A Gordonia sp. B210 and its applications

Abstract

The present invention relates to the field of microbial technology, and particularly relates to a strain of Gordonia fridii B210 and its application. The strain B210 of the present invention was isolated by the applicant from the rhizosphere soil of pears, and has good abilities of producing IAA, decomposing organic phosphorus, dissolving phosphorus, decomposing potassium, and inhibiting the growth of Ralstonia solanacearum ( Ralstonia solanacearum ). When the bacterial suspension and fermentation broth of this strain were used in plant pot experiments, it was found that the volatile organic compounds and metabolites produced by this strain could effectively promote the development of Arabidopsis thaliana roots and shoots. Through pot experiments, it was found that B210 could significantly improve the root development morphology of tomato and Pyrus betulaefolia seedlings, and enhance the root uptake of K

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a Gordonia fridii B210 and its application. Background Art

[0002] Potassium is one of the essential macronutrients for plant growth and development. It is also the most abundant cation in plant cells and participates in various 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 soil, potassium exists in four different forms, among which soluble potassium is the 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 soil potassium content. In agricultural production, long-term and large-scale absorption of potassium by crops, combined with limited application of potassium fertilizers, leads to a decrease in the availability of potassium in the soil, and ultimately results in smaller crop seeds, lower yields, and increased susceptibility to pests and diseases.

[0003] In the traditional agricultural production mode, the long-term excessive application of chemical fertilizers has caused many negative impacts on the ecological environment and seriously threatens the sustainable development of agriculture. At present, when green agriculture has become an inevitable trend in the industry, using beneficial microorganisms to maintain the healthy growth of plants, improve the nutrient absorption efficiency, and increase crop yields has become an effective means to replace the traditional fertilization method. As an important group of microorganisms, many bacteria in the Bacillaceae family have the function of plant growth-promoting rhizobacteria. They can provide necessary nutrients for plant growth through various direct mechanisms, such as synthesizing and secreting hormones required for plant growth, dissolving insoluble phosphorus in the soil, decomposing and activating potassium elements 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 for ecological niches with pathogenic bacteria to prevent the invasion of pathogenic bacteria on plants, and inducing plants to produce systemic resistance to enhance the plant's own immunity. It is particularly worth mentioning that many bacteria in the Bacillaceae family can convert insoluble potassium in the soil environment into available potassium that plants can absorb and utilize by secreting organic acids, producing extracellular polysaccharides, and specific enzymes. Therefore, this family may be the core microbial group closely related to improving plant potassium nutrition.

[0004] The genus Gordonia is an emerging genus under the Bacillaceae family, which was separated and established from the genus Bacillus by Gupta RS et al. in 2020 based on phylogenetic and molecular evidence. However, at present, the research on the mechanism of action of Gordonia in potassium solubilization and growth promotion of fruit trees is still relatively scarce, 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, with the expectation of improving the utilization range of the new strains and promoting the growth and disease resistance of plants, providing new ideas for the effective utilization of biological bacterial fertilizers.

[0006] To achieve the above object, the present invention has screened out a new strain: Gordonia sp. strain B210, whose taxonomic name is: Gottfriedia sp. Gottfriedia sp. B210, and its Chinese taxonomic name is: Gordonia B210, and the preservation number is CCTCC NO: M2025285; this strain is preserved 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 a bacterial agent containing the above-mentioned Gordonia Gottfriedia sp. strain B210.

[0008] The present invention also includes the application of the above-mentioned Gordonia Gottfriedia sp. strain B210 or the above-mentioned bacterial agent in the preparation of biological organic fertilizers.

[0009] The present invention also includes the application of the above-mentioned Gordonia Gottfriedia sp. strain B210 or the above-mentioned bacterial agent in promoting plant growth and / or inhibiting the growth of Ralstonia solanacearum ( Ralstonia solanacearum ).

[0010] Furthermore, the plants are Arabidopsis thaliana, tomato and / or Pyrus betulifolia.

[0011] The present invention also includes the application of the above-mentioned Gordonia Gottfriedia sp. strain B210 or the above-mentioned bacterial agent in the production of IAA, phosphorus solubilization, phosphorus dissolution and / or potassium dissolution.

[0012] The present invention also includes a method for planting plants with the above-mentioned Gordonia Gottfriedia sp. strain B210 or the above-mentioned bacterial agent, which is characterized in that the method is: irrigating the root rhizosphere of plant seedlings with the bacterial suspension and / or fermentation broth of Gordonia Gottfriedia sp. strain B210.

[0013] Furthermore, the preparation method of the fermentation broth is: inoculating Gordonia Gottfriedia sp. strain B210 in TSB medium, centrifuging to extract the supernatant after 24 hours, rotary evaporating the supernatant, and then diluting it with sterile water to obtain it.

[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 abilities of producing IAA, decomposing organic phosphorus, dissolving phosphorus, decomposing potassium and inhibiting the growth of Ralstonia solanacearum ( Ralstonia solanacearum ). When the bacterial suspension and fermentation broth of this strain are used in plant pot experiments, it is found that: The volatile organic compounds and metabolites produced by this strain can effectively improve the development of Arabidopsis thaliana roots and shoots. Through pot experiments, it is found that B210 can significantly improve the root development morphology of tomato and Pyrus betulifolia seedlings, increase the absorption rate of K + by the roots, increase the seedling biomass, and has a good growth promotion effect on plants. After inoculating with the B210 strain, it can replace chemical potassium fertilizer to a certain extent and reduce the input of chemical fertilizers. Description of the Drawings

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

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

[0017] Figure 3 is the potassium decomposition ability result diagram of strain B210.

[0018] Figure 4 is the inhibition result diagram of strain B210 against different pathogenic bacteria.

[0019] Figure 5 is the morphological result diagram of the overall growth of Arabidopsis thaliana seedlings by VOC (volatile organic compounds) of strain B210.

[0020] Figure 6 is the result diagram of the growth of Arabidopsis thaliana seedlings by VOC (volatile organic compounds) of strain B210; in the figure, A is the result of the dry weight of the seedling roots, B is the result of the dry weight of the shoots, and C is the result of the total dry weight.

[0021] Figure 7 is the morphological result diagram of the root growth of Arabidopsis thaliana seedlings by VOC (volatile organic compounds) of strain B210.

[0022] Figure 8 is the influence result diagram of VOC (volatile organic compounds) of strain B210 on the roots of Arabidopsis thaliana seedlings; in the figure, A is the result of the root length, B is the result of the root surface area, and C is the result of the number of root tips.

[0023] Figure 9 is the phenotypic result of the growth of Arabidopsis thaliana seedlings by different concentrations of metabolites of strain B210.

[0024] Figure 10Results of metabolites at different concentrations of strain B210 on the growth of Arabidopsis thaliana seedlings; in the figure, A is the result of root dry weight, B is the result of shoot dry weight, and C is the result of total dry weight.

[0025] Figure 11 Results of metabolites at different concentrations of strain B210 on the root growth of Arabidopsis thaliana seedlings; in the figure, A is the result of root length, B is the result of root surface area, and C is the result of the number of root tips.

[0026] Figure 12 Phenotype result diagram of the growth of strain B210 on tomato potted seedlings.

[0027] Figure 13 Result diagram of the growth of strain B210 on tomato seedlings; in the figure, A is the result of the dry weight of the seedling roots, B is the result of the dry weight of the shoots, and C is the result of the total dry weight.

[0028] Figure 14 Phenotype result diagram of the effect of strain B210 on tomato roots.

[0029] Figure 15 Results of the growth of strain B210 on tomato roots; in the figure, A is the result of root length, B is the result of root surface area, and C is the result of the number of root tips.

[0030] Figure 16 Phenotype result diagram of the growth of strain B210 on Pyrus betulifolia Bunge potted seedlings.

[0031] Figure 17 Result diagram of the growth of strain B210 on Pyrus betulifolia Bunge seedlings; in the figure, A is the result of the dry weight of the seedling roots, B is the result of the dry weight of the shoots, and C is the result of the total dry weight.

[0032] Figure 18 Phenotype result diagram of the effect of strain B210 on Pyrus betulifolia Bunge roots.

[0033] Figure 19 Results of the growth of strain B210 on Pyrus betulifolia Bunge roots; in the figure, A is the result of root length, B is the result of root surface area, and C is the result of the number of root tips.

[0034] Figure 20 For the results of the K + absorption rate of strain B210 on Pyrus betulifolia Bunge roots.

[0035] Biological material preservation information

[0036] The strain information preserved in this application is: Gordonia sp. strain B210, and its taxonomic name is: Gottfriedia sp. strain B210, and its taxonomic name is: Gottfriediasp. B210, named in Chinese classification as: Gordonia B210, with the preservation number of CCTCC NO: M2025285; this strain is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is February 24, 2025. Detailed implementation manners

[0037] All the features disclosed in this specification, or the steps in all the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any manner.

[0038] Any feature disclosed in this specification (including any additional claims, abstract) is, unless otherwise stated, only an example of a series of equivalent or similar features. Example 1

[0039] This example is for the isolation and identification of Gordonia B210.

[0040] 1. Isolation and purification of strain B210: 1 g of rhizosphere soil was collected from the rhizosphere of pear trees in Lishui District, Nanjing City, where potassium fertilizer has not been applied for a long time, and added to a 50 ml triangular flask containing glass beads and 9 ml of SM buffer solution. The mixture was shaken at 170 rpm at 30 °C for 30 min to obtain a soil suspension, and then gradually diluted with sterile water to a concentration suspension of 10 -5 ~10 -7 . The suspension was spread on TSA medium and cultured at 30 °C for 48 h. Then, single colonies were picked by the streak plate method to purify the strain.

[0041] 2. Identification of strain B210: ① The specific operation of morphological identification is as follows: As Figure 1 shown, the B210 strain was inoculated on TSA medium and placed in a constant temperature incubator for culture. After 24 h of culture, round colonies appeared on the culture dish, which were light yellow, relatively moist, with a smooth surface and regular edges. This conforms to the morphological characteristics of Gordonia; ② After extracting the DNA of the strain and sequencing, molecular identification was carried out. The DNA sequence of the strain was amplified, and the phylogenetic evolution tree of the B210 strain was analyzed and identified based on the DNA sequence (as Figure 2 shown). It was determined that the strain has a relatively close genetic relationship with Gottfriedia sp., and the phylogenetic tree is shown in Figure 2 . Combining morphological identification, this strain was classified and named as Gottfriedia sp. Example 2

[0042] This example is for the determination of the growth-promoting function of strain B210.

[0043] 1. Qualitative determination of the IAA-producing function of the strain: The strain B210 was added to the TSB medium containing L-tryptophan at an inoculation amount of 1%, and shaken at 170 r / min at 30°C for 48 h. Then, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. 100 μL of the supernatant was mixed with an equal volume of Salkowski colorimetric solution, and 100 μL of the uninoculated medium was mixed with an equal volume of Salkowski colorimetric solution as a blank control. The reaction was carried out for 30 min under light-shielded conditions. If the solution turned red, it indicated that the strain had the ability to produce IAA. -1 After 48 hours of shaking at 170 r / min at 30°C, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. 100 μL of the supernatant was mixed with an equal volume of Salkowski colorimetric solution, and 100 μL of the uninoculated medium was mixed with an equal volume of Salkowski colorimetric solution as a blank control. The reaction was carried out for 30 min under light-shielded conditions. If the solution turned red, it indicated that the strain had the ability to produce IAA.

[0044] 2. Determination of the phosphorus-solubilizing and potassium-dissolving abilities of the strain: 10 μL of the activated B210 bacterial suspension was added to the Mengjinna organic phosphorus medium, PKO inorganic phosphorus medium, and silicate solid medium with potassium feldspar replacing the potassium source, with three replicates for each. The cultures were incubated in an incubator at 28°C for 5 days, and the presence of halos or transparent zones on the medium was observed. The ability of the strain to dissolve organic phosphorus, inorganic phosphorus, and insoluble potassium was judged based on the ratio of the halo or transparent zone (D) to the colony diameter (d). Meanwhile, the strain B210 was added to the silicate liquid medium with potassium feldspar replacing the potassium source at an inoculation amount of 1%, with three replicates for each. The cultures were incubated in an incubator at 28°C for 2 days, and the potassium ion content in the supernatant was measured using a flame spectrophotometer.

[0045] 3. Determination of the ability of the strain to antagonize pathogenic bacteria: 10 μL of the activated B210 bacterial suspension was added to the NA and PDA media respectively. The bacterial wilt pathogen was spotted at the center of the NA medium, and Alternaria alternata, Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. cubense, and Gibberella fujikuroi were spotted at the center of the PDA medium. After 5 days of incubation, the growth of the pathogenic bacteria was observed to judge the antagonistic ability of the strain.

[0046] The results of the above growth-promoting property determinations are shown in Table 1 and Figures 3 - 4 as follows.

[0047]

[0048] As can be seen from Table 1, the strain B210 has the abilities to produce IAA, dissolve organic phosphorus, dissolve insoluble potassium, and antagonize the bacterial wilt pathogen.

[0049] As Figure 3 shown, the strain B210 has a good potassium-dissolving ability; as Figure 4 shown, the strain B210 has no inhibitory effect on Alternaria alternata, Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. cubense, and Gibberella fujikuroi, but has a good inhibitory ability against the bacterial wilt pathogen. Example 3

[0050] This example is a study on the growth-promoting effect of the strain B210 on Arabidopsis thaliana.

[0051] (1)Arabidopsis thaliana seedling raising: Arabidopsis thaliana seeds were placed in sterile water and soaked for 24 h, then soaked in 75% alcohol for 30 s, rinsed with sterile water 3 - 5 times, then soaked in 2% sodium hypochlorite for 5 min, and finally rinsed clean with sterile water. The disinfected seeds were spread out on a medium with only agar and placed in an incubator at a constant temperature of 28 °C for 3 - 5 days for germination. The germinated seeds were selected for transplantation.

[0052] (2)Investigation on the growth - promoting effect of B210 VOC (volatile organic compound): The prepared B210 bacterial suspension (final concentration of 10 8 CFU / mL) was inoculated on one side of a divided plate, and 3 Arabidopsis thaliana seedlings were transplanted on the other side. Using no bacterial suspension as a control, there were 12 replicates for each treatment. After 20 d of cultivation, samples were collected to measure the phenotypic traits of Arabidopsis thaliana. (Since the bacterial suspension was not directly irrigated to the roots of the plants but inoculated on the other side of the divided plate, we believe that the influence of this strain on plants is mainly brought about by the volatile organic compounds (VOC) produced during the growth process of the strain).

[0053] (3)Investigation on the growth - promoting effect of B210 metabolites: B210 was inoculated in TSB medium, and TSB medium without inoculating B210 was used as a control treatment. It was cultured on a shaker at 28 °C and 170 rpm for 24 h, then centrifuged at 5000 g to extract the supernatant. The supernatant was rotary evaporated in units of 1 ml, serially diluted 10 2 、10 3 and 10 4 and added to the medium. At the same time, 5 Arabidopsis thaliana seedlings were transplanted into the medium. There were 12 replicates for each treatment. After 20 d of cultivation, samples were collected to measure the phenotypic traits of Arabidopsis thaliana.

[0054] Test results: 1. As Figure 5 shown, the VOC (volatile organic compound) of B210 can significantly promote the growth of Arabidopsis thaliana seedlings; as Figure 6 shown, compared with the control, the root dry weight, shoot dry weight and total dry weight of Arabidopsis thaliana seedlings treated with B210 inoculation increased by 1036.00%, 234.29% and 293.24% respectively.

[0055] 2. As Figure 7 shown, the VOC (volatile organic compound) of B210 can improve the root morphology of Arabidopsis thaliana; as Figure 8 shown, the root length, root surface area and root tip number of the VOC experimental group of B210 increased by 353.13%, 439.50% and 163.16% respectively.

[0056] 3. As Figure 9 shown, metabolites of different concentrations of B210 can significantly promote the growth of Arabidopsis thaliana seedlings and root morphology; as Figure 10and Figure 11 As shown, compared with the control, the dry weight of roots, the dry weight of shoots and the total dry weight of Arabidopsis thaliana seedlings treated with B210 metabolites were significantly increased. The root length, root surface area and the number of root tips were also significantly increased. However, when the concentration of B210 metabolites reached 10 4 After that, the growth-promoting ability of the dry weight of roots, the dry weight of shoots and the total dry weight of Arabidopsis thaliana seedlings decreased significantly. Example 4

[0057] This example is a study on the growth-promoting effect of strain B210 on Pyrus betulifolia seedlings and tomato seedlings.

[0058] (1) Tomato and Pyrus betulifolia seeding: Place the corresponding plant seeds in sterile water and soak for 24 h. Then soak in 75% alcohol for 30 s, and then rinse with sterile water for 3 - 5 times. Then add 2% sodium hypochlorite and soak for 20 min. Finally, wash with sterile water until clean. The disinfected seeds are spread out on a culture medium padded with filter paper moistened with sterile water, and placed in a constant temperature incubator at 28 °C for germination for 3 - 5 days. Select the germinated seeds and sow them in the sterilized substrate for seeding. When the seedlings in the substrate reach 2 - 3 true leaves, select the healthy and consistent seedlings and transplant them into sterilized substrate flower pots.

[0059] (2) Inoculation: Inoculate the prepared B210 bacterial suspension (final concentration of 10 7 CFU / g soil) into the rhizosphere of the seedlings. Use non-inoculation as the control treatment. Each treatment has 12 replicates. After culturing for 60 d, collect samples to measure the phenotypic traits of tomatoes and Pyrus betulifolia.

[0060] (3) K + Flow velocity measurement: Select the plants with consistent growth and good condition in the group. Use filter paper strips and resin blocks to fix the intact roots at the bottom of the culture dish, exposing the root tip part. Add the test solution (0.05 mM KCl, 0.2 mM MES, pH 5.8) into the culture dish to submerge the roots. After standing for 30 minutes, load the sample for detection. Find the root measurement site to be measured under the microscope (the point on the root surface 1200 μm away from the root tip vertex, belonging to the elongation zone). Place the K + flow velocity microsensor 5 μm away from the measurement site to be measured and not in contact with the sample, and start detection. Record the data for 5 minutes for each sample, and detect 6 biological replicates for each group. Directly read and output the K + flow velocity data through the imFluxes V3.0 software. The flow velocity unit is pico mol • cm -2 • s -1 . The positive and negative values of the flow velocity only represent the K + transport direction. The negative value indicates that K + flows from outside the root cells to outside the root cells; the positive value is the opposite.

[0061] Test results: 1. As Figure 12As shown, B210 can significantly promote the growth of tomato seedlings; as Figure 13 shown, compared with the control, the root, shoot dry weight and total dry weight of tomato seedlings treated with B210 inoculation were significantly increased by 50.00%, 25.75% and 28.08% respectively.

[0062] As Figure 14 shown, inoculation with B210 can improve the root morphology of tomatoes; as Figure 15 shown, the root surface area and the number of root tips were increased by 35.18% and 23.43% respectively.

[0063] 3. As Figure 16 shown, B210 can significantly promote the growth of Pyrus betulifolia seedlings; as Figure 17 shown, compared with the control, the root, shoot dry weight and total dry weight of Pyrus betulifolia seedlings treated with B210 inoculation were significantly increased by 119.28%, 82.57% and 89.51% respectively.

[0064] 4. As Figure 18 shown, inoculation with B210 can improve the root morphology of Pyrus betulifolia; as Figure 19 shown, the root length, root surface area and the number of root tips were increased by 96.76%, 103.71% and 212.13% respectively.

[0065] 5. As Figure 20 shown, inoculation with B210 can significantly increase the K + absorption rate of Pyrus betulifolia roots by 1989.10%.

[0066] In summary, the Gordonfrieda B210 screened by the applicant of the present invention has good abilities of producing IAA, potassium solubilization, dissolving organic phosphorus and antagonizing Ralstonia solanacearum. Plate tests found that the volatile organic compounds and metabolites produced by it can effectively improve the development of Arabidopsis thaliana roots and shoots. Through pot experiments, it was found that B210 can significantly improve the root development morphology of tomato and Pyrus betulifolia seedlings, increase the root absorption rate of K + , improve the seedling biomass, and have a good growth-promoting effect on plants. After inoculating with strain B210, it can replace chemical potassium fertilizer to a certain extent and reduce fertilizer input. It shows that the strain of this application can be promoted and utilized as a biological organic fertilizer material.

[0067] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended 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 Application in growth; the plant is Arabidopsis thaliana, tomato and / or Pyrus betulaeformis.

5. 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, decomposition of organic phosphorus and / or potassium.

6. Use of the Gordon Friedman bacteria as claimed in claim 1 Gottfriedia sp. strain B210 or the method for promoting plant growth 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; the plants are Arabidopsis thaliana, tomato and / or Pyrus betulaeformis.

7. The method according to claim 6, 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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