A strain of Pantoea YNK-FB0056 and its application

Abstract

The present invention relates to the technical field of microorganisms and discloses a strain of Pantoea ( Pantoea endophytica ) YNK‑FB0056 and its application. The Pantoea provided by the present invention has a preservation number of CCTCC NO: M 2025644, which has excellent sulfur oxidation, nitrogen fixation, phosphate solubilization, zinc solubilization, as well as the ability to produce iron carriers and high-yield indoleacetic acid. It can regulate the nutritional structure of the soil and transform substances in the soil that are difficult for plants to utilize, thereby promoting plant growth and improving the stress resistance of plants. In addition, the sulfur-oxidizing bacteria YNK‑FB0056 provided by the present invention is a strain isolated from rapeseed farmland soil. Compared with strains screened by extreme environments and laboratory mutagenesis, it has better adaptability to planting environments, can stably colonize in the rhizosphere and soil of plants, and thus play a long-term and stable role in promoting growth. It is safe and environmentally friendly and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically to a strain of Pantoea ( Pantoea endophytica )YNK-FB0056 and its applications. Background Art

[0002] The core contradiction facing modern agricultural development lies in balancing the need for high yields with ecological sustainability. Currently, soil ecosystem degradation has become a key bottleneck hindering high-quality agricultural development, manifesting primarily in reduced microbial diversity, impeded nutrient cycling, and pollutant accumulation. Traditional agronomic practices struggle to maintain soil health while ensuring yields, necessitating innovative solutions.

[0003] In recent years, microbiome-based ecological regulation technologies have provided new insights into soil remediation. Among them, plant growth promoting rhizobacteria (PGPR) have become a research hotspot due to their unique niche adaptability and multifunctionality. These microorganisms not only improve nutrient utilization efficiency through biological nitrogen fixation, organophosphate mineralization, and sulfur oxidation, but also synthesize active substances such as siderophores and indoleacetic acid, alleviating abiotic stresses on plants. More importantly, some PGPR strains can form biofilms through quorum sensing, establishing stable functional microbial communities in the rhizosphere. This characteristic gives them significant advantages in field applications. However, existing commercial microbial agents generally suffer from poor environmental adaptability and limited functionality, and their practical application is limited by multiple factors, including soil type, climatic conditions, and crop variety.

[0004] To address these challenges, this technology, through the development of an efficient PGPR screening system, aims to identify dominant strains with broad-spectrum growth-promoting properties and strong environmental adaptability. This research not only provides core bacterial strain resources for the development of new agricultural biostimulants but also offers technical support for establishing an eco-agricultural model centered around microorganisms, thus having significant practical value in promoting the green transformation of agriculture. Summary of the Invention

[0005] The present invention aims to solve the problem that the prior art lacks effective plant growth-promoting bacteria and their products in practical applications, and provides a Pantoea aeruginosa ( Pantoea endophytica ) YNK-FB0056 and its application. The present invention provides Pantoea ( Pantoea endophytica ) YNK-FB0056 was isolated from rapeseed farmland soil in Qujing City, Yunnan Province. The bacterium has the characteristics of sulfur oxidation, nitrogen fixation, zinc solubilization, organic and inorganic phosphorus solubilization, and iron carrier production. At the same time, when 0.1g / L tryptophan was added, the IAA production was as high as 66.89mg / L.

[0006] To achieve the above object, the present invention provides a first aspect of a Pantoea strain ( Pantoea endophytica ) YNK-FB0056, the deposit number of this strain is CCTCC NO: M 2025644.

[0007] The second aspect of the present invention provides a bacterial agent, the active ingredient of which is the Pantoea described in the first aspect.

[0008] The third aspect of the present invention provides the use 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.

[0009] A fourth aspect of the present invention provides a method for producing indoleacetic acid, comprising: Pantoea endophytica ) YNK-FB0056 was fermented and the culture products were collected.

[0010] The fifth aspect of the present invention provides a composite bacterial agent, comprising Pantoea ( Pantoea endophytica ) YNK-FB0056 and Burkholderia gladiolus ( Burkholderia gladioli ); wherein the preservation number of the gladiolus Burkholderia is CCTCC NO: M 20242288.

[0011] The sixth aspect of the present invention provides a method for promoting plant growth, the method comprising: Pantoea endophytica ) YNK-FB0056, the bacterial agent described in the second aspect, or the composite bacterial agent described in the fifth aspect is applied to the rhizosphere of plants.

[0012] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0013] (1) Pantoea provided by the present invention ( Pantoea endophytica ) YNK-FB0056 has excellent sulfur oxidation, nitrogen fixation, zinc solubilization, organic and inorganic phosphorus decomposition, and siderophore production functions, and can produce high amounts of indoleacetic acid, providing new materials and methods for the production of indoleacetic acid.

[0014] (2) Pantoea provided by the present invention ( Pantoea endophytica YNK-FB0056 can effectively promote crop growth. Experimental results showed that 28 days after application of this strain to tomato seedlings, plant height, stem diameter, aboveground fresh weight, root length, underground fresh weight, and chlorophyll content were significantly improved compared to the control group (CK1) treated with pure water and the control group (CK2) treated with NB culture medium, demonstrating that this strain has a strong plant growth-promoting effect.

[0015] (3) Pantoea provided by the present invention ( Pantoea endophytica ) YNK-FB0056 is a natural strain isolated from rapeseed farmland soil. Compared with laboratory-induced mutant strains, it is easier to colonize and is safer and more environmentally friendly. It has the potential to be developed and applied as a new type of microbial strain resource. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the Pantoea in Example 1 ( Pantoea endophytica ) Colony morphology of YNK-FB0056 (A) and scanning electron microscopy images (B) and (C);

[0017] Figure 2 is the Pantoea in Example 1 ( Pantoea endophytica ) Phylogenetic tree of YNK-FB0056;

[0018] Figure 3 is the Pantoea in Example 2 ( Pantoea endophytica ) Effect diagram of YNK-FB0056 in promoting nutrient cycling: (A) Sulfur oxidation effect diagram; (B) Nitrogen fixation effect diagram; (C) Zinc dissolution effect diagram; (D) Organic phosphorus decomposition effect diagram; (E) Inorganic phosphorus decomposition effect diagram; (F) Siderophore production effect diagram.

[0019] Figure 4 is the Pantoea in Example 2 ( Pantoea endophytica ) Standard curve plotted for the quantification of the sulfur oxidation effect of YNK-FB0056.

[0020] Figure 5 is the Pantoea in Example 3 ( Pantoea endophytica ) Effect diagram of IAA production by YNK-FB0056: (A) Qualitative color development of IAA production; (B) Quantitative changes of IAA production over 7 consecutive days.

[0021] Figure 6 is the Pantoea in Example 3 ( Pantoea endophytica ) Standard curve plotted for the quantification of IAA production by YNK-FB0056.

[0022] Figure 7 is the Pantoea in Example 5 ( Pantoea endophytica ) YNK-FB0056 and Burkholderia gladiolus ( Burkholderia gladioli ) Schematic diagram of the compatibility results of CCTCC NO: M20242288.

[0023] Figure 8 is the Pantoea in Example 6 ( Pantoea endophytica ) The growth-promoting effect of YNK-FB0056 on plant seedlings: (A) The growth-promoting effect of YNK-FB0056 liquid inoculant; (B) The growth-promoting effect of mixed liquid inoculants.

[0024] Figure 9 is the Pantoea in Example 6 ( Pantoea endophytica ) Analysis of YNK-FB0056's growth-promoting effects on plant seedlings; (A) Growth-promoting effect of YNK-FB0056 liquid microbial agent; (B) Growth-promoting effect of mixed liquid microbial agents.

[0025] Biological Deposits

[0026] The present invention provides Pantoea ( Pantoea endophytica )YNK-FB0056, classified as: Pantoea endophytica YNK-FB0056 was deposited in the China Center for Type Culture Collection on March 31, 2025, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 2025644.

[0027] The gladiolus Burkholderia provided by the present invention Burkholderia gladioli ) YNK-FB0053, classified as Burkholderia gladioli YNK-FB0053 was deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on October 21, 2024, with the deposit number CCTCC NO: M 20242288. This strain has been disclosed in the invention patent application number: CN202411827636.3. DETAILED DESCRIPTION

[0028] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0029] The beneficial functions of PGPR on soil and plants make it one of the main raw materials for agricultural microbial preparations. The development and use of PGPR make up for the problems of pesticide residues, environmental pollution and food safety caused by chemical control, and are in line with the principles of sustainable development of agriculture, environmental friendliness and safety of humans and animals. In order to improve development efficiency, the current methods for developing PGPR strains with better efficacy in this field mainly rely on artificial mutagenesis or genetic engineering modification, so as to screen and obtain new strains with excellent plant growth promotion and / or disease prevention effects. However, the test strains obtained by artificial mutagenesis or genetic engineering modification are usually bred in a laboratory environment, and have poor adaptability to the natural environment. In addition, the colonization of the strains in plants or in the soil is difficult to be well verified in the laboratory, resulting in many high-quality PGPR bacteria developed in laboratories being difficult to show the same level of growth promotion or disease prevention effects in actual applications as in research.

[0030] During the research, the inventors of the present invention isolated a sulfur-oxidizing bacterium from rapeseed farmland soil in Qujing City, Yunnan Province, and identified it as Pantoea ( Pantoea endophytica The inventors discovered that this strain possesses excellent sulfur oxidation capabilities, as well as the ability to fix nitrogen, dissolve zinc, and dissolve organic and inorganic phosphorus, as well as produce siderophores. Furthermore, it can efficiently ferment and produce indoleacetic acid (IAA) in culture medium. Further research has also revealed that when applied to plants such as tomatoes through root irrigation, this strain can effectively increase the height and dry weight of seedlings, demonstrating a significant plant growth-promoting effect, thereby increasing agricultural productivity and promoting the green and sustainable development of ecological agriculture.

[0031] Based on the above findings, the present invention provides a strain of Pantoea ( Pantoea endophytica )YNK-FB0056, the deposit number of this strain is CCTCC NO: M 2025644.

[0032] The second aspect of the present invention provides the Pantoea ( Pantoea endophytica ) Application of YNK-FB0056 in sulfur oxidation, nitrogen fixation, zinc dissolution, organic and inorganic phosphorus decomposition, and siderogen production or indoleacetic acid production.

[0033] In the present invention, "nitrogen fixation" and "phosphate solubilization" refer to the nitrogen fixation and phosphate solubilization of the Pantoea ( Pantoea endophytica YNK-FB0056 increases the amount of N and P available to plants in the soil. For example, it converts inorganic phosphorus components (such as Ca3(PO4)2 and FePO4) and organic phosphorus components (such as lecithin and inositol hexaphosphate) in the soil into forms that can be used by plants, thereby promoting their absorption and utilization of phosphorus in the soil.

[0034] The third aspect of the present invention provides a method for sulfur oxidation and indoleacetic acid production, the method comprising the steps of: Pantoea endophytica ) YNK-FB0056 was cultured and the culture products were collected.

[0035] Any species capable of fermenting and culturing Pantoea ( Pantoea endophytica ) YNK-FB0056, and the methods and conditions for sulfur oxidation thereof are all applicable to the present invention.

[0036] According to a preferred embodiment of the present invention, the fermentation culture conditions include: culture temperature 25-37°C, culture time 24-120h, and initial pH 6-8.

[0037] According to a preferred embodiment of the present invention, when the fermentation culture is carried out in a shaking table fermentation manner, the fermentation culture process can be carried out at a shaking table rotation speed of 120-200 rpm.

[0038] In the present invention, there is no particular limitation on the culture medium used for the fermentation culture, and any medium that can be used for Pantoea ( Pantoea endophytica ) The culture medium for YNK-FB0056 fermentation culture can be applied to the present invention.

[0039] According to a preferred embodiment of the present invention, the culture 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).

[0040] The fourth aspect of the present invention provides a bacterial agent having the function of promoting plant growth, wherein the active ingredient in the bacterial agent includes the Pantoea ( Pantoea endophytica )YNK-FB0056.

[0041] In the present invention, there is no particular limitation on the specific formulation of the microbial agent, and any microbial agent formulation commonly used in agricultural microbial preparations in the art can be applied to the present invention.

[0042] According to a preferred embodiment of the present invention, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0043] Preferably, in the liquid bacterial agent, the Pantoea ( Pantoea endophytica )The content of 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.

[0044] Preferably, in the solid bacterial agent, the Pantoea ( Pantoea endophytica )The effective viable count of 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.

[0045] In order to increase the content of Pantoea ( Pantoea endophytica To enhance the activity and stability of YNK-FB0056, or to make the inoculum more convenient to transport, store, and use, according to a preferred embodiment of the present invention, the inoculum may further contain an adjuvant. Any adjuvant known in the art for use in agricultural inoculum products is suitable for use in the present invention. Preferably, the adjuvant is selected from at least one of an excipient (e.g., tapioca starch, sorbitol, etc.), a protective agent (e.g., a carbohydrate protective agent, sodium glutamate, calcium chloride, etc.), and a buffer (e.g., phosphate buffer, sodium bicarbonate, magnesium oxide, etc.).

[0046] During the long-term research process, the inventors found that if only a single strain of bacteria is present in a microbial preparation, it is easy to have a single effect and unstable results. By adding excipients and / or other strains to the microbial preparation, the function can be made more comprehensive, the effect can be better and more stable.

[0047] During the research process, the inventor accidentally discovered that the Pantoea provided by the present invention ( Pantoea endophytica ) YNK-FB0056 and certain Burkholderia species ( Burkholderia ) bacteria (e.g. Burkholderia gladioli ( Burkholderia gladioli )CCTCC NO: M 20242288) can co-exist on NA medium. Further research has found that when they are mixed and used, they have a synergistic effect, which not only enables the Pantoea ( Pantoea endophytica ) The properties of YNK-FB0056 are more stable and can further enhance the growth-promoting effect on plants.

[0048] According to some preferred embodiments of the present invention, the bacterial agent further comprises auxiliary bacteria, and the auxiliary bacteria is Burkholderia gladiolus ( ).

[0049] Preferably, the deposit number of the Burkholderia gladiolus is CCTCC NO: M 20242288.

[0050] Preferably, in the bacterial agent, Pantoea (Burkholderia gladioli ) The ratio of the viable bacterial counts of YNK-FB0056 and Burkholderia gladiolus was 1:1.

[0051] In the bacterial agent provided by the present invention, the auxiliary bacteria can be packaged separately and used together with the Pantoea ( Pantoea endophytica ) YNK-FB0056, or they can be mixed during preparation and applied directly to plants when used.

[0052] The fifth aspect of the present invention provides the Pantoea ( Pantoea endophytica ) YNK-FB0056, or, the use of the bacterial agent described in the fourth aspect in promoting plant growth.

[0053] In the present invention, the “promoting plant growth” may include increasing the growth rate of plants, increasing yield, improving the quality of agricultural products (eg, increasing the weight of a single fruit, etc.), etc.

[0054] A sixth aspect of the present invention provides a method for promoting plant growth, comprising: Pantoea endophytica ) YNK-FB0056 or the fungal agent described in the fourth aspect is applied to plants.

[0055] According to a preferred embodiment of the present invention, wherein the Pantoea ( Pantoea endophytica )The dosage of YNK-FB0056 is not less than 1×10 8 CFU / strain / time, preferably 1×10 9 -1×10 12 CFU / strain / time.

[0056] According to a preferred embodiment of the present invention, the amount of the bacterial agent is such that Pantoea ( Pantoea endophytica Pantoea )The dosage of 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.

[0057] Preferably, the Pantoea ( endophytica ) The application frequency of YNK-FB0056 or fungal agent is 1-3 times per crop.

[0058] According to a preferred embodiment of the present invention, the dosage of the metabolite is not less than 100 mL / plant / time, preferably 100-200 mL / plant / time.

[0059] Preferably, the plant is selected from the Solanaceae family, preferably tomatoes.

[0060] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0061] In the following examples, unless otherwise specified, all reagents and materials used were purchased from regular chemical / biological reagent or material suppliers, and all reagents were of analytical grade.

[0062] In the following examples, unless otherwise specified, the operating temperature is room temperature (25±5°C).

[0063] Example 1

[0064] This example is used to illustrate the Pantoea endophytica ) Acquisition, identification and deposit of YNK-FB0056 CCTCC NO: M2025644.

[0065] (I) Strain isolation and purification

[0066] Thiosulfate agar medium (MST medium) was used during strain isolation and purification. The preparation method was as follows: 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 were weighed and added to 1000 mL of water. The pH was adjusted to 8 ± 0.1, and then 20 g of agar was added. The mixture was autoclaved at 121°C for 25 min.

[0067] A bacterial strain named YNK-FB0056 was isolated and purified from rapeseed soil samples collected from Qujing City, Yunnan Province by using the dilution spread plate method and the streak plate method.

[0068] (2) Strain identification

[0069] 1. Identification of bacterial morphological, physiological and biochemical characteristics

[0070] Physiological and biochemical tests were performed on strain YNK-FB0056 according to the Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria, and the colony morphology characteristics were described.

[0071] Colony and strain morphology: Pantoea endophytica (A) shows the colony morphology of strain YNK-FB0056 on MST medium. As can be seen from the figure, the colony of this strain is nearly circular, with relatively neat edges, bright yellow color, and a convex center, moist and shiny. Electron microscopy observation ( Figure 1 (B) (C) The strain is a rod-shaped bacterium without flagella and spores.

[0072] Physiological and biochemical characteristics: Strain YNK-FB0056 is Gram-positive, facultative anaerobic, and has positive reactions to catalase, glucose, sucrose, mannitol, arabinose, xylose, maltose, and glycerol; it has negative reactions to oxidase, urease, gelatin liquefaction, starch hydrolysis, lactose, dulcitol, and inositol.

[0073] Molecular identification: Total DNA of strain YNK-FB0056 was extracted by Chelex extraction method and used as template. 27F (5'AGAGTTTGATCCTGGCTCAG-3') was used as upstream primer and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') was used as downstream primer. 16S rRNA was amplified using the reaction system and conditions listed in Table 1.

[0074] Table 1 PCR system and conditions

[0075]

[0076]

[0077] Figure 1 The phylogenetic tree of strain YNK-FB0056 is shown, from which it can be seen that YNK-FB0056 is closely related to Pantoea ( Figure 2 )596 KR610525 has the highest homology.

[0078] 3. Identification results

[0079] Combined with the molecular detection results of strain YNK-FB0056 and the results of bacterial morphological and physiological and biochemical characteristics, the strain was identified as Pantoea ( Pantoea endophytica Pantoea endophytica ).

[0080] (3) Strain preservation

[0081] The Pantoea ( Pantoea endophytica ) YNK-FB0056 was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on March 31, 2025, with the deposit number CCTCC NO: M 2025644.

[0082] Example 2

[0083] This example is used to illustrate the Pantoea endophytica )YNK-FB0056 CCTCC NO: M2025644 has the effects of sulfur oxidation, nitrogen fixation, zinc dissolution, organic and inorganic phosphorus decomposition, and siderophore production.

[0084] (1) Sulfur oxidation effect test

[0085] 1. Qualitative testing

[0086] MST (Modified Thiosulfate) Medium (g / L): Weigh 5.0 g of sodium thiosulfate, 0.1 g of potassium dihydrogen 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 and dissolve in 1000 mL of water. Add 0.008 g of bromocresol purple as a color developer, adjust the pH to 8 ± 0.1, and add 20 g of agar. Autoclave at 121°C for 20 min.

[0087] Strain YNK-FB0053 obtained in Example 1 was inoculated into MST medium supplemented with a colorimetric reagent using a four-point inoculation method. Each treatment was repeated three times. The culture was placed in a 30°C incubator and continuously observed for the formation of a yellow halo around the colonies. The ratio of the diameter of the resulting halo (D) to the colony diameter (d) was also measured.

[0088] Figure 3(A) Shows the sulfur oxidation activity of strain YNK-FB0056. As can be seen, a yellow halo forms in the culture medium surrounding the colonies, indicating that the strain is capable of sulfur oxidation. The size of the halo was measured using the cross-hatch method, and the ratio of the halo diameter (D) to the colony diameter (d) was calculated (D / d = 2.823 ± 0.022).

[0089] 2. Quantitative detection

[0090] 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 to 1000 mL of water and dissolve. Adjust the pH to 8 ± 0.1, and sterilize by autoclaving at 121°C for 20 min.

[0091] Preparation of stabilizing solution: Dissolve 75g analytical grade NaCl in 300ml water, add 30ml analytical grade concentrated HCl, 50ml glycerol and 100ml 95% ethanol, and mix well.

[0092] Preparation of sulfate standard stock solution: Weigh 1.814 g of anhydrous potassium sulfate (K2SO4 analytical grade, pre-heat at 105 o C for two hours), dissolve in a small amount of pure water, transfer to a 1000ml volumetric flask, and dilute to the mark with pure water. 1.00ml of this solution contains 1.00mg of sulfate (SO4 2- ).

[0093] Preparation of sulfate standard solution: Accurately pipette 10.00ml of the above stock solution into a 100ml volumetric flask and dilute to the mark with pure water. 1.00ml of this solution contains 100ug sulfate (SO4 2- ).

[0094] Method for drawing a standard curve: Weigh several portions of 0.1g BaCl2 crystals for later use, accurately add 0.00, 0.30, 0.50, 1.00, 1.25, 1.50, 2.00 and 2.50ml of sulfate standard working solution into a series of 50ml beakers, add distilled water to the final volume of 5.00ml, and then add 1ml of stabilizer to each beaker. Place the 50ml beakers configured into the standard series one by one on a magnetic stirrer, quickly add 0.1g BaCl2 crystals while stirring, stir for 1 minute, let it stand for 15 minutes, and then use a 2cm cuvette to measure the absorbance at a wavelength of 420nm. Use distilled water as a control, and draw a standard curve.

[0095] Figure 4 The standard curve obtained by the above steps is shown in the figure: y = 0.0019x - 0.0002, R² = 0.9947, where x is SO42- content (mg / L), y is the corresponding absorbance value at this time.

[0096] The bacterial liquid was cultured in MST liquid medium and inoculated with Pantoea ( Pantoea endophytica ) YNK-FB0056 was then added, and the bacterial suspension and blank MST liquid culture medium were used as controls. The culture was cultured in a shaking incubator at 180 rpm at 30°C for 7 days. The culture medium samples were centrifuged at 12,000 rpm for 10 min, and the supernatant was collected.

[0097] Turbidimetric (absorbance) determination: Add 0.1g BaCl2 to 1ml of stabilizing solution, pipette 250μL of the supernatant from the bacterial culture to be tested, and add to a small beaker to make a 5ml system. Stir for 1 minute on a magnetic stirrer. Let stand for 15 minutes. Measure the absorbance at 420nm using a UV-visible spectrophotometer. Repeat three times. Zero the system by adding blank MST medium and compare to the standard curve.

[0098] The sulfate production rate was determined by spectrophotometry. The total amount of sulfate ions produced by the sulfur-oxidizing bacteria isolate was 60.456±1.403 mg / L.

[0099] (2) Nitrogen fixation effect test

[0100] Preparation 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, dissolve in 1000 mL of water, and add 20 g of agar. Autoclave at 121°C for 20 min.

[0101] The strain YNK-FB0056 obtained in Example 1 was inoculated onto Aspergillus nitrogen-free culture plates using a four-point plate inoculation method. Three plates were inoculated. After inoculation, the plates were placed in a 30°C incubator for 5 days. Bacterial growth and the formation of clear zones around the colonies were observed daily during the incubation period.

[0102] Figure 3 (B) shows the nitrogen-fixing ability of strain YNK-FB0056. As shown, strain YNK-FB0056 produces a clearing zone when cultured on Aspergillus nitrogen-free medium, indicating nitrogen fixation. The clearing zone size was measured using the cross-hatch method, and the ratio of the clearing zone diameter (D) to the colony diameter (d) was calculated to be D / d = 4.476 ± 0.235, indicating that strain YNK-FB0056 has strong nitrogen-fixing ability.

[0103] (3) Zinc dissolving effect test

[0104] Preparation of solid culture medium: Weigh 1 g zinc oxide, 10 g glucose, 0.2 g potassium chloride, 1 g ammonium sulfate, 0.2 g magnesium sulfate, and 0.1 g dipotassium hydrogen phosphate, add to 1000 mL water to dissolve, adjust the pH to 7.3 ± 0.1, add 20 g agar, and sterilize by autoclaving at 121°C for 20 min.

[0105] The strain YNK-FB0056 obtained in Example 1 was inoculated onto zinc-soluble culture plates using the four-point plate inoculation method. Three plates were inoculated. After inoculation, the plates were placed in a 30°C incubator for 5 days. Bacterial growth and the formation of clear zones around the colonies were observed daily during the incubation period.

[0106] Figure 3 (C) shows the zinc solubilization ability of strain YNK-FB0056. As can be seen, strain YNK-FB0056 produces a clearing zone when cultured on a zinc-solubilizing plate, demonstrating its zinc solubility. The size of the clearing zone was measured using the cross-hatch method, and the ratio of the clearing zone diameter (D) to the colony diameter (d) was calculated: D / d = 5.087 ± 0.3021.

[0107] (IV) Phosphate solubilization effect test

[0108] 1. Organophosphorus

[0109] To prepare organophosphorus 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. Dissolve in 1000 mL of water, adjust the pH to 7.3 ± 0.2, and add 18 ± 2 g of agar. Autoclave at 121°C for 20 min.

[0110] The strain YNK-FB0056 obtained in Example 1 was inoculated onto three organophosphate culture plates using the four-point plate inoculation method. After inoculation, the plates were placed in a 30°C incubator for 5 days. Bacterial growth and the formation of clear zones around the colonies were observed daily during the incubation period.

[0111] Figure 3 (D) shows the organophosphate degrading activity of strain YNK-FB0056. As can be seen, strain YNK-FB0056 produces a clearing zone when cultured on an organophosphate plate, demonstrating its organophosphate degrading ability. The clearing zone size was measured using the cross-hatch method, and the ratio of the clearing zone diameter (D) to the colony diameter (d) was calculated (D / d = 3.687 ± 0.251).

[0112] 2. Inorganic phosphorus

[0113] Preparation 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. Dissolve in 1000 mL of water, adjust the pH to 7.3 ± 0.1, and add 20 g of agar. Autoclave at 121°C for 20 min.

[0114] Using the four-point plate inoculation method, strain YNK-FB0056 obtained in Example 1 was inoculated onto three inorganic phosphate culture plates. After inoculation, the plates were placed in a 30°C incubator for 5 days. Bacterial growth and the formation of clear zones around the colonies were observed daily during the incubation period.

[0115] Figure 3 (E) shows the inorganic phosphate solubilization ability of strain YNK-FB0056. As can be seen, strain YNK-FB0056 produces a clearing zone when cultured on an inorganic phosphate plate, demonstrating its ability to solubilize inorganic phosphate. The size of the clearing zone was measured using the cross-hatch method, and the ratio of the clearing zone diameter (D) to the colony diameter (d) was calculated (D / d = 1.997 ± 0.174).

[0116] (V) Siderophore production effect test

[0117] Iron-free Czapek medium (g / L): 30 g glucose, 3 g sodium nitrate, 1 g potassium dihydrogen phosphate, 0.5 g potassium chloride, 0.5 g magnesium sulfate, 20 g agar, 1000 mL water.

[0118] CAS test solution: Solution A: 60.5 mg chrome azurol S and 10 ml Fe 3+ Solution B: Add 72.9 mg of hexadecylaminoalkylammonium bromide to 40 ml of water. Mix solution A and solution B thoroughly.

[0119] Water agar: 18g agar, 1000ml water.

[0120] CAS lower layer chromogenic assay medium: Evenly add 10% CAS assay solution to a 50-60°C water agar medium. Pour a larger amount of the medium as the lower layer. After the water agar medium cools and solidifies, pour iron-free Czapek medium into the medium to form a double-layer plate.

[0121] Strain YNK-FB0056 obtained in Example 1 was inoculated into a CAS chromogenic double-layer culture medium using a four-point inoculation method. Each treatment was repeated three times. The culture was placed in a 30°C incubator and continuously observed for the formation of a color zone around the colony. The ratio of the diameter of the color zone (D) to the colony diameter (d) was also measured.

[0122] Figure 3 (F) shows the siderophore production of strain YNK-FB0056. As can be seen, an orange-red halo forms in the culture medium surrounding the colonies, indicating that the strain produces siderophores. The size of the halo was measured using the cross-hatch method, and the ratio of the halo diameter (D) to the colony diameter (d) was calculated (D / d = 1.769 ± 0.068).

[0123] Example 3

[0124] This example is used to illustrate the Pantoea endophytica ) Effect of YNK-FB0056 CCTCC NO: M2025644 on the secretion of indoleacetic acid.

[0125] 1. Qualitative testing

[0126] KB medium: Dissolve 20 g of peptone, 15 ml of glycerol, 1.5 g of K₂HPO₄, 1.5 g of MgSO₄·7H₂O, and 0.1 g of tryptophan in 1000 mL of water and adjust the pH to 7.2 ± 0.2. Sackowcki's color developer: Slowly add 150 mL of concentrated sulfuric acid to 250 mL of deionized water while stirring. After the solution cools, add 7.5 mL of 0.5 mol / L FeCl₃·6H₂O solution.

[0127] A single colony of strain YNK-FB0056 obtained in Example 1 was inoculated into KB medium and cultured at 30°C and 180 rpm / min for 24 h. 1 mL of the fermentation broth was aseptically pipetted into a centrifuge tube and quickly mixed with 4 mL of Sackowcki's color developer. The tube was incubated in the dark at room temperature for 40 min to develop color. The color change was observed and recorded. A pink color was considered positive, indicating that the strain could secrete IAA.

[0128] Figure 5 (A) shows the color development of strain YNK-FB0056, where pink color indicates IAA production.

[0129] 2. Quantitative detection

[0130] Accurately weigh 10 mg of pure IAA, first dissolve it with a small amount of ethanol, and then dilute to 100 mL with distilled water to prepare an IAA mother solution with a concentration of 100 mg / L. Then dilute the IAA mother solution to IAA standard solutions according to 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 of different concentrations, mix them with 1 mL of Salkowski reagent, react at room temperature in the dark for 40 minutes, 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, a standard curve is drawn as shown below. Figure 6 As shown (y = 0.0068x + 0.0549, R 2 =0.9971).

[0131] After culturing the strain in NB medium for 24 hours, a seed solution was prepared and inoculated into KB liquid medium containing L-tryptophan at a 1% inoculum. The culture was placed in a 30°C constant temperature shaker at 180 rpm. Every day, 4 ml of the supernatant was aspirated and mixed with 4 ml of Sackowcki's colorimetric reagent. After standing in the dark for 40 minutes, the OD value was measured at a wavelength of 535 nm. The absorbance value obtained was substituted into the standard curve for calculation to obtain the indoleacetic acid content of the strain. Figure 5 (B) shows the production of indoleacetic acid by strain YNK-FB0056 for 7 consecutive days, with the highest indoleacetic acid content reaching 66.89±0.26 mg / L on the 4th day.

[0132] Example 4

[0133] This example is used to illustrate the Pantoea endophytica ) YNK-FB0056 CCTCC NO: M2025644 promotes germination of rapeseed, tomato, cucumber and rye seeds.

[0134] Experimental group settings: 2 control groups (CK1: sterile water, CK2: nutrient broth NB medium) and 4 treatment groups (T1: 1×10 8 CFU / mL of the 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 and 180 rpm for 24 h); T2: 1×10 7 CFU / mL of the fermentation broth; T3: 1×10 6 CFU / mL of the fermentation broth; T4: 1×10 5 CFU / mL of the fermentation supernatant of the strain.

[0135] Specific implementation method: Rapeseed, tomato, cucumber, and rye seeds were selected as experimental subjects. The seeds were soaked in 70% alcohol for 10 minutes and then rinsed three times with sterile water to complete disinfection. The seeds that settled under the water were taken and soaked in water at room temperature for 12 hours. After that, the seeds of each group were placed in a 9.00 cm transparent Petri dish lined with two layers of sterilized filter paper. Each dish contained 15 seeds per treatment, and the experiment was repeated three times. 3 mL of liquid from each group was pipetted to moisten the filter paper. The seeds were placed in a 26°C artificial climate chamber with alternating light and dark cycles of 16 hours and 8 hours. 2.00 mL of sterile water was added regularly every day to keep the filter paper moist. After 7 days, germination was recorded and root length and overall plant length were measured.

[0136] Table 2 Seed germination data results

[0137]

[0138] The data show that in this experiment, compared with pure water, NB medium inhibited the germination of rapeseed, tomato, cucumber and rye seeds. However, after adding strain YNK-FB0056, the seed germination rate and the whole plant length were improved, indicating that strain YNK-FB0056 can promote seed germination.

[0139] Example 5

[0140] This example is used to illustrate the Pantoea endophytica ) YNK-FB0056 and Burkholderia gladiolus ( Burkholderia gladioli ) compatibility test.

[0141] Our laboratory isolated and purified a bacterial strain from a rapeseed soil sample collected in Dali, Yunnan Province, and named it YNK-FB0053. The strain was identified as Burkholderia gladioli ( Burkholderia gladioli The strain was deposited on October 21, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M20242288. The strain has been disclosed in the invention patent application number: CN2024118276363.

[0142] During the experiment, it was accidentally discovered that YNK-FB0056 and YNK-FB0053 could survive together on nutrient agar medium. In order to further test whether these two strains could be mixed for application, strain compatibility tests were performed on them separately.

[0143] In a sterile Petri dish with a diameter of 9 cm, three sterilized Oxford cups were placed vertically on the surface of a 2% water agar (WA) medium. Each Oxford cup was 2.5 cm apart. 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 into the NA medium and cooled to 45°C. ◦ C. Mix the mixture quickly and immediately pour it evenly onto the WA plate with the Oxford cup. After the plate is completely solidified, remove the Oxford cup with sterilized tweezers and make a small hole with a diameter of 6 mm. Burkholderia gladioli ) CCTCC NO: M20242288 was used as the challenge strain, and 20 μL of the previously prepared suspension (1×10 8 CFU / mL), at 28 ◦ Incubate at 48°C for 48 h. For the blank control, add the same amount of sterile water and repeat each treatment 3 times. Then exchange the indicator bacteria with the challenge strain and repeat the above test. If a transparent inhibition zone (such as Figure 7 If the two groups are similar (as shown in (C)), it is considered that there is antagonism; otherwise, it is considered to be compatible.

[0144] Figure 7 Strain YNK-FB0056 is shown to be related to Burkholderia gladioli ( Burkholderia gladioli ) compatibility. No clear inhibition zone was observed around YNK-FB0056 when used as a challenge, nor was a clear inhibition zone observed around YNK-FB0053 when used as a challenge. Therefore, these two strains are feasible for co-administration.

[0145] Example 6

[0146] This example is used to illustrate the Pantoea endophytica ) The growth-promoting effect of YNK-FB0056 on plant seedlings.

[0147] Preparation of inoculant:

[0148] (1) Pantoea Pantoea endophytica ) YNK-FB0056 liquid bacterial agent

[0149] The strain YNK-FB0056 obtained in Example 1 was inoculated on NA solid medium and cultured at 30°C for 24 h. After that, a single colony was picked and inoculated into NB liquid medium and cultured at 30°C with shaking at 180 rpm for 72 h. The obtained culture was diluted to a viable count of about 1×10 8 CFU / mL, and the dilution liquid is the liquid inoculum of strain YNK-FB0056.

[0150] (2) Mixed liquid microbial agent

[0151] The strain YNK-FB0056 obtained in Example 1 was inoculated on NA solid medium, cultured at 30°C for 24 hours, and then a single colony was picked and inoculated into NB liquid medium. The culture was shaken at 180 rpm at 30°C for 72 hours to obtain YNK-FB0056 bacterial solution. The same method and conditions were used to culture the gladiolus Burkholderia YNK-FB0053 to obtain YNK-FB0053 bacterial solution. The YNK-FB0056 bacterial solution and the YNK-FB0053 bacterial solution were mixed at a volume ratio of 1:1. The resulting mixed culture was diluted to a total viable count of approximately 1×10 8 CFU / mL of this mixture is Pantoea ( Pantoea endophytica ) A mixed liquid inoculum of YNK-FB0056 and Burkholderia gladiolus YNK-FB0053.

[0152] Plant planting: Add the same weight of soil to each pot, randomly group 6 pots per treatment, and plant one tomato seedling of similar growth in each pot.

[0153] Experimental group settings: CK1: sterile water; CK2: sterile NB liquid culture medium; T1: concentration of approximately 1×10 8 CFU / mL of mixed liquid inoculum; T2: concentration is about 1×10 9 CFU / mL of mixed liquid inoculum; T3: concentration is about 1×10 10 CFU / mL of mixed liquid bacterial agent.

[0154] Verification of the growth-promoting effect: On the 8th day after the tomato seedlings were transplanted into the pots, the roots of the tomato seedlings were irrigated with 200 mL / plant of the above-mentioned mixed liquid fungus agent. At the same time, the same treatment was performed with an equal amount of sterile water as the control group 1 (CK1), and the same treatment was performed with an equal amount of NB culture medium as the control group 2 (CK2).

[0155] After root irrigation, the tomato seedlings were placed in a greenhouse and allowed to grow naturally. Twenty-eight days after root irrigation, the tomato plants were measured for their aboveground fresh and dry weights, underground (root) fresh and dry weights, stem diameter, root length, and plant height.

[0156] The specific measurement method is as follows:

[0157] Aboveground fresh and dry weight: Cut the tomato plant above the root base and weigh the aboveground portion on an analytical balance to obtain the fresh weight, rounding to one decimal place. Place the weighed aboveground portion in a paper bag and place in an oven at 100±5°C for 10 minutes. Then, lower the oven temperature to approximately 75±5°C and dry to a constant weight. Weigh the aboveground dry weight on an analytical balance to obtain the dry weight, rounding to one decimal place.

[0158] Fresh and dry weight of underground: Cut the tomato plant below the root base and weigh the underground portion fresh weight on an analytical balance to one decimal place. Place the weighed underground portion in a paper bag and place in an oven at 100±5°C for 10 minutes. Then, lower the oven temperature to approximately 75±5°C and dry to a constant weight. Weigh the underground portion dry weight on an analytical balance to one decimal place.

[0159] Stem Diameter: Use a vernier caliper to measure the diameter of the thickest stem part of the plant.

[0160] Root length: Straighten the roots and measure their length using a ruler.

[0161] Plant height: Straighten the plant and use a ruler to measure the length of the above-ground part from the highest point of the leaves.

[0162] Chlorophyll measurement: A value obtained by measuring the same part of each tomato plant using a SPAD instrument. This value indicates the relative chlorophyll content in the leaves. A higher SPAD value indicates a higher chlorophyll content in the leaves, and a greener leaf.

[0163] Figure 8 (A) and (B) show the growth of tomato plants 28 days after irrigation with YNK-FB0056 liquid inoculant and mixed liquid inoculant at different concentrations.

[0164] Figure 9 (A) and (B) show that 28 days after root irrigation with YNK-FB0056 liquid inoculant and mixed liquid inoculant, tomato plant height, stem diameter, aboveground fresh weight, root length, belowground fresh weight, and chlorophyll content were significantly increased compared to the control group (CK1) treated with pure water and the control group (CK2) treated with NB medium. This indicates that the application of strain YNK-FB0056 and mixed inoculants significantly promoted the growth of tomato seedlings.

[0165] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

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

2. A bacterial agent, the active ingredient of which is the Pantoea according to claim 1.

3. The Pantoea ( Pantoea endophytica ) Use of YNK-FB0056 or the bacterial agent described in 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 comprises the steps of: Pantoea endophytica ) YNK-FB0056 was fermented and the culture products were collected.

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

6. A composite bacterial agent, characterized in that: Including Pantoea with an effective viable bacterial count ratio of 1:1 ( Pantoea endophytica ) YNK-FB0056 and Burkholderia gladiolus ( Burkholderia gladioli ); wherein the preservation number of the gladiolus Burkholderia is CCTCC NO: M 20242288, Pantoea ( Pantoea endophytica ) The deposit number of YNK-FB0056 is CCTCC NO: M 2025644.

7. The Pantoea ( Pantoea endophytica ) Use of YNK-FB0056, the bacterial agent described in claim 2, or the composite bacterial agent described in claim 6 in promoting plant growth.

8. A method for promoting plant growth, characterized in that: The method comprises the steps of: Pantoea endophytica ) YNK-FB0056, the bacterial agent according to claim 2 or the composite bacterial agent according to claim 6 is applied to the rhizosphere of plants.

9. The method according to claim 8, wherein: The Pantoea ( Pantoea endophytica ) The use of YNK-FB0056, bacterial agents or compound bacterial agents makes Pantoea ( Pantoea endophytica )The dosage of YNK-FB0056 is not less than 1×10 8 CFU / plant / time; the application frequency is 1-3 times per crop; the plant is selected from the Solanaceae family.

10. The method according to claim 9, characterized in that: The Solanaceae plant is tomato.

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