Ralstonia sp. Regulated by quorum sensing and application of Ralstonia sp.

Abstract

The invention provides a strain of Ralstonia sp. Regulated by quorum sensing and application thereof, the strain is Ralstonia sp., is named as YTR-R242, has been preserved in China General Microbiological Culture Collection Center (CGMCC) on October 21, 2024, and has a preservation number of CGMCC32269. The strain can generate N-acyl-homoserine lactone, the structure of the strain is C4-HSL, a phosphate-solubilizing ring proves that the Ralstonia sp. Strain YTR-R242 has a phosphate-solubilizing characteristic, and when the Ralstonia sp. Strain YTR-R242 is prepared into a bacterial agent for use, the content of available phosphorus in red soil is remarkably increased, and the growth condition of corn is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Ralstonia regulated by quorum sensing and application thereof. Background Art

[0002] Quorum sensing (QS) is an important bacterial communication mechanism that can change the interactions between microorganisms, thereby triggering a series of cascade reactions and gene expressions. Studies have shown that QS plays an important role in the adaptive survival and collective activities of bacterial communities. Gram-negative bacteria usually use N-acetyl-L-homoserine lactones (AHLs) to complete intercellular communication and information transmission, and the concentration of signal molecules released depends on the population density of bacteria. As the density of microorganisms increases, AHLs in the QS system will gradually accumulate and activate the expression of related genes in bacteria. Based on this, some studies have found that the phosphate solubilization ability of soil microorganisms can be improved by strengthening QS.

[0003] Phosphorus (P) is essential for crop growth. Several key plant processes of crop growth rely heavily on the adequate absorption of phosphorus by crops. Effective use of phosphorus is crucial to achieving global food security. Red soil is widely distributed in tropical and subtropical regions in southern and central China, and the distribution area accounts for 11.8% of the country's land area. It is rich in water and heat resources and has huge production potential. It is an important grain and cash crop production area in my country. There are a large number of active iron ions and aluminum ions in red soil, which can adsorb and fix phosphorus into ineffective phosphorus. Therefore, phosphorus deficiency is a common problem in red soil areas. Soil microorganisms are key participants in the dynamic changes of soil phosphorus. Phosphorus-solubilizing microorganisms can convert ineffective phosphorus in the soil into effective phosphorus that can be absorbed by plants (such as HPO4 2- or H2PO4 -), thereby effectively increasing the content of available phosphorus in the soil. This not only reduces the use of chemical phosphorus fertilizers, but also significantly enhances the absorption and utilization efficiency of crops for phosphorus and promotes crop growth and development. In this context, the QS system, as a natural bacterial regulation mechanism, may provide a new solution for improving soil phosphorus utilization. Through in-depth understanding and optimization of the QS regulation mechanism, more efficient phosphate-solubilizing microbial strains can be selected and transformed to increase the available phosphorus content in the soil. In addition, through the regulation of the QS mechanism, the synergy between microbial communities can be promoted, further improving the dynamic circulation efficiency of phosphorus in the soil. In short, as an essential element for crop growth, its effective utilization is crucial to achieving global food security. Due to its special soil characteristics, red soil areas generally face the problem of phosphorus deficiency, and the application of phosphate-solubilizing microorganisms provides an effective way to solve this problem. By strengthening the QS system, the function of phosphate-solubilizing microorganisms can be further improved, providing new technical support for reducing the application of chemical fertilizers, improving agricultural production efficiency, and protecting the environment. This research direction not only has important theoretical significance, but also has broad application prospects. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a Ralstonia regulated by quorum sensing, which has excellent phosphate solubilization ability.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A Ralstonia pickettii strain YTR-R242, which is regulated by the AHL-QS system and has the ability to solubilize phosphate, was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 21, 2024, with the deposit number CGMCC No.32269.

[0007] The above Ralstonia was isolated from the rhizosphere soil of corn in the long-term organic fertilization experiment plot of the Red Soil Ecological Experimental Station of the Chinese Academy of Sciences. After soil suspension dilution, inoculation and separation and purification in LB medium, a Ralstonia sp. strain was obtained and named YTR-R242.

[0008] The biological properties of the strain are as follows:

[0009] (1) Morphological characteristics: Form milky white oval colonies on LB solid medium, with smooth surface, neat and convex edges, and Gram-negative staining ( Figure 1 )

[0010] (2) Culture characteristics: The optimal growth temperature range is 25-30°C, aerobic, and grown in LB medium. Medium ingredients: Trptone 10.0 g / L, Yeast Extract 5.0 g / L, NaCl 10.0 g / L.

[0011] Preferably, the 16S rRNA sequence of strain YTR-R242 is shown as SEQ ID NO.1.

[0012] Preferably, the type of AHL produced by strain YTR-R242 is C4-HSL.

[0013] The second object of the present invention is to provide the use of the above Ralstonia pickettii strain YTR-R242.

[0014] Preferably, it is used for corn cultivation.

[0015] Preferably, the bacterial agent is added to the roots of the corn plants at a concentration of 1×10 7 CFU mL -1 .

[0016] The third object of the present invention is to provide a microbial agent containing the Ralstonia pickettii strain YTR-R242.

[0017] The fourth object of the present invention is to provide a method for preparing a microbial preparation of Ralstonia, which comprises culturing YTR-R242 bacterial liquid in LB medium for 24 hours, and when OD 600 When the value reaches 1.0, the bacterial solution is centrifuged, the precipitate is washed three times with 10 mL of 0.9% saline, and finally the bacteria are suspended in sterile water to prepare a bacterial agent.

[0018] The present invention has the following beneficial effects:

[0019] The invention screens out a quorum sensing bacterium from the rhizosphere soil of corn in the long-term organic fertilization test plot of the red soil ecological experimental station of the Chinese Academy of Sciences. The quorum sensing bacterium can produce N-acyl-homoserine lactone with a structure of C4-HSL. The phosphate-solubilizing circle proves that the Ralstonia sp. strain YTR-R242 has the property of solubilizing phosphate. The strain is prepared into a bacterial agent for use, which significantly increases the effective phosphorus content of the red soil and improves the growth condition of corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0021] Figure 1The colony characteristics of strain YTR-R242 isolated by the present invention on LB solid medium;

[0022] Figure 2 Color reaction for detecting reporter bacteria in strains YTR-R242 and CV026;

[0023] Figure 3 is the growth curve of strain YTR-R242 at 28°C;

[0024] Figure 4 This is a phylogenetic tree constructed based on 16S rDNA sequences;

[0025] Figure 5 Generate characteristic result graph for AHLs of strain YTR-R242;

[0026] Figure 6 This is the phosphosphere picture of strain YTR-R242;

[0027] Figure 7 This is a diagram of root growth under the inoculation of strain YTR-R242;

[0028] Figure 8 Rhizosphere available phosphorus content and plant biomass under the control and inoculated strain YTR-R242. DETAILED DESCRIPTION

[0029] The present invention is further explained below in conjunction with embodiments. According to the following embodiments, the present invention can be better understood. However, it is easy for those skilled in the art to understand that the specific material proportions, process conditions and results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0030] Example 1: Screening of YTR-R242 strain

[0031] 1. Screening of strains

[0032] (1) Collect the maize root zone soil from each plot at a depth of 0-15 cm, shake off the loosely adhering soil, and collect the tightly adhering rhizosphere soil with a brush. Weigh 1 g of maize rhizosphere soil into a centrifuge tube, add 9 mL of 0.9% sterile saline to make 10 -1 The soil suspension was placed in an oscillating box at 180 r / min for 10 min, and then taken out and allowed to stand for 1 h. 5 μL of the soil suspension after standing was taken out and added with 45 μL of sterile water to make 10 -2 Soil suspension. Dilute the suspension into 10 -3 , 10 -4 , 10 -5 Then take 5mL 10 -5The soil suspension was added to 50 mL and 500 mL of LB medium to make 10 -6 and 10 -7 250 μL of 10 -6 and 10 -7 The gradient culture medium was added to the 96-well plate. Finally, the 96-well plate was placed in an ELISA reader and the absorbance OD was set at 28°C. 600 Determination of the growth status of YTR-R242 ( Figure 3 ). Streak the YTR-R242 in the 96-well plate on LB solid medium, pick a single colony and enrich it in LB liquid medium, pipette 200 μL of bacterial solution into a preservation tube, add 200 μL of glycerol and store it in a -80°C refrigerator. All subsequent experiments are based on the preserved bacterial solution.

[0033] (2) Chromobacterium violaceum CV026 does not produce N-acyl-homoserine lactone (AHLs) signal molecules by itself. Only when exogenous AHLs signal molecules appear can AHLs activate the quorum sensing regulatory system of Chromobacterium violaceum and induce the expression of violacein genes. Only then can the strain produce violacein and detect the signal molecules C4-HSL to C8-HSL in the environment. Based on this characteristic, we activated and cultured the test bacteria CV026 and YTR-R242 in LB medium overnight, and then streaked the test bacteria CV026 and YTR-R242 in a "T" shape on the LB solid plate, keeping them as close as possible but not touching. The two were cultured at a constant temperature of 30°C for 24 hours to observe whether the test bacteria CV026 showed purple and the degree of color development. At the same time, a control group with only CV026 and YTR-R242 was set up. Judging from this color reaction, YTR-R242 can produce AHLs ( Figure 2 ).

[0034] Example 2: Identification of strains

[0035] 1. Morphological characteristics identification

[0036] 2 μL of the bacterial solution stored at -80℃ was taken and placed in fresh LB medium for overnight activation culture. A small amount of YTR-R242 overnight culture solution was dipped into a spreader stick and streaked back and forth on the LB agar plate medium in a zigzag pattern. The plate was sealed with a breathable membrane and placed in a 30℃ incubator. Photos were taken after 24 hours of culture. Figure 1 As shown, the YTR-R242 colony was milky white and oval in shape, with a smooth surface and neat, convex edges.

[0037] 2. Molecular Biology Identification

[0038] The bacterial 16S rRNA universal primer 27F / 1492R was used to perform PCR amplification on YTR-R242 stored at -80°C and activated overnight. PCR amplification conditions: 94°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, denaturation, annealing and extension for 35 cycles, and 72°C extension for 10 min before terminating the reaction. The effective sequences obtained by sequencing were compared on NCBI. Figure 4 As shown, the strain obtained by the invention has the highest homology with Ralstonia sp. The measured 16SrDNA sequence is shown in SEQ ID NO.1 below:

[0039] SEQ ID NO.1:

[0040]

[0041] Example 3: AHLs production characteristics of YTR-R242

[0042] Pick a single colony of YTR-R242 in LB medium, culture overnight at 30°C, and centrifuge to separate. Take 20 mL of the supernatant and add an equal volume of ethyl acetate (containing 0.5% acetic acid) in a 50 mL centrifuge tube. After oscillating and extracting for 2 hours, transfer 15 mL of the upper layer of ethyl acetate to 10 g of anhydrous Na2SO4, vortex mix for 2 minutes, and swirl at 4000 r min. -1 Centrifuge for 20 minutes, remove 10 mL of the upper organic phase, concentrate to the minimum volume in a rotary evaporator, dry and blow dry with a nitrogen evaporator, add 1 mL of methanol to dissolve, filter with a 0.22 μm polytetrafluoroethylene membrane into a chromatographic injection bottle to obtain the YTR-R242 concentrated extract. Analyze the extract with high performance liquid chromatography / tandem mass spectrometry (LC-MS / MS), compare the peak time with the C4-HSL, C6-HSL, and C8-HSL standard solutions, and determine the type of AHLs produced by YTR-R242. Among them:

[0043] LC-MS model: Shimadzu AB 5500 LC-MS / MS liquid chromatography-triple quadrupole mass spectrometer

[0044] Chromatographic column: Waters, BEH C18, 100mm×2.1mm, 1.7μm

[0045] Mobile phase B: acetonitrile + A: 0.1% formic acid aqueous solution

[0046] Flow rate: 0.28mLmin -1

[0047] Injection volume: 5 μL for standard sample, 5 μL for sample

[0048] Autosampler temperature: 15°C

[0049] The results are as follows Figure 5 As shown, the peak times of C4-HSL, C6-HSL, and C8-HSL standard solutions were 1.32 min, 2.81 min, and 4.38 min, respectively, and the peak time of the YTR-R242 extract was 1.32 min. Therefore, it was judged that the type of AHL produced by YTR-R242 was C4-HSL.

[0050] Example 4: Phosphorus dissolving properties of YTR-R242

[0051] The phosphate solubilization function was identified using an inorganic phosphorus bacterial culture medium plate, which was divided into four areas. 2 μL of activated YTR-R242 was dropped in the center of each area and cultured in a 28°C incubator for 3 days to observe whether the strain grew and whether a phosphate solubilization zone was formed.

[0052] The culture medium formula is as follows:

[0053] Inorganic phosphorus bacterial culture medium (1L): glucose 10.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4 0.03 g, MnSO4·H2O 0.03 g, KCl 0.3 g, CaCO3 1.0 g, Ca3(PO4) 25.0 g, agar 20 g, pH 7.0.

[0054] The results of the dephosphorization cycle are shown in Figure 6 The results showed that YTR-R242 could grow colonies normally on the organophosphate bacteria culture medium plate and form obvious circles, indicating that YTR-R242 has the ability to solubilize phosphate.

[0055] Table 1. Phosphorus dissolution characteristics of YTR-R242

[0056]

[0057] Example 5

[0058] Corn pot experiment

[0059] (1) This experiment has two treatments: adding YTR-R242 (10 mL of bacterial agent, concentration of 1×10 7 CFU mL -1 ) and a control treatment without application of bacterial agent (10 mL sterile water), and each treatment was repeated 3 times.

[0060] (2) Soil and fertilizer dosage: According to the organic fertilizer content (mass content) in the soil, organic fertilizer was mixed with red soil (Quaternary red clay) from Yingtan, Jiangxi Province. The initial physical and chemical properties of the soil were: pH 4.73, organic carbon 2.52 g kg -1 , total nitrogen 0.40g kg -1 , total phosphorus 0.23g kg -1 , available phosphorus 0.76 mg kg -1 , cation exchange capacity 12.12 cmol kg -1 ) are thoroughly mixed, sterilized, and placed in plastic pots with an upper diameter of 15.5 cm, a lower diameter of 12 cm, and a height of 15 cm, with 1000 g of soil matrix per pot.

[0061] (3) Seedling cultivation: The corn seeds used were Suyu No. 29. 1) Seed disinfection: First, disinfect with 70% alcohol for 1 min, then disinfect with 1% sodium hypochlorite for 15 min, and finally rinse with sterile water five times. Place the disinfected seeds on sterile water and place them in a 28°C incubator in the dark to wait for germination; 2) On the fifth day of seed germination, select 3 seedlings with the same growth and transplant them into 3 plastic pots filled with soil.

[0062] (4) Preparation and addition of bacterial solution: 100 mL of YTR-R242 bacterial solution was cultured in shake flasks in LB medium. After 24 h of culture, the OD values ​​of the three bottles of fermentation solution were measured. 600 Value, when OD 600 The value reaches 1.0 (the bacterial concentration is 1×10 9 CFU mL -1 ) was centrifuged, the precipitate was washed three times with 10 ml of 0.9% saline, and finally the bacteria were suspended in 10 mL of sterile water to prepare a bacterial agent (the concentration of the bacterial agent was 1×10 7 CFU mL -1 ).

[0063] On the third day after the corn seedlings were transplanted, 10 mL of the prepared inoculant was added to each pot at a concentration of 1 × 10 7 CFU mL -1 ), use a pipette to add the bacterial agent to the roots of corn plants (within 2 cm of the roots), and use an equal amount of sterile water instead of the bacterial solution as a control. Each treatment was repeated 3 times.

[0064] Repeat the above operation on the 15th day after transplanting the corn seedlings and add YTR-R242 inoculant again.

[0065] (5) After 30 days of potted experiment, corn plants were harvested and their roots were scanned. The alkaline phosphatase in the rhizosphere soil and the biomass of corn plants were measured for each treatment. Figure 7 and 8 As shown in Figure 2, inoculation with the fungus YTR-R242 changed the root structure of corn, making the main root and lateral roots of corn more developed; the available phosphorus in the control and YTR-R242 inoculation were 55.28 mg kg -1 ,67.72mg kg -1 It increased by 22.5% compared with the control; the corn biomass of the control and YTR-R242 inoculation were 5.13g and 11.44g, respectively, which increased by 1.23 times compared with the control.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Ralstonia characterized by: The strain is Ralstonia, named YTR-R242, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024, with the deposit number CGMCC No.32269.

2. The Ralstonia according to claim 1, characterized in that: The 16S rRNA sequence of strain YTR-R242 is shown in SEQ ID NO.

1.

3. The Ralstonia according to claim 1, characterized in that: The type of AHL produced by strain YTR-R242 is C4-HSL.

4. The use of Ralstonia according to claim 1, characterized in that: Used for corn cultivation.

5. The use of Ralstonia according to claim 4, characterized in that: The inoculant was added to the roots of the corn plants at a concentration of 1×10 7 CFU mL -1 .

6. A microbial preparation containing the Ralstonia according to claim 1.

7. A method for preparing a microbial preparation containing the Ralstonia according to claim 1, characterized in that: YTR-R242 bacterial suspension was cultured in LB medium in a shake flask for 24 h. 600 When the value reaches 1.0, the bacterial solution is centrifuged, the precipitate is washed three times with 10 mL of 0.9% saline, and finally the bacteria are suspended in sterile water to prepare a bacterial agent.

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