Pantoea sp. YNK-FB0056 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a strain of Pantoea endophytic bacteria YNK-FB0056 and an application of the Pantoea endophytic bacteria YNK-FB0056. The collection number of the pantoea is CCTCC NO: M 2025644, and the pantoea has good capacity of sulfur oxidation, nitrogen fixation, phosphorus dissolving, zinc dissolving, siderophore production and high yield of indoleacetic acid, and can regulate the nutrient structure of soil and convert substances which are difficult to utilize by plants in the soil, thereby promoting the growth of the plants and improving the stress resistance of the plants. Besides, the sulfur-oxidizing bacterium YNK-FB0056 provided by the invention is a bacterium separated from oilseed rape farmland soil, has better adaptability when being applied to a planting environment compared with a strain which is subjected to mutagenesis screening in an extreme environment and a laboratory, and can be stably colonized in plant rhizosphere and soil, so that the growth promoting effect is stably exerted for a long time, and the sulfur-oxidizing bacterium YNK-FB0056 is safe and environment-friendly and has a wide application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly to a Pantoea sp. ( Pantoea endophytica ) YNK-FB0056 and its applications. Background Art

[0002] The core contradiction faced by the development of modern agriculture lies in the balance between the demand for high yields and ecological sustainability. Currently, the degradation of the soil ecosystem has become a key bottleneck restricting the high-quality development of agriculture, mainly manifested as problems such as reduced microbial diversity, blocked nutrient cycling, and pollutant accumulation. Traditional agronomic measures are difficult to maintain soil health while ensuring yields, and innovative solutions are urgently needed. In recent years, ecological regulation technologies based on the microbiome have provided new ideas for soil remediation. Among them, plant growth promoting rhizobacteria (PGPR) have become a research hotspot due to their unique niche adaptability and multifunctionality. These microorganisms can not only improve nutrient use efficiency through biological nitrogen fixation, organic phosphorus mineralization, and sulfur oxidation, but also synthesize active substances such as siderophores and indole acetic acid to alleviate abiotic stress on plants. More importantly, some PGPR strains can form biofilms through quorum sensing regulation and establish a stable functional microecosystem in the rhizosphere, which gives them significant advantages in field applications. However, existing commercial microbial agents generally have problems such as poor environmental adaptability and single functions, and their actual application effects are restricted by multiple factors such as soil type, climate conditions, and crop varieties. To address the above problems, this technology aims to obtain superior strains with both broad-spectrum growth promotion characteristics and strong environmental adaptability by constructing an efficient PGPR screening system. This research not only provides core bacterial resources for the development of new agricultural inputs of bio-stimulants, but also provides technical support for the establishment of a microbiome-centered ecological agriculture model, and has important practical value for promoting the green transformation of agriculture. Summary of the Invention

[0003] The present invention aims to solve the problem of the lack of effective plant growth promoting bacteria and their products in the actual application of the existing technology, and provides a Pantoea sp. ( Pantoea endophytica ) YNK-FB0056 and its applications. The Pantoea sp. ( Pantoea endophytica ) YNK-FB0056 provided by the present invention was isolated from the soil of rapeseed farmland in Qujing City, Yunnan Province. This bacterium has the characteristics of sulfur oxidation, nitrogen fixation, zinc solubilization, decomposition of organic and inorganic phosphorus, and production of siderophores. At the same time, when 0.1 g / L of tryptophan is added, the production of IAA is as high as 66.89 mg / L.

[0004] To achieve the above object, the first aspect of the present invention provides a Pantoea sp. ( Pantoea endophytica )YNK-FB0056, the preservation number of this strain is CCTCC NO: M 2025644.

[0005] The second aspect of the present invention provides a bacterial agent, and the active ingredient of the bacterial agent is the Pantoea described in the first aspect.

[0006] The third aspect of the present invention provides the application of the Pantoea described in the first aspect and the bacterial agent described in the second aspect in sulfur oxidation, zinc dissolution, and / or IAA production, wherein sulfur oxidation refers to the oxidation of sodium thiosulfate to produce sulfate ions; zinc dissolution refers to the dissolution of zinc oxide.

[0007] The fourth aspect of the present invention provides a method for producing indole acetic acid, and the method includes fermenting and culturing the Pantoea described in the first aspect ( Pantoea endophytica ) YNK-FB0056 and collecting the culture product.

[0008] The fifth aspect of the present invention provides a compound bacterial agent, including Pantoea ( Pantoea endophytica ) YNK-FB0056 and Burkholderia gladioli ( Burkholderia gladioli ) with an effective viable count ratio of 1:1; wherein, the preservation number of the Burkholderia gladioli is CCTCC NO: M 20242288.

[0009] The sixth aspect of the present invention provides a method for promoting plant growth, and the method includes applying the Pantoea described in the first aspect ( Pantoea endophytica ) YNK-FB0056, the bacterial agent described in the second aspect, or the compound bacterial agent described in the fifth aspect to the rhizosphere of plants.

[0010] Through the above technical solutions, the present invention can at least achieve the following beneficial effects: (1) The Pantoea ( Pantoea endophytica ) YNK-FB0056 provided by the present invention has good functions of sulfur oxidation, nitrogen fixation, zinc dissolution, decomposition of organic phosphorus and inorganic phosphorus, and production of siderophores, and can produce high yields of indole acetic acid, providing new materials and methods for the production of indole acetic acid.

[0011] (2) The Pantoea ( Pantoea endophytica ) YNK-FB0056 provided by the present invention can effectively promote crop growth. Through experimental verification, 28 days after applying this strain to tomato seedlings, the plant height, stem diameter, above-ground fresh weight, root length, underground fresh weight, and chlorophyll of tomato plants are significantly increased compared with the control group (CK1) applying clear water and the control group (CK2) applying NB medium, indicating that this strain has good plant growth-promoting effects.

[0012] (3) The Pantoea ( Pantoea endophytica )YNK-FB0056 is a natural strain isolated from rapeseed farmland soil. It is more likely to colonize compared to laboratory-induced mutant strains, and is also safer, more environmentally friendly, and has the potential for development and application as a new type of microbial strain resource. Description of the Drawings

[0013] 图1 is the colony morphology diagram (A), SEM images (B) and (C) of Pantoea sp. Pantoea endophytica )YNK-FB0056 in Example 1; 图2 is the phylogenetic tree of Pantoea sp. Pantoea endophytica )YNK-FB0056 in Example 1; 图3 is the effect diagram of promoting nutrient cycling of Pantoea sp. Pantoea endophytica )YNK-FB0056 in Example 2: (A) sulfur oxidation effect diagram; (B) nitrogen fixation effect diagram; (C) zinc solubilization effect diagram; (D) organic phosphorus solubilization effect diagram; (E) inorganic phosphorus solubilization effect diagram; (F) siderophore production effect diagram.

[0014] 图4 is the standard curve drawn in the quantitative analysis of sulfur oxidation effect of Pantoea sp. Pantoea endophytica )YNK-FB0056 in Example 2;

[0015] 图5 is the effect diagram of IAA production by Pantoea sp. Pantoea endophytica )YNK-FB0056 in Example 3: (A) qualitative color development of IAA production; (B) quantitative change of IAA production for 7 consecutive days.

[0016] 图6 is the standard curve drawn in the quantitative analysis of IAA production effect of Pantoea sp. Pantoea endophytica )YNK-FB0056 in Example 3;

[0017] 图7 is the schematic diagram of the compatibility result between Pantoea sp. Pantoea endophytica )YNK-FB0056 and Burkholderia gladioli Burkholderia gladioli )CCTCC NO: M20242288 in Example 5;

[0018] 图8 is the growth promotion effect of Pantoea sp. Pantoea endophytica )YNK-FB0056 on plant seedlings in Example 6: (A) growth promotion effect of YNK-FB0056 liquid inoculant; (B) growth promotion effect of mixed liquid inoculant.

[0019] 图9 is the Pantoea sp. Pantoea endophytica )Analysis chart of growth-promoting indicators of YNK-FB0056 on plant seedlings; (A) Growth-promoting effect of YNK-FB0056 liquid microbial inoculum; (B) Growth-promoting effect of mixed liquid microbial inoculum.

[0020] Biological deposit The Pantoea provided by the present invention ( Pantoea endophytica ), YNK-FB0056, is classified and named as: Pantoea endophytica YNK-FB0056 was deposited at the China Center for Type Culture Collection on March 31, 2025. The address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025644.

[0021] The Burkholderia gladioli provided by the present invention ( Burkholderia gladioli ), YNK-FB0053, is classified and named as Burkholderia gladioli YNK-FB0053 was deposited at the China Center for Type Culture Collection on October 21, 2024. The address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242288. This strain has been disclosed in the invention patent with the application number: CN202411827636.3. Detailed implementation manners

[0022] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0023] The beneficial functions of PGPR on soil and plants make it one of the main raw materials for agricultural microbial agents. The development and use of PGPR make up for the problems such as pesticide residues, environmental pollution, and food safety brought by chemical control, and conform to the principles of sustainable agricultural development, environmental friendliness, and human and livestock safety. In order to improve the development efficiency, currently, the methods for developing PGPR strains with good utility in this field mainly rely on artificial mutagenesis or genetic engineering transformation to screen and obtain new strains with excellent plant growth-promoting and / or disease-preventing effects. However, the test strains obtained by artificial mutagenesis or genetic engineering transformation are usually selected and bred in the laboratory environment, and their adaptability to the natural environment is poor. Moreover, the colonization of the strains in plants or soil is also difficult to be well verified in the laboratory, resulting in many high-quality PGPR bacteria developed in the laboratory being difficult to show the same level of growth-promoting or disease-preventing effects as in the research in actual applications.

[0024] During the research process, the inventors of the present invention isolated a sulfur-oxidizing bacterium from the soil of rapeseed fields in Qujing City, Yunnan Province. After detection and identification, it was found to be Pantoea sp. ( Pantoea endophytica ). The inventors discovered that this strain has good sulfur-oxidizing ability, and at the same time has the properties of nitrogen fixation, zinc solubilization, decomposition of organic and inorganic phosphorus, and production of siderophores, and can efficiently ferment and produce indole-3-acetic acid (IAA) in the culture medium. Further research also found that when this strain is applied to plants such as tomatoes by root irrigation, it can effectively increase the plant height and dry weight of plant seedlings, showing good plant growth-promoting effects, thereby improving agricultural productivity and promoting the green and sustainable development of ecological agriculture.

[0025] Based on the above findings, on the one hand, the present invention provides a strain of Pantoea sp. ( Pantoea endophytica ), namely YNK-FB0056, and the preservation number of this strain is CCTCC NO: M 2025644.

[0026] On the second aspect, the present invention provides the application of the Pantoea sp. ( Pantoea endophytica ), namely YNK-FB0056 described in the first aspect, in sulfur oxidation, nitrogen fixation, zinc solubilization, decomposition of organic and inorganic phosphorus, production of siderophores or production of indole-3-acetic acid.

[0027] In the present invention, "nitrogen fixation" and "phosphorus decomposition" refer to the increase in the contents of N and P available for plants in the soil through the action of the Pantoea sp. ( Pantoea endophytica ), namely YNK-FB0056 provided by the present invention. For example, it promotes the conversion of inorganic phosphorus components (such as Ca3(PO4)2, FePO4, etc.) and organic phosphorus components (such as lecithin, inositol hexaphosphate, etc.) in the soil into forms that can be utilized by plants, thereby promoting the absorption and utilization of phosphorus elements in the soil by plants.

[0028] On the third aspect, the present invention provides a method for sulfur oxidation and production of indole-3-acetic acid, which includes culturing the Pantoea sp. ( Pantoea endophytica ), namely YNK-FB0056 described in the first aspect, and collecting the culture product.

[0029] Any method and conditions capable of fermenting and culturing the Pantoea sp. ( Pantoea endophytica ), namely YNK-FB0056, and enabling it to perform sulfur oxidation can be applied to the present invention.

[0030] According to a preferred embodiment of the present invention, the conditions for the fermentation culture include: culture temperature 25 - 37 °C, culture time 24 - 120 h, and initial pH 6 - 8.

[0031] According to a preferred embodiment of the present invention, when the fermentation culture is carried out by the method of shaking table fermentation, the fermentation culture process can be carried out under the condition of a shaking table speed of 120 - 200 rpm.

[0032] In the present invention, there is no particular limitation on the medium used for the fermentation culture, and any medium that can be used for the fermentation culture of Pantoea ( Pantoea endophytica ) YNK-FB0056 in the art can be applicable to the present invention.

[0033] According to a preferred embodiment of the present invention, the medium is at least one of nutrient agar medium (NA medium), sodium thiosulfate medium, Luria-Bertani medium (LB medium), and tryptic soy agar medium (TSA medium).

[0034] The fourth aspect of the present invention provides a bacterial agent having the function of promoting plant growth, and the active ingredient in the bacterial agent includes Pantoea ( Pantoea endophytica ) YNK-FB0056 described in the first aspect.

[0035] In the present invention, there is no particular limitation on the specific dosage form of the bacterial agent, and any dosage form of the bacterial agent commonly used in agricultural microbial agents in the art can be applicable to the present invention.

[0036] According to the preferred embodiment of the present invention, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0037] Preferably, in the liquid bacterial agent, the content of Pantoea ( Pantoea endophytica ) YNK-FB0056 is not less than 1×10 5 CFU / mL, preferably 1×10 6 -1×10 10 CFU / mL. More preferably 1×10 8 -1×10 9 CFU / mL.

[0038] Preferably, in the solid bacterial agent, the effective viable count of Pantoea ( Pantoea endophytica ) YNK-FB0056 is not less than 1×10 5 CFU / g, preferably 1×10 6 -1×10 10 CFU / g. More preferably 1×10 8 -1×10 9 CFU / g.

[0039] In order to increase the Pantoea ( Pantoea endophytica The activity and stability of YNK-FB0056, or to make the bacterial agent more convenient for transportation, storage and use. According to the preferred embodiment of the present invention, the bacterial agent may further contain excipients. Any excipients that can be used in agricultural bacterial agents in the art are applicable to the present invention. Preferably, the excipients are selected from at least one of excipients (such as tapioca starch, sorbitol, etc.), protectants (such as sugar protectants, sodium glutamate, calcium chloride, etc.) and buffers (such as phosphate buffer, sodium bicarbonate, magnesium oxide, etc.).

[0040] During the long-term research process, the inventors found that in microbial preparations, if only a single strain exists, it is easy to have problems such as single function and unstable effects. By adding excipients and / or other strains to the microbial preparation, its functions can be more comprehensive, and the use effect can be better and more stable.

[0041] During the research process, the inventors accidentally discovered that the Pantoea ( Pantoea endophytica ), YNK-FB0056, can coexist with certain bacteria of the genus Burkholderia ( Burkholderia ), such as Burkholderia gladioli ( Burkholderia gladioli ), CCTCC NO: M 20242288, on NA medium. After further research, it was found that when they are used in combination, they have a synergistic effect, which can not only make the properties of the Pantoea ( Pantoea endophytica ), YNK-FB0056 provided by the present invention more stable, but also further improve the growth-promoting effect on plants.

[0042] According to some preferred embodiments of the present invention, the bacterial agent further includes an auxiliary bacterium, and the auxiliary bacterium is Burkholderia gladioli ( Burkholderia gladioli ).

[0043] Preferably, the preservation number of the Burkholderia gladioli is CCTCC NO: M 20242288.

[0044] Preferably, in the bacterial agent, the viable cell count ratio of Pantoea ( Pantoea endophytica ), YNK-FB0056 to Burkholderia gladioli is 1:1.

[0045] In the bacterial agent provided by the present invention, the auxiliary bacterium can be separately packaged and co-applied with the Pantoea ( Pantoea endophytica ), YNK-FB0056 provided by the present invention during use, or the two can be mixed during preparation and directly applied to plants during use.

[0046] The fifth aspect of the present invention provides the Pantoea ( Pantoea endophytica )YNK-FB0056, or the use of the microbial agent described in the fourth aspect in promoting plant growth.

[0047] In the present invention, the "promoting plant growth" may include increasing the growth rate of plants, increasing the yield, improving the quality of agricultural products (such as increasing the weight of single fruits, etc.).

[0048] The sixth aspect of the present invention provides a method for promoting plant growth, which includes applying the Pantoea ( Pantoea endophytica )YNK-FB0056 or the microbial agent described in the fourth aspect to plants.

[0049] According to a preferred embodiment of the present invention, the dosage of the Pantoea ( Pantoea endophytica )YNK-FB0056 is not less than 1×10 8 CFU / strain / time, preferably 1×10 9 -1×10 12 CFU / strain / time.

[0050] According to a preferred embodiment of the present invention, the dosage of the microbial agent is such that the application amount of Pantoea ( Pantoea endophytica )YNK-FB0056 is not less than 1×10 8 CFU / strain / time, preferably 1×10 9 -1×10 12 CFU / strain / time. More preferably 1×10 9 -1×10 10 CFU / strain / time.

[0051] Preferably, the Pantoea ( Pantoea endophytica )YNK-FB0056 or the microbial agent is applied at a frequency of 1-3 times per crop.

[0052] According to a preferred embodiment of the present invention, the dosage of the metabolite is not less than 100 mL / strain / time, preferably 100-200 mL / strain / time.

[0053] Preferably, the plant is selected from Solanaceae plants, preferably tomatoes.

[0054] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention by way of example, and are not used to limit the present invention.

[0055] In the following examples, unless otherwise specified, the reagents and materials used are commercially available products purchased from regular chemical / biological reagent or material suppliers, and the reagents are all of analytical grade.

[0056] In the following examples, unless otherwise specified, the operating temperature is room temperature (25 ± 5 °C).

[0057] Example 1 This example is used to illustrate the acquisition, identification, and preservation of Pantoea ( Pantoea endophytica ) YNK-FB0056 CCTCC NO: M2025644.

[0058] (I) Strain isolation and purification During the strain isolation and purification process, thiosulfate agar medium (MST medium) was used, and the preparation method is as follows: Weigh 5 g of sodium thiosulfate, 0.1 g of dipotassium hydrogen phosphate, 0.2 g of sodium bicarbonate, 0.1 g of ammonium chloride, 5 g of glucose, and 5 g of yeast extract powder, add them to 1000 mL of water, adjust the pH to 8 ± 0.1, then add 20 g of agar, and autoclave at 121 °C for 25 min.

[0059] A strain of bacteria was isolated and purified from rapeseed planting soil samples collected from Qujing City, Yunnan Province by the dilution plating method and the streak plate method, and named YNK-FB0056.

[0060] (II) Strain identification 1. Identification of bacterial morphological characteristics and physiological and biochemical characteristics Refer to "Bergey's Manual of Determinative Bacteriology" and "Manual of Systematic Identification of Common Bacteria" to conduct physiological and biochemical assays on strain YNK-FB0056 and describe the colony morphological characteristics.

[0061] Colony and strain morphology: 图1 (A) shows the colony morphology of strain YNK-FB0056 on MST medium. As can be seen from the figure, the colonies of this strain are nearly circular, with relatively neat edges, the colony color is yellow, the color is bright, the middle of the colony is convex, moist, and shiny. Observed by electron microscopy ( 图1 (B)(C)), this strain is a rod-shaped bacterium without flagella and spores.

[0062] Physiological and biochemical characteristics: Strain YNK-FB0056 is Gram-positive, facultatively anaerobic, and shows positive reactions to catalase, glucose, sucrose, mannitol, arabinose, xylose, maltose, and glycerol; it shows negative reactions to oxidase, urease, gelatin liquefaction, starch hydrolysis, lactose, dulcitol, and inositol.

[0063] Molecular identification: The total DNA of strain YNK-FB0056 was extracted using the Chelex extraction method as a template. 27F (5'AGAGTTTGATCCTGGCTCAG-3’) was used as the upstream primer, and 1492R (5'-TACGGCTACCTTGTTACGACTT-3’) was used as the downstream primer. 16S rRNA amplification was carried out using the reaction system and conditions in Table 1.

[0064] Table 1 PCR system and conditions

[0065] 图2 The phylogenetic tree of the drawn strain YNK-FB0056 is shown, from which it can be seen that YNK-FB0056 has the highest homology with Pantoea ( Pantoea endophytica ).596 KR610525

[0066] 3. Identification results Combining the molecular detection results of strain YNK-FB0056 and the detection results of bacterial morphological characteristics and physiological and biochemical characteristics, this strain was identified as Pantoea ( Pantoea endophytica ).

[0067] (III) Strain preservation The obtained Pantoea ( Pantoea endophytica ) YNK-FB0056 was deposited at the China Center for Type Culture Collection on March 31, 2025. The address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025644. Example 2 This example is used to illustrate the sulfur oxidation, nitrogen fixation, zinc dissolution, organic and inorganic phosphorus solubilization, and siderophore production effects of Pantoea ( Pantoea endophytica ) YNK-FB0056 CCTCC NO: M2025644.

[0068] (I) Sulfur oxidation effect test 1. Qualitative detection MST (modified thiosulfate) medium (g / L): Weigh 5.0 g of sodium thiosulfate, 0.1 g of dipotassium hydrogen phosphate, 0.2 g of sodium bicarbonate, 0.1 g of ammonium chloride, 5.0 g of glucose, and 5.0 g of yeast extract powder, add them to 1000 mL of water and dissolve. 0.008 g of bromocresol purple is used as the color indicator, the pH is adjusted to 8 ± 0.1, and 20 g of agar is added. Autoclave at 121 °C for 20 min.

[0069] The strain YNK-FB0053 obtained in Example 1 was inoculated into the MST medium with the color indicator by the 4-point inoculation method. Each treatment was repeated 3 times and placed in an incubator at 30 °C. Continuously observe whether a yellow halo is produced around the colony. And measure the ratio of the diameter (D) of the color development circle to the diameter (d) of the colony.

[0070] Figure 3 (A) shows the sulfur oxidation effect of strain YNK-FB0056. It can be seen from the figure that a yellow halo is produced in the medium around the colony, indicating that this strain can carry out sulfur oxidation. The size of the color development circle was measured by the cross method, and the ratio of the diameter (D) of the color development circle to the diameter (d) of the colony was calculated as D / d = 2.823 ± 0.022.

[0071] 2. Quantitative detection MST liquid medium (g / L): Weigh 5.0 g of sodium thiosulfate, 0.1 g of dipotassium hydrogen phosphate, 0.2 g of sodium bicarbonate, 0.1 g of ammonium chloride, 5.0 g of glucose, and 5.0 g of yeast extract powder, add them to 1000 mL of water and dissolve. Adjust the pH to 8 ± 0.1, and autoclave at 121 °C for 20 min.

[0072] Preparation of stabilizing solution: Dissolve 75 g of analytical pure NaCl in 300 ml of water, add 30 ml of analytical pure concentrated HCl, 50 ml of glycerol, and 100 ml of 95% ethanol, and mix well.

[0073] Preparation of sulfate standard stock solution: Weigh 1.814 g of anhydrous potassium sulfate (analytical pure K2SO4, dried at 105 o °C for two hours in advance), dissolve it in a small amount of pure water, then transfer it to a 1000 ml volumetric flask, and make up the volume to the mark with pure water. 1.00 ml of this solution contains 1.00 mg of sulfate (SO4 2- ).

[0074] Preparation of sulfate standard solution: Accurately pipette 10.00 ml of the above stock solution into a 100 ml volumetric flask, and dilute it to the mark with pure water. 1.00 ml of this solution contains 100 μg of sulfate (SO4 2- ).

[0075] Method for drawing standard curve: Weigh several portions of 0.1 g of BaCl2 crystals for standby. Accurately add sulfate standard working solution, 0.00, 0.30, 0.50, 1.00, 1.25, 1.50, 2.00, and 2.50 ml, to a series of 50 ml beakers, add distilled water to a final volume of 5.00 ml, then add 1 ml of stabilizer respectively. Place the 50 ml beakers configured into a standard series on a magnetic stirrer one by one. Quickly add 0.1 g of BaCl2 crystals during stirring, time the stirring for 1 min, let it stand for 15 min, then use a 2 cm colorimetric cell to measure the absorbance value at a wavelength of 420 nm, using distilled water as a control, and draw the standard curve.

[0076] Figure 4 The standard curve drawn through the above steps is shown as y = 0.0019x - 0.0002, R² = 0.9947, where x is the SO4 2- content (mg / L), and y is the corresponding absorbance value at this time.

[0077] Cultivate the bacterial liquid with MST liquid medium, inoculate Pantoea Pantoea endophyticaAfter YNK-FB0056, the bacterial suspension and blank MST liquid culture medium were used as controls, and cultured on a shaker at 180 rpm at 30 °C for 7 days. The obtained culture medium sample was centrifuged at 12000 rpm for 10 min, and the supernatant was taken.

[0078] Measurement by turbidimetry (absorbance): Take 0.1 g of BaCl2 and 1 ml of stable solution, aspirate 250 μL of the supernatant of the bacterial solution to be measured, add it to a small beaker to make a 5 ml system, stir for 1 min on a magnetic stirrer, and let it stand for 15 min. Measure the absorbance at a wavelength of 420 nm with a UV-visible spectrophotometer, repeat 3 times, zero with the treatment adding blank MST medium, and compare the drawn standard curve.

[0079] The sulfate production rate was measured by a spectrophotometer. The total amount of sulfate ions produced by the sulfur-oxidizing bacterial isolate was 60.456 ± 1.403 mg / L.

[0080] (II) Nitrogen fixation effect test Preparation method of Asbhy nitrogen-free medium: Weigh 10 g of glucose, 0.2 g of dipotassium hydrogen phosphate, 0.2 g of sodium chloride, 0.2 g of magnesium sulfate monohydrate, 0.2 g of potassium sulfate, and 5 g of calcium carbonate, add them to 1000 mL of water and dissolve, then add 20 g of agar. Autoclave at 121 °C for 20 min.

[0081] Using the plate four-point inoculation method, inoculate the strain YNK-FB0056 obtained in Example 1 on the Asbhy nitrogen-free culture plate, and repeat inoculation on 3 plates. After inoculation, place the plates in a constant temperature incubator at 30 °C for 5 days, and observe the bacterial growth status and the generation of transparent circles around the colonies every day during the culture period.

[0082] Figure 3 The nitrogen fixation effect of the strain YNK-FB0056 is shown in (B). It can be seen from the figure that the strain YNK-FB0056 can produce a transparent circle when cultured on the Asbhy nitrogen-free medium, indicating that this strain has nitrogen fixation ability. Using the cross method to measure the size of the transparent circle, the ratio of the diameter of the transparent circle (D) to the diameter of the colony (d) D / d = 4.476 ± 0.235 is calculated, indicating that the strain YNK-FB0056 has strong nitrogen fixation ability.

[0083] (III) Zinc dissolution effect test Preparation method of solid medium: Weigh 1 g of zinc oxide, 10 g of glucose, 0.2 g of potassium chloride, 1 g of ammonium sulfate, 0.2 g of magnesium sulfate, and 0.1 g of dipotassium hydrogen phosphate, add them to 1000 mL of water and dissolve, adjust the pH to 7.3 ± 0.1, add 20 g of agar, and autoclave at 121 °C for 20 min.

[0084] Using the four-point inoculation method on a flat plate, inoculate the strain YNK-FB0056 obtained in Example 1 onto a zinc-dissolving culture plate, and repeat the inoculation on 3 plates. After inoculation, place the plates in an incubator at a constant temperature of 30°C for 5 days, and observe the bacterial growth status and the formation of clear zones around the colonies every day during the cultivation period.

[0085] Figure 3 (C) shows the zinc-dissolving effect of the strain YNK-FB0056. As can be seen from the figure, when the strain YNK-FB0056 is cultured on a zinc-dissolving culture plate, a clear zone can be produced, indicating that this strain has the ability to dissolve zinc. Use the cross-cross method to measure the size of the clear zone, and calculate the ratio of the clear zone diameter (D) to the colony diameter (d), D / d = 5.087 ± 0.3021.

[0086] (IV) Test of phosphorus-solubilizing effect 1. Organic phosphorus Preparation method of organic phosphorus solid medium: Weigh 10 g of glucose, 0.5 g of ammonium sulfate, 0.5 g of yeast extract powder, 0.3 g of sodium chloride, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate, 0.03 g of ferrous sulfate, 0.03 g of manganese sulfate, 0.2 g of lecithin, and 1 g of calcium carbonate, add them to 1000 mL of water and dissolve, adjust the pH to 7.3 ± 0.2, and add 18 ± 2 g of agar. Autoclave at 121°C for 20 min.

[0087] Using the four-point inoculation method on a flat plate, inoculate the strain YNK-FB0056 obtained in Example 1 onto an organic phosphorus culture plate, and repeat the inoculation on 3 plates. After inoculation, place the plates in an incubator at a constant temperature of 30°C for 5 days, and observe the bacterial growth status and the formation of clear zones around the colonies every day during the cultivation period.

[0088] Figure 3 (D) shows the organic phosphorus-solubilizing effect of the strain YNK-FB0056. As can be seen from the figure, when the strain YNK-FB0056 is cultured on an organic phosphorus culture plate, a clear zone can be produced, indicating that this strain has the ability to solubilize organic phosphorus. Use the cross-cross method to measure the size of the clear zone, and calculate the ratio of the clear zone diameter (D) to the colony diameter (d), D / d = 3.687 ± 0.251.

[0089] 2. Inorganic phosphorus Preparation method of inorganic phosphorus solid medium: Weigh 10 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of sodium chloride, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate heptahydrate, 0.03 g of ferric sulfate heptahydrate, 0.03 g of manganese sulfate tetrahydrate, and 2 g of tricalcium phosphate, add them to 1000 mL of water and dissolve, adjust the pH to 7.3 ± 0.1, and add 20 g of agar. Autoclave at 121°C for 20 min.

[0090] Using the flat plate four-point inoculation method, inoculate the strain YNK-FB0056 obtained in Example 1 on an inorganic phosphorus culture plate, and repeat the inoculation on 3 plates. After inoculation, place the plates in an incubator at 30°C for 5 days, and observe the bacterial growth status and the formation of clear zones around the colonies every day during the cultivation period.

[0091] Figure 3 The effect of strain YNK-FB0056 on inorganic phosphorus solubilization is shown in (E). It can be seen from the figure that when the strain YNK-FB0056 is cultured on an inorganic phosphorus culture plate, a clear zone can be produced, indicating that this strain has the ability to dissolve inorganic phosphorus. The size of the clear zone was measured by the cross-cross method, and the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated as D / d = 1.997 ± 0.174.

[0092] (V) Test for the effect of siderophore production Czapek-Dox medium without iron (g / L): glucose 30 g, sodium nitrate 3 g, dipotassium hydrogen phosphate 1 g, potassium chloride 0.5 g, magnesium sulfate 0.5 g, agar 20 g, 1000 mL of water.

[0093] CAS detection solution: Solution A: 60.5 mg of chrome azurol S and 10 ml of Fe 3+ solution (1 mmol / L FeCl3·6H2O, 10 mmol / L HCl) added to 50 ml of water; Solution B: 72.9 mg of hexadecyltrimethylammonium bromide added to 40 ml of water. After preparing Solutions A and B, mix them evenly.

[0094] Water agar: agar 18 g, 1000 ml of water.

[0095] CAS lower layer color development detection medium: Uniformly add 10% of the CAS detection solution to the water agar medium at 50 - 60°C, and pour a relatively large amount of the medium as the lower layer medium. After the water agar medium cools and solidifies, pour the Czapek-Dox medium without iron to form a double-layer plate.

[0096] Inoculate the strain YNK-FB0056 obtained in Example 1 on the CAS color development double-layer medium by the 4-point inoculation method, repeat each treatment 3 times, place it in an incubator at 30°C, and continuously observe whether a color development zone is produced around the colonies. And measure the ratio between the diameter (D) of the color development zone produced and the colony diameter (d).

[0097] Figure 3 The effect of siderophore production by strain YNK-FB0056 is shown in (F). It can be seen from the figure that an orange-red halo is produced in the medium around the colonies, indicating that this strain can produce siderophores. The size of the color development zone was measured by the cross-cross method, and the ratio of the color development zone diameter (D) to the colony diameter (d) was calculated as D / d = 1.769 ± 0.068.

[0098] Example 3 This example is used to illustrate the effect of Pantoea ( Pantoea endophytica ) YNK-FB0056 CCTCC NO: M2025644 in secreting indole-3-acetic acid.

[0099] 1. Qualitative detection KB medium: 20 g of peptone, 15 ml of glycerol, 1.5 g of K2HPO4, 1.5 g of MgSO4·7H2O, 0.1 g of tryptophan, added to 1000 mL of water and dissolved, and the pH was adjusted to 7.2 ± 0.2. Sackowcki's color reagent: 150 mL of concentrated sulfuric acid was slowly added to 250 mL of deionized water while stirring, and after the solution cooled, 7.5 mL of 0.5 mol / L FeCl3·6H2O solution was added.

[0100] A single colony of strain YNK-FB0056 obtained in Example 1 was picked and inoculated into KB medium, and cultured with shaking at 30 °C and 180 rpm / min for 24 h. Under sterile conditions, 1 mL of the fermentation broth was aspirated into a centrifuge tube and quickly mixed with 4 mL of Sackowcki's color reagent, and left to develop color in the dark at room temperature for 40 min. The color change was observed and recorded. If it turned pink, it was positive, indicating that the strain could secrete IAA.

[0101] Figure 5 (A) shows the color development of strain YNK-FB0056, and the pink color development indicates the production of IAA.

[0102] 2. Quantitative detection Precisely weigh 10 mg of pure IAA, first dissolve it with a small amount of ethanol, and then make up the volume to 100 mL with distilled water to prepare an IAA stock solution with a concentration of 100 mg / L. Then the IAA stock solution was diluted into IAA standard solutions with concentrations of 0, 4 mg / L, 8 mg / L, 12 mg / L, 16 mg / L, 20 mg / L, and 24 mg / L. Take 1 mL of each IAA standard solution with different concentrations, mix them with 1 mL of Salkowski reagent respectively, react in the dark at room temperature for 40 min, and measure the OD535 value. According to the measurement results, with the OD535 value as the y-axis and the IAA content (mg / L) as the x-axis, the standard curve was plotted as Figure 6 shown (y = 0.0068x + 0.0549, R 2 = 0.9971).

[0103] After culturing the strain in NB medium for 24 h, a seed solution was prepared and inoculated into KB liquid medium containing L-tryptophan at an inoculation amount of 1%. After culturing in a constant temperature shaker at 30 °C with a shaking speed of 180 r / min, 4 ml of the supernatant was taken every day and mixed with 4 ml of Sackowcki's chromogenic reagent. After standing for 40 min in the dark, the OD value was measured at a wavelength of 535 nm. The obtained absorbance value was substituted into the standard curve for calculation to obtain the indole-3-acetic acid content produced by the strain. Figure 5 (B) shows the indole-3-acetic acid production of the strain YNK-FB0056 for 7 consecutive days, with the highest indole-3-acetic acid content of 66.89 ± 0.26 mg / L on the 4th day.

[0104] Example 4 This example is used to illustrate the germination-promoting effect of Pantoea ( Pantoea endophytica ) YNK-FB0056 CCTCC NO: M2025644 on rape, tomato, cucumber and rye seeds.

[0105] Experimental group setting: 2 control groups (CK1: applying sterile water, CK2: applying nutrient broth NB medium) and 4 treatment groups (T1: 1×10 8 CFU / mL of the strain fermentation broth (obtained by inoculating the strain YNK-FB0056 obtained in Example 1 into NB medium and culturing in a constant temperature shaker at 30 °C with a shaking speed of 180 r / min for 24 h); T2: 1×10 7 CFU / mL of the strain fermentation broth; T3: 1×10 6 CFU / mL of the strain fermentation broth; T4: 1×10 5 CFU / mL of the strain fermentation supernatant.

[0106] Specific implementation method: Select rape, tomato, cucumber and rye seeds as experimental objects. The seeds were soaked in 70% alcohol for 10 min and then washed 3 times with sterile water for disinfection. Take the seeds that sank to the bottom of the water, soak them in water at room temperature for 12 h, and then place the seeds of each group in a 9.00 cm transparent culture dish lined with 2 layers of sterilized filter paper. There were 15 seeds per dish for each treatment, and it was repeated 3 times. Use a pipette to take 3 mL of each group of liquid to moisten the filter paper, and place it in an artificial climate chamber at 26 °C. The light and dark alternation time was 16 h and 8 h for culturing. Thereafter, 2.00 mL of sterile water needed to be added regularly every day to keep the filter paper always moist. After 7 d, record the germination situation and measure the root length and the whole plant length.

[0107] Table 2 Results table of seed germination data Data shows that in this experiment, compared with clear water, NB medium inhibited the germination of rapeseed, tomato, cucumber, and rye seeds. However, after adding strain YNK-FB0056, the seed germination rate and whole plant length increased, indicating that strain YNK-FB0056 can promote seed germination.

[0108] Example 5 This example is used to illustrate the compatibility test between Pantoea ( Pantoea endophytica ), strain YNK-FB0056, and Burkholderia gladioli ( Burkholderia gladioli ).

[0109] A bacterium was isolated and purified from rapeseed planting soil samples collected from Dali City, Yunnan Province in this laboratory and named YNK-FB0053. After identification, this strain was Burkholderia gladioli ( Burkholderia gladioli ). This strain was deposited at the China Center for Type Culture Collection on October 21, 2024, with the address of Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242288. This strain has been disclosed in the invention patent with the application number: CN2024118276363.

[0110] During the experiment, it was accidentally found that YNK-FB0056 and YNK-FB0053 could coexist on nutrient agar medium. In order to further detect whether these two strains can be mixed and applied, a strain compatibility test was carried out on them respectively.

[0111] In a sterile petri dish with a diameter of 9 cm, three sterilized Oxford cups were vertically placed on the surface of 2% water agar (WA) medium. Each Oxford cup was 2.5 cm apart from each other. The strain YNK-FB0056 obtained in Example 1 was selected as the indicator strain. A 1% (v / v) suspension of the indicator strain was inoculated in NA medium and cooled to 45 ◦ °C. The mixture was quickly mixed and immediately poured evenly onto the WA plate with Oxford cups. After the plate was completely solidified, the Oxford cups were removed with sterilized forceps to form small holes with a diameter of 6 mm. Burkholderia gladioli ( Burkholderia gladioli ), CCTCC NO: M20242288, was used as the challenge strain, and 20 µL of the previously prepared suspension (1×10 8 CFU / mL) was added to each well and incubated at 28 ◦ °C for 48 h. As a blank control, the same amount of sterile water was added, and each treatment was repeated 3 times. Then the indicator bacterium and the challenge strain were exchanged, and the above test was repeated. If a clear inhibition zone was observed around the challenge strain (as shown in Figure 7 (C)), it was considered that there was an antagonistic effect; otherwise, it was considered to be compatible.

[0112] Figure 7 shows the compatibility of strain YNK-FB0056 with Burkholderia gladioli ( Burkholderia gladioli ). When YNK-FB0056 was used as the inoculum, no clear inhibition zone was observed around it. When YNK-FB0053 was used as the inoculum, no clear inhibition zone was observed around it either. Therefore, the co-application of these two strains is feasible.

[0113] Example 6 This example is used to illustrate the growth-promoting effect of Pantoea ( Pantoea endophytica ) YNK-FB0056 on plant seedlings.

[0114] Preparation of bacterial agent: (1) Liquid bacterial agent of Pantoea ( Pantoea endophytica ) YNK-FB0056 The strain YNK-FB0056 obtained in Example 1 was inoculated on NA solid medium and cultured at 30 °C for 24 h. Then, single colonies were picked and inoculated into NB liquid medium, and cultured with shaking at 180 rpm at 30 °C for 72 h. The obtained culture solution was diluted to a viable cell count of about 1×10 8 CFU / mL, and this dilution was the liquid bacterial agent of strain YNK-FB0056.

[0115] (2) Mixed liquid bacterial agent The strain YNK-FB0056 obtained in Example 1 was inoculated on NA solid medium and cultured at 30 °C for 24 h. Then, single colonies were picked and inoculated into NB liquid medium, and cultured with shaking at 180 rpm at 30 °C for 72 h to obtain the YNK-FB0056 bacterial liquid. Burkholderia gladioli YNK-FB0053 was cultured in the same manner and conditions to obtain the YNK-FB0053 bacterial liquid. The YNK-FB0056 bacterial liquid and the YNK-FB0053 bacterial liquid were mixed by volume at a ratio of 1:1. The obtained mixed culture solution was diluted to a total viable cell count of about 1×10 8 CFU / mL, and this mixture was the mixed liquid bacterial agent of Pantoea ( Pantoea endophytica ) YNK-FB0056 and Burkholderia gladioli YNK-FB0053.

[0116] Planting: The same weight of soil was added to each flowerpot, and randomly grouped into 6 pots for each treatment. One tomato seedling with similar growth vigor was planted in each pot.

[0117] Experimental group settings: CK1: Sterilized water; CK2: Sterilized NB liquid medium; T1: Mixed liquid bacterial agent with a concentration of about 1×10 8 CFU / mL; T2: Mixed liquid bacterial agent with a concentration of about 1×10 9 CFU / mL; T3: Mixed liquid bacterial agent with a concentration of about 1×1010 A mixed liquid bacterial agent with CFU / mL.

[0118] Verification of growth promotion effect: On the 8th day after transplanting tomato seedlings into flower pots, the above-mentioned mixed liquid bacterial agent at 200 mL / plant was used for root irrigation of tomato seedlings. At the same time, the same treatment was carried out with an equal amount of sterile water as control group 1 (CK1), and the same treatment was carried out with an equal amount of NB medium as control group 2 (CK2).

[0119] The tomato seedlings after root irrigation treatment were placed in the greenhouse to grow naturally. 28 days after the root irrigation treatment, the above-ground fresh weight and dry weight, underground (root) fresh weight and dry weight, stem diameter, root length and plant height of the tomato plants were measured.

[0120] The specific measurement methods are as follows: Above-ground fresh weight & dry weight: Cut off the part above the root base of the tomato plant and weigh the above-ground fresh weight on an analytical balance, retaining 1 decimal place. The above-ground part of the plant whose fresh weight has been weighed is put into a paper bag and placed in an oven. It is blanched at 100 ± 5 °C for 10 min, then the temperature of the oven is reduced to about 75 ± 5 °C and dried to a constant weight. The above-ground dry weight is weighed on an analytical balance, retaining 1 decimal place.

[0121] Underground fresh weight & dry weight: Cut off the part below the root base of the tomato plant and weigh the underground fresh weight on an analytical balance, retaining 1 decimal place. The underground part of the plant whose fresh weight has been weighed is put into a paper bag and placed in an oven. It is blanched at 100 ± 5 °C for 10 min, then the temperature of the oven is reduced to about 75 ± 5 °C and dried to a constant weight. The underground dry weight is weighed on an analytical balance, retaining 1 decimal place.

[0122] Stem diameter: Use a vernier caliper to measure the diameter of the thickest part of the plant stem.

[0123] Root length: After straightening the root, use a ruler to measure the root length.

[0124] Plant height: After straightening the plant, use a ruler to measure the above-ground part length from the highest leaf.

[0125] Chlorophyll measurement value: A value obtained by measuring the same part of each tomato plant through a SPAD instrument. This value refers to the relative content of chlorophyll in the leaves. The higher the SPAD value, the higher the chlorophyll content in the leaves and the greener the leaves.

[0126] Figure 8 (A) and (B) respectively show the growth conditions of tomato plants after 28 days of inoculation with YNK-FB0056 liquid bacterial agent and mixed liquid bacterial agent at different concentrations.

[0127] Figure 9(A) and (B) show that 28 days after the root irrigation treatment with YNK-FB0056 liquid inoculant and mixed liquid inoculant, the plant height, stem diameter, above-ground fresh weight, root length, underground fresh weight and chlorophyll of tomato plants are significantly increased compared with the control group (CK1) treated with clear water and the control group (CK2) treated with NB medium. This shows that the application of strain YNK-FB0056 inoculant and mixed inoculant has a significant promoting effect on the growth of tomato seedlings.

[0128] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including the combination of each technical feature in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A Pantoea sp. Pantoea endophytica ), YNK-FB0056, characterized in that The preservation number of this strain is CCTCC NO: M 2025644.

2. A microbial agent, wherein the active ingredient of the microbial agent is the Pantoea as claimed in claim 1.

3. Use of Pantoea sp. Pantoea endophytica ) YNK-FB0056 or the bacterial agent according to claim 2 in sulfur oxidation, zinc dissolution, and / or IAA production, wherein sulfur oxidation refers to the oxidation of sodium thiosulfate to produce sulfate ions; zinc dissolution refers to the dissolution of zinc oxide.

4. A method for producing indoleacetic acid, characterized in that, The method includes fermenting and culturing Pantoea sp. Pantoea endophytica YNK-FB0056 described in claim 1, and collecting the culture product.

5. The method according to claim 4, wherein The conditions for the fermentation culture include: a culture temperature of 25 - 37 °C, a culture time of 24 - 120 h, and an initial pH of 6 - 8.

6. A composite microbial agent, characterized in that: including Pantoea ( Pantoea endophytica ) YNK-FB0056 and Burkholderia gladioli ( Burkholderia gladioli ); wherein, the preservation number of the Burkholderia gladioli is CCTCC NO: M 20242288.

7. Use of Pantoea sp. Pantoea endophytica YNK-FB0056 described in claim 1, the bacterial agent described in claim 2, or the compound bacterial agent described in claim 6 in promoting plant growth.

8. A method for promoting plant growth, characterized in that, The method includes applying Pantoea sp. YNK-FB0056 described in claim 1, the microbial agent described in claim 2, or the compound microbial agent described in claim 6 to the rhizosphere of plants. Pantoea endophytica ​ 9. The method according to claim 8, wherein: The use of the Pantoea sp. ( Pantoea endophytica ), the bacterial agent or the compound bacterial agent enables the dosage of the Pantoea sp. to be not less than 1×10 8 CFU / strain / time; the application frequency is 1-3 times per crop; the plant is selected from the Solanaceae plants.

10. The method according to claim 8, characterized in that: The Solanaceae plant is tomato.

Images