weight of Pyrus betulaeformis seedlings inoculated with B210 increased significantly by 119.28%, 82.57% and 89.51%, respectively.

[0063] 4. If Fig.18 As shown in Figure 2, inoculation with B210 can improve the root morphology of Pyrus betula; Fig.19 As shown, root length, root surface area and number of root tips increased by 96.76%, 103.71% and 212.13%, respectively.

[0064] 5. If Fig. 20 As shown in the figure, inoculation with B210 could significantly increase the root K + Absorption rate 1989.10%.

[0065] In summary, the Gordon Friedman B210 screened by the applicant of the present invention has good ability to produce IAA, dissolve potassium, dissolve organic phosphorus and antagonize Ralstonia solanacearum. Plate tests found that the volatile organic compounds and metabolites produced by it can effectively improve the root system and aboveground development of Arabidopsis thaliana. Potted plant experiments found that B210 can significantly improve the root development morphology of tomato and pear seedlings, and enhance the root resistance to K + The absorption rate can be increased, the biomass of seedlings is improved, and it has a good growth-promoting effect on plants. After inoculation with the B210 strain, it can replace chemical potassium fertilizer to a certain extent and reduce fertilizer input. This shows that the strain of the present application can be promoted and utilized as a biological organic fertilizer material.

[0066] 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. Gordonia Gottfriedia sp. strain B210, whose deposit number is CCTCC NO: M2025285.

2. Containing the Gordon Friedman bacteria according to claim 1 Gottfriedia sp. strain B210.

3. Gordon Friedman's bacteria as claimed in claim 1 Gottfriedia sp. strain B210 or the bacterial agent as claimed in claim 2 in the preparation of biological organic fertilizer.

4. Gordon Friedman's bacteria as claimed in claim 1 Gottfriedia sp. strain B210 or the bacterial agent as claimed in claim 2 in promoting plant growth and / or inhibiting bacterial wilt Ralstonia solanacearum Growing applications.

5. The use according to claim 4, characterized in that: The plant is Arabidopsis thaliana, tomato and / or Pyrus betulaeformis.

6. Gordon Friedman's bacteria as claimed in claim 1 Gottfriedia sp. strain B210 or the bacterial agent as claimed in claim 2 in the production of IAA, phosphate solubilization, phosphate dissolution and / or potassium solubilization.

7. Use of the Gordon Friedman bacteria as described in claim 1 Gottfriedia sp. strain B210 or the method for planting plants with a bacterial agent as claimed in claim 2, characterized in that, The method comprises: injecting Gordon Friedman's bacteria Gottfriedia sp. strain B210 bacterial suspension and / or fermentation liquid is poured into the rhizosphere of plant seedlings.

8. The method according to claim 7, characterized in that The preparation method of the fermentation liquid is as follows: Gottfriedia sp. strain B210 was inoculated in TSB medium, and the supernatant was extracted by centrifugation after 24 hours. The supernatant was rotary evaporated and then diluted with sterile water.

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