Caviriavidus for producing ACC deaminase as well as application and use method of Caviriavidus

Abstract

The invention relates to the field of microorganisms, in particular to variovoraxparadoxus JKM1 capable of producing ACC (1-aminocyclopropanecarboxylic acid) deaminase as well as an application and a use method of the variovoraxparadoxus JKM1. The strain is preserved in the China General Microbiological Culture Collection Center on October 15, 2024, and the preservation number is CGMCC NO: 32218. According to the present invention, the variovorax paradoxus JKM1 capable of producing the ACC deaminase can be used for relieving the soil compaction stress so as to promote the plant growth; according to the application and the use method of the Variovoxparadoxus JKM1 capable of producing ACC deaminase, the Variovoxparadoxus JKM1 can degrade ethylene to synthesize a precursor 1-aminocyclopropane-1-carboxylic acid (ACC), so that the content of ethylene in a plant root zone under soil compaction stress is effectively reduced, plant growth is promoted, the content or synthesis of IAA and ABA of a root system under soil compaction stress can be increased, the shape of the root system of the plant is adjusted, and the growth of the plant is promoted. The tolerance of the plants to the soil compaction stress is enhanced.

Description

Technical Field

[0001] The invention relates to the field of agricultural microorganisms, and relates to a phage producing ACC deaminase, and a use and a use method thereof. Background Art

[0002] Soil compaction refers to the phenomenon that soil bulk density increases or porosity decreases under internal or external pressure. Soil compaction is affected by many factors, including natural factors such as soil texture and dry-wet alternation, as well as human factors such as field management and agricultural machinery operations. The compaction of soil by agricultural machinery is the main cause of soil compaction stress. The harm of soil compaction stress to crop growth and agricultural production mainly includes: 1. Destroying soil structure, reducing the number and size of soil pores, increasing soil bulk density and penetration resistance, producing excessive mechanical impedance, and inhibiting root penetration in the soil. 2. Reducing soil hydraulic conductivity and water infiltration rate, reducing soil aeration, and inhibiting the transport of soil nutrients and water. 3. Restricting crop root growth, resulting in a decrease in root length and an increase in root diameter, restricting the root system's absorption of nutrients and water, and then inhibiting the growth of the aboveground part of the crop, resulting in a serious reduction in crop yields. In addition, soil compaction stress also affects the physical and chemical conditions for the production of greenhouse gases, causing N 2 The increase in greenhouse gas emissions such as O further harms the ecosystem. With the increasing degree of mechanization in modern agriculture, soil compaction stress has become one of the most common problems of soil quality degradation, seriously restricting the sustainable development of agriculture.

[0003] In the prior art, soil compaction stress is mainly alleviated by improving the physical properties of the soil. For example, CN115039533 A is a method for improving soil compaction in an orchard, and rhamnolipids are spread in combination with watering; its specification states: The results of Examples 1 to 6 show that soil bulk density and compaction are decreasing with the increase of rhamnolipid concentration. The reason for this trend of change may be that rhamnolipid increases the water-stable aggregates in the soil, and the water-stable aggregates enhance the overall aggregation of the soil and improve the ability of the soil to permeate water and air. On the other hand, the root system tends to be loose in the soil during growth, and the gaps between the aggregates are conducive to the growth of the root system. The interlacing of the root system to form a complex network structure of gaps also promotes the decrease in soil bulk density and compaction; the decrease in soil bulk density and compaction is conducive to the improvement of root indicators, because rhamnolipids can form small vesicles in the soil, and the soil becomes loose locally. In the stress of soil compaction, the root system of plants will automatically grow towards the area with low mechanical pressure, and the root system grows faster at the site where the soil is relatively loose. It is through the tiny vesicles that the roots have loose space to grow. In this document, "spreading rhamnolipids in conjunction with watering" achieves "not only can it be applied to the soil under compaction stress to exert its effect, but also has the function of promoting plant growth." There is also the improvement of soil structure by constructing complex plant communities, for example; CN 105921508A is a method for configuring plant communities for compacted soil repair. It utilizes the growth characteristics of plant roots and the requirements of plants for the absorption of organic matter, nitrogen, phosphorus, potassium and other elements to construct a plant community structure that can be used for compacted soil repair. It not only creates a stable plant community landscape on compacted soil, but also reshapes the soil aggregate structure and improves the sustainable utilization level of compacted soil. The above patents or technologies promote plant growth by improving the physical structure of the soil to make the soil loose.

[0004] Recent cutting-edge research has found that the plant hormone ethylene is a key factor that restricts plant root growth under soil compaction stress. The reduction of air pores in compacted soil limits the outward diffusion of ethylene produced by the roots, resulting in excessive accumulation of ethylene in the area near the roots. Excessive ethylene content triggers the hormone response of the roots, specifically inhibiting the growth of roots in compacted soil. However, the above-mentioned public technologies all ignore the importance of the soil-root interaction process in which ethylene in the root zone inhibits root growth. They only improve the physical properties of the soil, cannot specifically reduce the production of ethylene in the roots under soil compaction stress, and cannot reduce the inhibitory effect of ethylene on root growth, limiting the further increase in the growth and yield of crops under soil compaction stress.

[0005] Even if the physical structure of the soil is improved, the high ethylene content in the soil will still inhibit the growth of plant roots, affect the plant's absorption of water and nutrients, and thus affect plant growth.

[0006] Therefore, there is an urgent need for a solution to reduce the ethylene content in compacted soil and thus promote plant growth. Summary of the invention

[0007] The purpose of the present invention is to provide a phage producing ACC deaminase and its uses and methods of use, which can reduce the ethylene content in the root zone of plants in compacted soil and promote plant growth in compacted soil; increase the content or synthesis of IAA and ABA in the roots under soil compaction stress, regulate the root morphology of plants under soil compaction stress, and enhance the tolerance of plants to soil compaction stress.

[0008] In order to solve the above technical problems, the ACC deaminase-producing phage, use and method of use provided by the present invention are achieved as follows:

[0009] A Variovorax paradoxus JKM1 that produces ACC deaminase was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 15, 2024, with the deposit number CGMCC NO: 32218.

[0010] Application of Variovorax paradoxus JKM1, an ACC deaminase-producing bacterium, in promoting plant growth in compacted soils.

[0011] Optionally, the ACC deaminase-producing Variovorax paradoxus JKM1 promotes plant growth by reducing ethylene production in plant roots under soil compaction stress.

[0012] Optionally, the reducing of ethylene in plant roots is through degrading ACC, a precursor of ethylene synthesis, thereby inhibiting the production of ethylene in plant roots in compacted soil.

[0013] Optionally, the ACC deaminase-producing Variovorax paradoxus JKM1 promotes plant growth by increasing the IAA and ABA content or synthesis in plant roots.

[0014] A method for using Variovorax paradoxus JKM1 producing ACC deaminase to promote plant growth in compacted soil comprises the following steps: inoculating the Variovorax paradoxus JKM1 producing ACC deaminase during the plant seedling stage, diluting the activated cultured bacteria in LB liquid culture medium to prepare an inoculation solution with an OD600 value of 0.5±0.02, and inoculating by root irrigation.

[0015] Optionally, the method for using the ACC deaminase-producing Variovorax paradoxus JKM1 to promote plant growth in compacted soil comprises the following steps: using a shovel to dig a hole next to the plant that is deep to the root of the plant, pouring 50 mL of bacterial solution into the hole, and covering the hole with soil to fill it up to complete the inoculation.

[0016] A method for using Variovorax paradoxus JKM1 producing ACC deaminase to promote plant growth in compacted soil, comprising germinating plant seeds, and soaking the seeds in a bacterial strain with an OD600 value of 0.5±0.02 before sowing after the seeds grow buds of about 1 cm. The bacterial strain OD600 value of 0.5±0.02 is obtained by diluting the bacterial strain under sterile water.

[0017] Optionally, the ACC deaminase-producing Variovorax paradoxus JKM1 is isolated from field rhizosphere soil under soil compaction stress.

[0018] A soil compacting conditioner comprising the Variovoraxparadoxus JKM1 producing ACC deaminase.

[0019] The strain disclosed in the present invention is isolated from a field test of soil compaction stress, has clear functions, and has strong colonization ability in the soil. The ACC deaminase-producing bacteria can reduce the production of ethylene in plants by degrading ACC, a precursor of ethylene synthesis, thereby reducing the ethylene content in the root zone and promoting the growth of plants in compacted soil; and affects the content and synthesis of plant root hormones. The Variovorax paradoxus JKM1 of the present invention can play a good role in promoting the growth of plants under abiotic stresses such as soil compaction stress.

[0020] The present invention separates the ACC deaminase-producing bacterium Variovorax paradoxus JKM1, and the ACC deaminase activity produced by the JKM1 of the present invention can reach 13.50±0.87μmol α-ketobutyric acid·mg -1 ·h -1 The present invention conducted field tests and soil column tests, inoculated corn under soil compaction stress, and measured corn biomass, root morphology indexes and phosphorus nutrition indexes, and found that under soil compaction stress, the Variovorax paradoxus JKM1 producing ACC deaminase of the present invention had a significant promoting effect on corn growth and promoted the absorption of phosphorus by plants.

[0021] The present invention passed field tests: under soil compaction stress, after inoculation with Variovorax paradoxus JKM1, the aboveground dry weight increased by 38.5%, the root length, root surface area and root volume in the 0-30 cm soil layer increased by 123.2%, 90.2% and 62.4% respectively, the specific root length increased by 36.1%, and the average root diameter decreased by 13.8%.

[0022] The present invention determines the ethylene content in the root zone, the root hormone content and the expression of its synthesis gene, and clarifies the mechanism by which ACC deaminase-producing bacteria affect the synthesis of root hormones and regulate root morphology. The final test results reveal the soil-root-microorganism interaction process in soil compaction stress, and explore the potential ability of microorganisms in helping plants alleviate soil compaction stress. The present invention sets up a greenhouse soil column test: under soil compaction stress, after inoculation with Variovorax paradoxus JKM1, the ethylene content in the root zone decreased by 44.1%, the IAA content in the root system increased by 40.0%, the ABA content in the root system increased by 60.1%, the relative expression of IAA-related synthesis genes (ZmYUC4 genes) increased by 151.9%, and the relative expression of ABA-related synthesis genes (NCED1 genes) increased by 144.1%.

[0023] The present invention sets up a greenhouse soil column test: under soil compaction stress, after inoculation with Variovorax paradoxus JKM1, the aboveground dry weight and root dry weight are increased by 42.6% and 41.7% respectively, the total root length, total root surface area and total volume are increased by 62.4%, 63.7% and 71.7%, the average root diameter is reduced by 5.1%, and the aboveground phosphorus content is also significantly increased.

[0024] The present invention sets a greenhouse soil column test: 30 days after inoculation of Variovorax paradoxus JKM1 under soil compaction stress, a comparison diagram of corn growth is shown; as shown in the figure, the growth of corn in compacted soil is the worst, the growth of corn in compacted soil but inoculated with JKM1 is second, the growth of corn in non-compacted soil is the best, but the growth of compacted soil inoculated with JKM1 is also significantly better than that of compacted soil not inoculated with JKM1.

[0025] The present invention also provides a soil compacting conditioner, including Variovorax paradoxus JKM1 producing ACC deaminase; although the present invention verifies that the use of the bacterium alone can also promote plant growth in compacted soil; however, the present invention is not limited to adding other preparations to make the bacterial agent of the present invention further promote plant growth, or make the bacterial agent suitable for storage at room temperature, which is conducive to promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The present invention is to discuss the strain morphology of Variovorax paradoxus JKM1 on LB solid medium;

[0027] Figure 2 This is a comparison chart of plant growth indicators after corn was inoculated with Variovorax paradoxus JKM1 under soil compaction stress in a field experiment of the present invention;

[0028] Figure 3 It is a comparative diagram of soil ethylene content in the root zone of the plant, IAA and ABA in the root system, and determination of genes related to their synthesis after corn was inoculated with Variovorax paradoxus JKM1 under soil compaction stress in the greenhouse soil column test of the present invention;

[0029] Figure 4 This is a comparison chart of plant growth indicators after corn was inoculated with Variovoraxparadoxus JKM1 under soil compaction stress in a greenhouse soil column experiment;

[0030] Figure 5 This is a comparison of corn growth after inoculation with Variovorax paradoxus JKM1 under soil compaction stress in a greenhouse soil column experiment. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail in the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] Description of reagents, consumables and instruments used in the embodiments of the present invention:

[0033] The reagents used for strain culture or functional identification in the experiment, including α-ketobutyric acid, tryptone broth (TSB), 2,4-dinitrophenylhydrazine, Coomassie Brilliant Blue G-250, bovine serum albumin (BSA), tris (hydroxymethylaminomethane) (Tris), phosphate buffered saline (PBS), and LB broth (LB liquid culture medium), were purchased from Aladdin Biochemical Technology Co., Ltd.

[0034] DF medium (without nitrogen source) and 0.5 MACC stock solution were purchased from Beijing Coolbo Technology Co., Ltd.

[0035] LB nutrient agar (LB solid medium) was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0036] Toluene, HCl, NaOH, glycerol, CaSO 4 , H 2 O2 , H 2 SO 4 ,use Brand, Sinopharm Chemical Reagent Co., Ltd.

[0037] Consumables used in the embodiments of the present invention

[0038] The vacuum tube used for collecting ethylene was a "Xinge" brand negative pressure blood collection vacuum tube, and the Rhizon sampler (SMS type) was purchased from Wuhan Hanlinyuan Technology Co., Ltd. In the steps of culturing strains, an inoculation loop was used, and the culture dish was a JET BIOFIL brand one.

[0039] Instruments used in the present invention:

[0040] Eppendorf, pipettes and centrifuges used in the experiment

[0041] BioTek Synergy H1, BioTek Instruments, Inc.

[0042] Japan Sanyo Company, Sanyo high pressure steam sterilizer (MLS-3781L)

[0043] Epson Corporation, root scanner (Epson Expression 1600pro)

[0044] Agilent, USA, gas chromatograph (7820A GC, Agilent)

[0045] The test methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer; for reagents without specifying the source, reagent products of the same specifications on the market can also be used, and the implementation of the present invention is not limited to the reagents with specified sources.

[0046] Example 1: Isolation of Variovoraxparadoxus JKM1 strain

[0047] The maize rhizosphere soil from the soil compaction long-term positioning experiment at the Shangzhuang Experimental Station of China Agricultural University in Beijing (116.1°E, 40.1°N) was selected as the soil material. After natural air drying, 5 g of rhizosphere soil was weighed and mixed with 45 mL of sterile water by oscillation. The supernatant was diluted to prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Concentration gradient dilution, and select 10 -3 , 10 -4 , 10 -5200 μL of the dilution was plated on LB solid medium and cultured at 30°C for 3 days. The isolated single colonies were picked up using an inoculation loop and the plate streaking method was used to purify the rhizosphere bacteria of corn. The purified strains were stored at -20°C and -80°C using glycerol preservation method for future use. The picture of Variovorax paradoxus JKM1 strain being streaked on LB solid medium is shown below. Figure 1 As shown, Figure 1 The C-7 in the left picture is the Variovoraxparadoxus JKM1 strain. Figure 1 The right picture is an enlarged view of the left picture.

[0048] Example 2: Screening and determination of ACC deaminase in strains

[0049] The ACC deaminase activity was determined as follows:

[0050] A small amount of the selected strain was inoculated into 20 mL of TSB liquid medium and incubated at 28°C and 200 r / min. -1 The culture was shaken for 12 h and then incubated at 4 °C and 8000 r / min -1 Centrifuge for 10 min and collect the bacterial precipitate. Resuspend the bacterial cells in 7.5 mL of DF medium without nitrogen source and add 45 μL of 0.5 mol·L sterilized by filtration. -1 ACC solution, so that the final concentration of ACC is 3.0mmol·L -1 Then at 28℃, 200r·min -1 The bacterial suspension was shaken and cultured for 24 h to induce the ACC deaminase activity of the bacteria. -1 Centrifuge for 10 min, remove the supernatant, collect the bacterial precipitate, and resuspend it in 5 mL of 0.1 mol·L -1 In Tris-HCl buffer (pH 7.6), at 4°C and 8000 r / min -1 After centrifugation for 10 min, the precipitate was collected and this step was repeated 3 times to completely remove the DF medium. 1 mL of 0.1 mol·L -1 Tris-HCl (pH 7.6) was used to suspend the cells, and then transferred to a 1.5 mL centrifuge tube and centrifuged at 16000 r·min -1 The cell pellet was resuspended in 600 μL 0.1 mol·L -1 Add 30 μL of toluene to Tris-HCl buffer (pH 8.5) and vortex for 30 seconds to break the cells. Pipette 200 μL of the broken cell suspension and add 20 μL of 0.5 mol·L -1ACC solution, mixed well and placed in a water bath at 30°C for 15 min. Then add 1 mL of 0.56 mol·L -1 HCl mixed, 16000r·min at room temperature -1 Centrifuge for 5 minutes. Take 1 mL of the supernatant and add 800 μL of 0.56 mol·L -1 HCl, mix well and then add 300μL 2,4-dinitrophenylhydrazine (2mol·L -1 HCl dissolved, mass concentration is 2g·L -1 ), and placed in a water bath at 30°C for 30 min. Then add 2 mL of 2 mol·L -1 NaOH was used for color development and the absorbance (OD540) was measured at 540 nm using a spectrophotometer. The treatment in which distilled water was used instead of bacterial suspension was used as a control.

[0051] Use 0.1 mol·L -1 Tris-HCl buffer (pH 8.5) was prepared at 0.2, 0.4, 0.6, 0.8 and 1.0 mmol·L -1 300 μL of 2,4-dinitrophenylhydrazine (2 mol·L -1 HCl dissolved, mass concentration is 2g·L -1 ), mix well and place in a water bath at 30°C for 30 min. Then add 2 mL of 2 mol·L -1 NaOH was used for color development, and the OD540 was measured after stabilization. Tris-HCl buffer (pH8.5) was used as a blank control. A standard curve was prepared according to the concentration of α-ketobutyric acid solution and its corresponding absorbance value, and its regression equation was calculated. The OD540 of the sample was substituted into the regression equation of the standard curve to obtain the content of α-ketobutyric acid, and the amount of substance (μmol) catalyzed by ACC deaminase to produce α-ketobutyric acid from ACC was calculated. The protein content (mg) in the remaining bacterial suspension was then determined using the Coomassie Brilliant Blue G-250 method, with bovine serum albumin as a control. The activity results were expressed as enzyme specific activity (μmol α-ketobutyric acid·mg -1 ·h -1 ) indicates that the ACC deaminase activity of the bacteria used in the conditioning agent was measured to be 13.50 ± 1.07 μmolα-ketobutyric acid·mg -1 ·h -1 , which is higher than the threshold of ACC deaminase activity (20 nmol α-ketobutyric acid mg -1 ·h -1 , Penrose and Glick, 2003)

[0052] The screened strain JKM1 was identified by bacterial 16S rDNA. After sequencing, the sequence was compared with the ribosomal RNA gene of Variovoraxparadoxus strain GU-NR516S in the NCBI database, and the partial sequence similarity reached 99%, and JKM1 was identified as Variovorax paradoxus. The Variovorax paradoxus JKM1 producing ACC deaminase of the present invention was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 15, 2024, with a deposit number of CGMCC NO: 32218.

[0053] Example 3: Field experiment to verify the promoting effect of Variovoraxparadoxus JKM1 on corn growth under soil compaction stress

[0054] 3.1 Treatment settings for field trials

[0055] The soil compaction long-term positioning test was conducted in June 2023 at the Quzhou Experimental Station of China Agricultural University in Handan, Hebei Province (115°0′E, 36°51′N). The soil type of the test site was salinized tidal soil, and the total nitrogen content of the soil was 1.09 g·kg -1 , available phosphorus (Olsen-P) is 24.0 mg kg -1 , the available potassium is 172 mg kg -1 , pH (soil-water ratio 1:5) is 8.26, and organic matter is 16.3 g·kg -1 The compaction treatment was performed by rolling the soil for six consecutive times with a Dongfanghong 1304 tractor (weight 5080 kg) in parallel wheel tracks. The nitrogen fertilizer application rate was 225 kg N ha -1 , the amount of phosphate fertilizer is 75kg P 2 O 5 ha -1 , the potassium fertilizer dosage is 80kg K 2 Oh -1 Before sowing, evenly spread 1 / 2 nitrogen fertilizer (urea, containing 46% N) and phosphate fertilizer (superphosphate, containing P 2 O 5 16%) and potash fertilizer (potassium sulfate, containing K 2O (48%), and then use a rotary tiller to rotate into the 0-10cm soil layer. The remaining 1 / 2 nitrogen fertilizer is applied as topdressing during the corn jointing stage (V6 period). The corn variety selected is MC812 (Zeamays L.cv MC812), the plant row spacing is 60cm, the plant spacing is 21cm, the strain is inoculated at the seedling stage (25 days after sowing), and the aboveground dry weight and the root morphology index of the 0-30cm soil layer are measured at the flowering stage (50 days after sowing). The LB liquid culture medium dilution of the corresponding concentration is used as the blank control, and 3 biological replicates are set for the inoculated strain treatment.

[0056] 3.2. Preparation and inoculation method of Variovorax paradoxus JKM1 inoculum

[0057] After the strain Variovorax paradoxus JKM1 was activated, an appropriate amount was selected and incubated in LB liquid medium at 28°C and 160 r·min -1 After shaking culture for 48 hours, dilute and adjust the OD600 value of the inoculum to 0.5±0.02 (blank calibration with pure LB liquid medium). Use the root irrigation method to inoculate the inoculum, that is, use a small shovel to dig a hole about 15 cm deep and 7 cm in diameter 15 cm away from the corn plant, pour 50 mL of the inoculum into the hole, and then cover the hole with soil to fill it.

[0058] 3.3 Determination of aboveground dry weight and root morphology

[0059] When corn enters the flowering period (50 days after sowing), the aboveground dry weight and root morphology indicators are obtained. Cut the corn plants at the base of the stem with scissors, put the aboveground part into a mesh bag, and put it in a 105℃ oven for half an hour, then dry it at 60-70℃ until constant weight and weigh it. The root morphology indicators are collected using the soil drill method. The soil drill is driven in 5 cm from the root base. The soil drill is a metal cylindrical drill with a diameter of 10 cm and a height of 15 cm. The soil drill is used to collect two layers of soil cores below the soil surface, that is, soil samples at a depth of 0-30 cm. After the soil taken out by the soil drill passes through a 2mm aperture sieve, the roots are picked out with tweezers and washed with deionized water. The root system was scanned by a root scanner (Epson Expression 1600pro), and the scanned images were analyzed and processed by WinRHIZO image analysis system (WinRHIZO Pro 2004b, Version 5.0, Canada) to measure root morphological parameters such as root length, root surface area, root volume, and average root diameter. After scanning, the roots were collected and dried at 70°C to constant weight, and the specific root length (cm / g) data was calculated by dividing the root length (cm) by the root dry weight (g).

[0060] The test comparison results are as follows Figure 2 As shown: Under soil compaction stress, after inoculation with Variovorax paradoxus JKM1, the aboveground dry weight increased by 38.5%, the root length, root surface area and root volume in the 0-30 cm soil layer increased by 123.2%, 90.2% and 62.4% respectively, the specific root length increased by 36.1%, and the average root diameter decreased by 13.8%.

[0061] Example 4: Conducting a greenhouse soil column experiment to verify the effect of Variovorax paradoxus JKM1 on promoting corn growth under soil compaction stress

[0062] 4.1 Soil column test setup

[0063] The test adopts the soil column culture method. The test container is an assembled PVC pipe cut along the cross-sectional diameter direction, with a diameter of 15 cm and a height of 35 cm. The bottom is sealed with a 200-mesh nylon mesh during assembly. Radiation sterilization is used for sterilization, and the radiation sterilization dose is 25,000 Gy. Non-compacted treatment: 6.9 kg of soil is filled into each pot, and the soil is placed in the container without squeezing. The bulk density after filling is 1.3 g cm -3 ; Compacting treatment: Each pot was filled with 8.5 kg of soil. After adding 5 cm of soil, a flat metal column with closed ends was placed on the soil surface and hammered with a hammer to compact the soil evenly. The soil bulk density after filling was 1.6 g cm -3 A Rhizon sampler (Rhizo SMS) was placed 10 cm away from the soil surface in the PVC soil column. The sampler contained a filter membrane with a pore size of 1.5 μm, which could be used to collect gas and solution in the pores.

[0064] The corn variety selected was Zhengdan 958 (Zea mays L.cv ZD958). When sowing, seeds of similar shape and size were selected and 10% H 2 O 2 After immersion and disinfection for 30 min, the seeds were rinsed with deionized water and then placed in a saturated CaSO 4The solution was ventilated and imbibed for 12 hours in the dark. The imbibed seeds were placed in a tray with moist filter paper and placed in a plant culture room in the dark for germination for 24 hours. During this period, deionized water was used to replenish water every 4 hours to keep the filter paper in the tray moist. After the seeds grew buds of about 1 cm, seeds with relatively close root lengths were selected. The seeds of each inoculated strain treatment were soaked in the corresponding bacterial solution (OD6000.5±0.02) for 2 hours before sowing, and the control treatment was soaked in sterile water instead of bacterial solution. Three seeds were sown per pot during sowing, and thinning was performed after emergence, retaining 2 seedlings with relatively consistent growth conditions with the rest of the repeated growth conditions. Deionized water was added from the bottom of the soil column during the experiment, and water was replenished by osmosis to keep the soil moisture content at 60-80% of the field water holding capacity during the experiment. During the growth period of the plants, the inoculated strain treatment was irrigated with bacterial solution every 7 days after sowing, and 40 mL was irrigated per pot. The plants were irrigated three times before harvest (30 days in total), and the control treatment was irrigated with sterile water instead of bacterial solution.

[0065] 15 days after corn sowing, ethylene in the root zone was collected using a 5 mL vacuum tube connected to a Rhizon sampler to collect gas for 15 minutes. Plant samples were collected 30 days after sowing to measure plant growth indicators such as aboveground dry weight, aboveground phosphorus content, and root morphology.

[0066] 4.2 Preparation and inoculation method of Variovorax paradoxus JKM1 inoculum

[0067] The strain Variovorax paradoxus JKM1 was activated and inoculated into LB liquid medium and heated at 28°C and 160 r·min -1 Shake culture for 48 h at 4 °C, 8000 r / min -1 After centrifugation, the bacterial precipitate was collected and dispersed in sterile water. The OD600 value of the suspension was measured and the OD600 value was diluted and adjusted to 0.5±0.02. The bacterial agent was used for inoculation of the greenhouse soil column test.

[0068] 4.3 Determination of ethylene content in root zone soil, root auxin IAA (indole-3-acetic acid) content and related synthesis genes, root ABA (abscisic acid) content and related synthesis genes

[0069] 4.3.1 Determination of ethylene content in root zone soil:

[0070] Fifteen days after sowing, the root zone gas was collected in a vacuum tube by a Rhizon sampler, and the ethylene content was subsequently determined by gas chromatography (7820A GC, Agilent) with an injector temperature of 100 °C, a column temperature of 70 °C, a detector temperature of 250 °C, and a nitrogen flow rate (tailing flow rate) of 5 mL min -1, Hydrogen flow rate: 40 mL min -1 , air flow rate 400mL·min -1 The sample ethylene peak area is measured, and the amount of sample ethylene substance = (ethylene peak area / 1 nmol ethylene standard peak area) * dilution factor, where the 1 nmol ethylene standard peak area is 282 and the dilution factor is 125. The final sample ethylene content is expressed as the amount of ethylene substance per unit volume.

[0071] 4.3.2 Determination of root auxin IAA (indole-3-acetic acid) content and related synthesis genes, root ABA (abscisic acid) content and related synthesis genes

[0072] IAA promotes root growth and root cell division; regulates root tropism, and the root tropism, such as hydrotropism and fertilization tropism, is also regulated by IAA; and promotes the growth of root lateral roots and root hairs.

[0073] ABA has a bidirectional regulatory effect on root growth. Low concentrations of ABA can promote root growth, especially the elongation of the taproot; enhance the stress resistance of the root system. ABA enhances its drought resistance by regulating the physiological activities of the root system, maintains the ion balance of the plant root system under salt stress; and regulates water absorption and transportation of the root system.

[0074] The determination of the relative expression levels of the IAA-related synthesis gene ZmYUC4 and the ABA-related synthesis gene NCED1 in the maize root system was entrusted to Nanjing Zhongding Biotechnology Co., Ltd. to perform real-time fluorescence quantitative PCR, using GADPH as the internal reference gene for relative quantitative detection.

[0075] The primer sequences used for real-time fluorescence quantitative PCR detection are as follows:

[0076] GADPH-F:5'-TCATGCCATCACTGCCACACAG-3'

[0077] GADPH-R:5'-CACGGAAGGACATACCAGTGTGC-3'

[0078] ZmYUC4-F:5'-ACCAACACAACAACATCT-3'

[0079] ZmYUC4-R:5'-TAGAAGAATGAGGAGGAGAA-3'

[0080] NCED1-F:5'-AGTTGTTGTCACCCAGTCCAG-3'

[0081] NCED1-R:5'-CACGCACCGATAGCCACA-3'

[0082] Subsequent adoption 2 -△△Ct The data were analyzed by relative quantitative method:

[0083] △Ct=Ct(target gene)-Ct(reference gene)

[0084] △△Ct=△Ct(test group)-△Ct(control group)

[0085] 2 -△△Ct =Relative expression of target gene in the detection group

[0086] The control group was treated with non-compacted control.

[0087] The determination of ABA and IAA content in corn roots was commissioned to Nanjing Ruiyuan Biotechnology Co., Ltd. using the double antibody sandwich method. The materials mainly included plant abscisic acid (ABA) enzyme-linked immunosorbent assay kit and plant auxin (IAA) enzyme-linked immunosorbent assay kit. The control group also used the control treatment under non-compacting treatment.

[0088] The results are as follows: root soil ethylene content control, IAA content and related gene expression control, ABA content and related gene expression control Figure 3 shown.

[0089] 4.4 Determination of aboveground dry weight, aboveground total phosphorus content and root morphology

[0090] 30 days after sowing, the plants were cut at the base of the stem with scissors and the aboveground parts were placed in envelopes for determination of aboveground dry weight and aboveground total phosphorus content. The aboveground dry weight determination method was the same as that of the field test. After the aboveground dry sample was ground into powder, it was first passed through H 2 SO 4 -H 2 O 2 The test solution was prepared by the digestion method, and then the total phosphorus content of the aboveground part was determined by the vanadium molybdenum yellow absorption spectrophotometry. After the aboveground indexes were collected, the assembled PVC pipe was disassembled, the soil column was taken out as a whole, the root system and the soil column were placed on kraft paper as a whole, the plant and the soil column were shaken lightly to peel off the loose soil as a whole, the rhizosphere soil adhering to the root surface was gently brushed off with a soft brush, the root samples that were peeled off without damage were washed with deionized water, and dried with absorbent paper. The root samples were subsequently used to determine the root morphology indexes, and the determination steps were the same as those in the field test.

[0091] 4.4 Data Analysis Methods

[0092] The experimental data were analyzed using SPSS software, and the bar graphs were drawn using GraphpadPrism 8 software. Figure 3 and Figure 4The data in the figure represent the mean value (± standard error) of three replicates. The t-test was used for significance analysis between the two groups of data, and * indicates significant difference. The differences between the three groups of data were analyzed by one-way ANOVA at a significant level of 5% using the LSD method, and lowercase letters represent significant differences between treatments.

[0093] Figure 3 The results showed that under soil compaction stress, after inoculation with Variovorax paradoxus JKM1, the ethylene content in the root zone decreased by 44.1%, the IAA content in the root system increased by 40.0%, the ABA content in the root system increased by 60.1%, the relative expression level of IAA-related synthesis genes (ZmYUC4 gene) increased by 151.9%, and the relative expression level of ABA synthesis-related synthesis genes (NCED1 gene) increased by 144.1%.

[0094] Figure 4 The results showed that under soil compaction stress, after inoculation with Variovorax paradoxus JKM1, the aboveground dry weight and root dry weight increased by 42.6% and 41.7% respectively, the total root length, total root surface area and total volume increased by 62.4%, 63.7% and 71.7%, the average root diameter decreased by 5.1%, and the aboveground phosphorus content also increased significantly.

[0095] Figure 5 This is a comparison of corn growth 30 days after inoculation of Variovorax paradoxus JKM1 under soil compaction stress in a greenhouse soil column experiment; as shown in the figure, the growth of corn in compacted soil is the worst, the growth of corn in compacted soil but inoculated with JKM1 is second, and the growth of corn in non-compacted soil is the best. However, the growth of compacted soil inoculated with JKM1 is also significantly better than that of compacted soil not inoculated with JKM1.

[0096] In Example 3 and Example 4 of the present invention, only a single factor (only one bacterium JKM1 was added) was compared with the plant growth and root conditions of non-compacted soil. The comparison results showed that although they were not as good as the non-compacted growth conditions, they were better than the compacted soil without JKM1 inoculation, indicating that JKM1 did play a role in promoting plant growth in compacted soil. On the basis of the present invention, physical measures such as deep plowing and application of organic fertilizers can be added to improve the soil structure of compacted soil and promote plant growth.

[0097] Example 4 of the present invention further analyzes the mechanism by which ACC deaminase bacteria Variovorax paradoxus JKM1 regulates root morphology by affecting root hormone synthesis, and promotes plant growth in compacted soil by reducing the ethylene content in the compacted soil in the root zone and increasing IAA and ABA and their synthesis genes.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Variovorax paradoxus JKM1 that produces ACC deaminase, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 15, 2024, with the deposit number CGMCC NO: 32218.

2. Use of the Variovorax paradoxus JKM1 producing ACC deaminase as claimed in claim 1 in promoting plant growth in compacted soil.

3. The use of Variovorax paradoxus JKM1 producing ACC deaminase according to claim 2, characterized in that: The ACC deaminase-producing bacterium Variovorax paradoxus JKM1 promotes plant growth by reducing the ethylene content in plant roots under soil compaction stress.

4. The use of Variovorax paradoxus JKM1 producing ACC deaminase according to claim 3, characterized in that: The method of reducing the ethylene content in the plant roots under soil compaction stress is to degrade ACC, a precursor of ethylene synthesis, and inhibit the production of ethylene in the plant roots in compacted soil.

5. The use of Variovorax paradoxus JKM1 producing ACC deaminase according to claim 2, characterized in that: The ACC deaminase-producing Variovorax paradoxus JKM1 promotes plant growth by increasing the IAA and ABA content or synthesis in plant roots.

6. The method for using the Variovorax paradoxus JKM1 producing ACC deaminase as claimed in claim 2 to promote plant growth in compacted soil, characterized in that: Variovorax paradoxus JKM1 producing ACC deaminase was inoculated at the plant seedling stage, and the activated cultured bacteria were diluted in LB liquid medium to prepare an inoculation solution with an OD600 value of 0.5±0.02, and inoculated using the root irrigation method.

7. The method for using the ACC deaminase-producing Variovorax paradoxus JKM1 in promoting plant growth in compacted soil according to claim 6, characterized in that: The root irrigation inoculation method is to use a small iron shovel to dig a hole next to the plant that is deep to the root of the plant, pour 50 mL of bacterial solution into the hole, cover the hole with soil to fill it up, and complete the inoculation.

8. The method for using the Variovorax paradoxus JKM1 producing ACC deaminase as claimed in claim 2 to promote plant growth in compacted soil, characterized in that: The plant seeds are germinated. After the seeds grow buds of about 1 cm, the seeds are soaked in an inoculum with an OD600 value of 0.5±0.02 before sowing; the inoculum with an OD600 value of 0.5±0.02 is obtained by diluting the strain under sterile water.

9. The Variovorax paradoxus JKM1 producing ACC deaminase as claimed in claim 1 is isolated from rhizosphere soil in the field under soil compaction stress.

10. A soil compacting agent, characterized in that: The invention relates to a Variovorax paradoxus JKM1 which produces ACC deaminase as claimed in claim 1.

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