A bacteriophage of contended voraciousness producing acc deaminase and methods of use and methods of use thereof

Abstract

This invention relates to the field of microbiology, specifically to a *Variovorax paradoxus* JKM1 strain that produces ACC deaminase, along with its uses and methods of application. This strain was deposited on October 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC NO: 32218. The *Variovorax paradoxus* JKM1 strain of this invention, which produces ACC deaminase, can be used to alleviate soil compaction stress and promote plant growth. The uses and methods of application of the *Variovorax paradoxus* JKM1 strain of this invention include: degrading the ethylene synthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC), thereby effectively reducing the ethylene content in the root zone of plants under soil compaction stress and promoting plant growth; increasing the content or synthesis of IAA and ABA in the roots under soil compaction stress; regulating root morphology; and enhancing the plant's tolerance to soil compaction stress.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to a controversial gluttonous bacterium that produces ACC deaminase, its uses, and methods of application. Background Technology

[0002] Soil compaction refers to the phenomenon where soil bulk density increases or porosity decreases under internal or external pressure. Soil compaction is influenced by various factors, including natural factors such as soil texture and wetting / drying cycles, as well as human factors such as field management and agricultural machinery operations. The compaction effect of agricultural machinery is the main cause of soil compaction stress. The harms of soil compaction stress to crop growth and agricultural production mainly include: 1. Damaging soil structure, reducing the number and size of soil pores, increasing soil bulk density and penetration resistance, generating excessively high mechanical resistance, and inhibiting root penetration into the soil. 2. Reducing soil hydraulic conductivity and water infiltration rate, decreasing soil aeration, and inhibiting the transport of soil nutrients and water. 3. Restricting crop root growth, resulting in reduced root length and increased root diameter, restricting root absorption of nutrients and water, and thus inhibiting aboveground growth of crops, leading to severe yield reduction. Furthermore, soil compaction stress also affects the physical and chemical conditions for greenhouse gas production, causing an increase in greenhouse gas emissions such as N2O, further harming the ecosystem. With the increasing 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 existing technologies, soil compaction stress is mainly alleviated by improving soil physical properties. For example, CN115039533 A describes a method for improving compacted soil in orchards, which involves applying rhamnolipin in conjunction with irrigation. The specification states that the results of Examples 1-6 show that soil bulk density and compaction decrease with increasing rhamnolipin concentration. This trend may be due to the increased water-stable aggregates in the soil caused by rhamnolipin, which enhances the overall soil cohesion and improves water and aeration. On the other hand, roots tend to grow towards looser soil areas. The gaps between aggregates are beneficial for root growth, and the complex network of voids formed by root penetration also contributes to a decrease in soil bulk density and compaction. Reduced soil bulk density and compaction improve root performance because rhamnolipin can form small vesicles in the soil, making localized areas looser. Under soil compaction stress, plant roots automatically grow towards areas of less mechanical pressure, resulting in faster root growth at looser soil sites. It is through these tiny vesicles that the root system gains a loose space for growth. The literature describes "combining watering with rhamnolipin application," which "not only works effectively under soil compaction stress but also promotes plant growth." There are also methods to improve soil structure by constructing complex plant communities. For example, CN 105921508A, "A Method for Plant Community Configuration for Compacted Soil Remediation," utilizes the characteristics of plant root growth and the plant's requirements for the absorption of organic matter, nitrogen, phosphorus, and potassium to construct a plant community structure suitable for compacted soil remediation. This not only creates a stable plant community landscape on compacted soil but also reshapes the soil aggregate structure, improving the sustainable utilization level of compacted soil. The aforementioned patents or technologies improve the physical structure of the soil, making it looser and thus promoting plant growth.

[0004] Recent cutting-edge research has found that the plant hormone ethylene is a key factor restricting plant root growth under soil compaction stress. Compacted soil has reduced aeration porosity, limiting the outward diffusion of ethylene produced by roots, leading to excessive accumulation of ethylene in the vicinity of the roots. Excessive ethylene content triggers a hormonal response in the roots, specifically inhibiting root growth in compacted soil. However, the aforementioned publicly available techniques all overlook the importance of the soil-root interaction process in which ethylene in the rhizosphere inhibits root growth. Improving only soil physical properties cannot specifically reduce root ethylene production under soil compaction stress, nor can it reduce the inhibitory effect of ethylene on root growth, thus limiting further increases in crop growth and yield 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 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, thereby promoting plant growth. Summary of the Invention

[0007] The purpose of this invention is to provide a toxic bacterium that produces ACC deaminase, along with its uses and methods of application. This bacterium can reduce the ethylene content in the root zone of plants in compacted soil, promote plant growth in compacted soil, increase the content or synthesis of IAA and ABA in roots under soil compaction stress, regulate the root morphology of plants under soil compaction stress, and enhance the plant's tolerance to soil compaction stress.

[0008] To solve the above-mentioned technical problems, the present invention provides a glutathione that produces ACC deaminase, along with its uses and methods of application, as follows:

[0009] A controversial glutathione (Variovorax paradoxus) JKM1, which produces ACC deaminase, was deposited on October 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 32218.

[0010] The application of Variovora paradoxus JKM1, a controversial ACC-producing deaminase bacterium, in promoting plant growth in compacted soil.

[0011] Optionally, the ACC-producing glutathione (Variovoraxparadoxus) JKM1 promotes plant growth by reducing ethylene production in plant roots under soil compaction stress.

[0012] Optionally, the reduction of ethylene in plant roots is achieved by degrading the ethylene synthesis precursor ACC, thereby inhibiting the generation of ethylene in plant roots in compacted soil.

[0013] Optionally, the ACC-producing glutathione (Variovoraxparadoxus) JKM1 promotes plant growth by increasing the content or synthesis of IAA and ABA in plant roots.

[0014] A method for using Variovora paradoxus JKM1, an ACC-producing deaminase-producing bacterium, to promote plant growth in compacted soil involves inoculating seedlings with Variovora paradoxus JKM1. The activated culture is diluted in LB liquid medium to prepare an inoculum with an OD600 value of 0.5 ± 0.02, and then inoculated using the root drenching method.

[0015] Optionally, the method of using the ACC-producing deaminase-producing *Variovorax paradoxus* JKM1 to promote plant growth in compacted soil involves digging a hole deep enough to reach the roots of the plant next to it with a shovel, pouring 50 mL of bacterial solution into the hole, and then filling the hole with soil to complete the inoculation.

[0016] A controversial method for using Variovora paradoxus JKM1, a bacterium that produces ACC deaminase, to promote plant growth in compacted soil involves germinating plant seeds. After the seeds have sprouted to about 1 cm, they are soaked in a solution with an OD600 value of 0.5 ± 0.02 before sowing. The OD600 value of 0.5 ± 0.02 is obtained by diluting the bacterium in sterile water.

[0017] Optionally, the ACC-producing glutathione (Variovoraxparadoxus) JKM1 was isolated from field rhizosphere soil under soil compaction stress.

[0018] A soil compacting agent comprising the ACC-producing glutathione (Variovoraxparadoxus) JKM1.

[0019] The strain disclosed in this invention was isolated from field trials under soil compaction stress. It has a clear function and strong colonization ability in soil. The ACC-producing deaminase bacteria can reduce the production of ethylene by degrading the ethylene precursor ACC, thereby reducing the ethylene content in the root zone and promoting plant growth in compacted soil. It also affects the content and synthesis of plant root hormones. The voracious bacterium (Variovorax paradoxus) JKM1 in this invention can play a good role in promoting plant growth under abiotic stresses such as soil compaction stress.

[0020] This invention isolates the ACC deaminase-producing bacterium *Variovorax paradoxus* JKM1. The ACC deaminase activity produced by JKM1 can reach 13.50 ± 0.87 μmol α-butanone·mg. -1 ·h -1 This invention conducted field trials and soil column experiments. Under soil compaction stress, maize was inoculated. By measuring maize biomass, root morphology indicators, and phosphorus nutrition indicators, it was found that the ACC-producing glutathione (Variovoraxparadoxus) JKM1 of this invention had a significant promoting effect on maize growth and promoted the absorption of phosphorus by the plant under soil compaction stress.

[0021] This invention was demonstrated through field trials: Under soil compaction stress, after inoculation with Variovora paradoxus JKM1, the aboveground dry weight increased by 38.5%, the root length, root surface area, and root volume in the 0-30cm soil layer increased by 123.2%, 90.2%, and 62.4%, respectively, the root length increased by 36.1%, and the average root diameter decreased by 13.8%.

[0022] This invention measures ethylene content, root hormone content, and the expression of their synthetic genes in the rhizosphere, clarifying the mechanism by which ACC-producing deaminase bacteria affect root hormone synthesis and regulate root morphology. The final experimental results reveal the soil-root-microbe interaction process under soil compaction stress, exploring the potential ability of microorganisms to help plants alleviate soil compaction stress. This invention sets up a greenhouse soil column experiment: under soil compaction stress, after inoculation with *Variovorax paradoxus* JKM1, the rhizosphere ethylene content decreased by 44.1%, the root IAA content increased by 40.0%, the root ABA content increased by 60.1%, the relative expression of the IAA-related synthetic gene (ZmYUC4 gene) was upregulated by 151.9%, and the relative expression of the ABA-related synthetic gene (NCED1 gene) was upregulated by 144.1%.

[0023] This invention sets up a greenhouse soil column test: Under soil compaction stress, after inoculation with Variovora 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%, respectively, the average root diameter decreased by 5.1%, and the phosphorus content of the aboveground parts also increased significantly.

[0024] This invention sets up a greenhouse soil column experiment: 30 days after inoculating the controversial glutathione (Variovorax paradoxus) JKM1 under soil compaction stress, the growth of maize is compared. As shown in the figure, the maize with compacted soil has the worst growth, the maize with compacted soil but inoculated with JKM1 has the second best growth, and the maize with non-compacted soil has the best growth. However, the growth of compacted soil inoculated with JKM1 is also significantly better than that of compacted soil without JKM1.

[0025] This invention also provides a soil conditioner for compacting soil, comprising the controversial ACC deaminase-producing variovoraxparadoxus JKM1. Although this invention has verified that the bacterium can promote plant growth in compacted soil when used alone, this invention does not limit the addition of other preparations to make the inoculum of this invention further promote plant growth, or to make the inoculum suitable for storage at room temperature, which is conducive to its widespread use. Attached Figure Description

[0026] Figure 1This invention discusses the morphology of the glutathione (Variovorax paradoxus) JKM1 strain on LB solid medium;

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

[0028] Figure 3 This is a comparative diagram showing the determination of ethylene content in the root zone soil, root IAA and ABA, and their synthesis-related genes in maize plants after inoculation with the controversial glutathione (Variovoraxparadoxus) JKM1 under soil compaction stress in a greenhouse soil column experiment of this invention.

[0029] Figure 4 This is a comparison chart of plant growth indicators after maize was inoculated with the controversial gluttonous bacterium Variovora x paradoxus JKM1 under soil compaction stress in a greenhouse soil column experiment.

[0030] Figure 5 This is a comparison chart of maize growth after inoculation with the controversial gluttonous bacterium Variovorax paradoxus JKM1 under soil compaction stress in a greenhouse soil column experiment. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

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

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

[0034] DF medium (nitrogen-free) and 0.5 MACC stock solution were purchased from Beijing Coolplay Technology Co., Ltd.

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

[0036] Toluene, HCl, NaOH, glycerol, CaSO4, H2O2, H2SO4, used Brand: Sinopharm Chemical Reagent Co., Ltd.

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

[0038] Ethylene was collected using "Xinge" brand negative pressure blood collection vacuum tubes, and the Rhizon sampler (SMS type) was purchased from Wuhan Hanlinyuan Technology Co., Ltd. Inoculation loops and culture dishes were used for steps such as bacterial strain cultivation.

[0039] Instruments used in this invention:

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

[0041] BioTek Synergy H1 ELISA reader, manufactured by BioTek Instruments, Inc.

[0042] Sanyo Corporation of Japan, Sanyo High-Pressure Steam Sterilizer (MLS-3781L)

[0043] Epson, Root Scanner (Epson Expression 1600pro)

[0044] Gas chromatograph (7820A GC) from Agilent Technologies, USA.

[0045] Unless otherwise specified, the test methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer; reagents of the same specifications available on the market can also be used if the source is not specified, and the implementation of this invention is not limited to reagents of the specified source.

[0046] Example 1: The Isolation of Variovorus paradoxus Strain JKM1

[0047] Maize rhizosphere soil from a long-term field experiment on soil compaction at the Shangzhuang Experimental Station of China Agricultural University (116.1°E, 40.1°N) in Beijing was selected as the soil material. After natural air drying, 5g of rhizosphere soil was weighed and mixed with 45mL of sterile water by shaking. The supernatant was then diluted to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 Concentration gradient dilution solution, and select 10 -3 10 -4 10 -5Diluted 200 μL of the solution was plated on LB agar plates and incubated at 30°C for 3 days. Single colonies isolated were picked using an inoculation loop, and the maize rhizosphere bacteria were purified using the streak plating method multiple times. The purified strains were preserved using glycerol at -20°C and -80°C until use. An image of the controversial *Variovorax paradoxus* JKM1 strain streaked on LB agar plates is shown below. Figure 1 As shown, Figure 1 The strain labeled C-7 in the left image is the controversial Variovora paradoxus JKM1. Figure 1 The image on the right is an enlarged version of the image on the left.

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

[0049] The method for determining ACC deaminase activity is as follows:

[0050] A small amount of the screened strain was inoculated into 20 mL of TSB liquid medium and incubated at 28 °C and 200 rpm. -1 Incubate under shaking conditions for 12 hours, then at 4℃ and 8000 r·min -1 Centrifuge for 10 min and collect the bacterial pellet. Resuspend the bacterial cells in 7.5 mL of nitrogen-free DF medium and add 45 μL of filtered and sterilized 0.5 mol·L⁻¹ hydrochloric acid. -1 Prepare ACC solution to achieve a final ACC concentration of 3.0 mmol·L⁻¹. -1 Then at 28℃ and 200 r·min -1 The bacterial suspension was cultured under shaking conditions for 24 hours to induce ACC deaminase activity. -1 Centrifuge for 10 min, remove the supernatant, collect the bacterial pellet, and resuspend in 5 mL of 0.1 mol·L⁻¹ solution. -1 In Tris-HCl buffer (pH 7.6), at 4°C and 8000 rpm -1 Centrifuge for 10 min and collect the precipitate. Repeat this step three times to completely remove DF medium. Add 1 mL of 0.1 mol·L⁻¹ to the bacterial precipitate. -1 The bacterial cells were suspended in Tris-HCl (pH 7.6) and then transferred to 1.5 mL centrifuge tubes and centrifuged at 16000 rpm. -1 Centrifuge for 5 min; then resuspend the bacterial pellet in 600 μL of 0.1 mol·L⁻¹ solution. -1 Add 30 μL of toluene to Tris-HCl buffer (pH 8.5) and vortex for 30 s to lyse the cells. Take 200 μL of the lysed cell suspension and add 20 μL of 0.5 mol·L⁻¹ HCl. -1The ACC solution was mixed well and then incubated in a water bath at 30°C for 15 minutes. Then, 1 mL of 0.56 mol·L⁻¹ solution was added. -1 Mix with HCl and incubate at 16000 rpm at room temperature. -1 Centrifuge for 5 min. Take 1 mL of the supernatant and add 800 μL of 0.56 mol·L⁻¹ solution. -1 After mixing with HCl, add 300 μL of 2,4-dinitrophenylhydrazine (using 2 mol·L⁻¹). -1 Dissolved in HCl, with a mass concentration of 2 g·L⁻¹ -1 Incubate in a water bath at 30°C for 30 minutes. Then add 2 mL of 2 mol·L⁻¹ water. -1 NaOH was used for color development, and the absorbance (OD540) was measured at 540 nm using a spectrophotometer. Distilled water was used as a control instead of the bacterial suspension.

[0051] Use 0.1 mol·L -1 Prepare Tris-HCl buffer (pH 8.5) at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L. -1 A standard α-butanone gradient solution was prepared, and 300 μL of 2,4-dinitrophenylhydrazine (using 2 mol·L⁻¹) was added to each solution. -1 Dissolved in HCl, with a mass concentration of 2 g·L⁻¹ -1 After mixing well, incubate in a water bath at 30°C for 30 minutes. Then add 2 mL of 2 mol·L⁻¹ water. -1 After NaOH color development and stabilization, the OD540 was measured, with Tris-HCl buffer (pH 8.5) as a blank control. A standard curve was constructed based on the concentration of α-butanone solution and its corresponding absorbance, and the regression equation was determined. The OD540 of the sample was substituted into the regression equation of the standard curve to obtain the content of α-butanone. The amount of α-butanone produced by ACC deaminase catalyzing ACC was calculated (μmol). Then, the protein content (mg) in the remaining bacterial suspension was determined using the Coomassie Brilliant Blue G-250 method, with bovine serum albumin as a control. The activity results are expressed as specific enzyme activity (μmol α-butanone·mg). -1 ·h -1 The measured ACC deaminase activity of the bacteria used in the conditioner was 13.50 ± 13.50. 1.07 μmol α-Butylene-2-mg -1 ·h -1 It is higher than the ACC deaminase activity threshold (20 nmol α-butanone·mg) for bacterial colonization and growth-promoting strains. -1 ·h -1 (Penrose and Glick, 2003)

[0052] The selected strain JKM1 was identified by bacterial 16S rDNA analysis. After sequencing, the sequence was compared with the 16S ribosomal RNAgene of Variovorax paradoxus strain GU-NR5 in the NCBI database. The partial sequence similarity reached 99%, confirming that JKM1 belongs to Variovorax paradoxus. The ACC deaminase-producing Variovorax paradoxus JKM1 of this invention was deposited at the China General Microbiological Culture Collection Center on October 15, 2024, with accession number CGMCC NO: 32218.

[0053] Example 3: Field trial to verify the promoting effect of the controversial gluttonous bacterium Variovora paradoxus JKM1 on maize growth under soil compaction stress.

[0054] 3.1 Treatment settings for field trials

[0055] The long-term fixed-location soil compaction test was conducted in June 2023 at the Quzhou Experimental Station of China Agricultural University in Handan City, Hebei Province (115°0′E, 36°51′N). The soil type at the experimental site was saline-alkali alluvial soil, and the total nitrogen content of the soil was 1.09 g·kg⁻¹. -1 The available phosphorus (Olsen-P) concentration was 24.0 mg / kg. -1 The available potassium concentration is 172 mg / kg. -1 The pH (soil-to-water ratio 1:5) was 8.26, and the organic matter content was 16.3 g / kg. -1 The compaction process involved using a Dongfanghong 1304 tractor (weighing 5080 kg) to continuously compact the soil six times in a flat, even-track manner. The experimental nitrogen fertilizer application rate was 225 kg N·m³. -1 The amount of phosphate fertilizer used is 75 kg of P2O5 ha -1 The amount of potassium fertilizer used is 80 kg K2O ha -1Before sowing, half of the nitrogen fertilizer (urea, 46% N), phosphate fertilizer (superphosphate, 16% P2O5), and potassium fertilizer (potassium sulfate, 48% K2O) were evenly applied as basal fertilizer, and then rotary tilled into the 0-10cm soil layer. The remaining half of the nitrogen fertilizer was applied as topdressing during the corn jointing stage (V6 stage). The corn variety used was MC812 (Zeamays L.cv MC812), with a row spacing of 60cm and a plant spacing of 21cm. The bacterial strain was inoculated at the seedling stage (25 days after sowing), and the aboveground dry weight and root morphology in the 0-30cm soil layer were measured at the flowering stage (50 days after sowing). A diluted LB broth medium of the corresponding concentration served as a blank control, and three biological replicates were set up for each bacterial strain treatment.

[0056] 3.2. Controversy over the preparation and inoculation method of Variovorus paradoxus JKM1 inoculum.

[0057] After activating the strain Variophora paradoxus JKM1, an appropriate amount was picked and placed in LB liquid medium at 28°C and 160 rpm. -1 After shaking culture for 48 hours, the inoculum was diluted and the OD600 value was adjusted to 0.5±0.02 (blank calibration was performed using pure LB liquid medium). The inoculum was inoculated using the root drenching method, which involved digging a hole about 15cm deep and about 7cm in diameter about 15cm away from the corn plant with a small shovel, pouring 50mL of inoculum solution into the hole, and then covering the hole with soil to fill it in.

[0058] 3.3 Methods for determining aboveground dry weight and root morphology indicators

[0059] The aboveground dry weight and root morphology indicators were obtained when the corn entered the flowering stage (50 days after sowing). The corn plants were cut at the base of the stem with scissors, and the aboveground parts were placed in a mesh bag and blanched in a 105℃ oven for half an hour. Then, they were dried at 60-70℃ until constant weight and weighed. Root morphology indicators were collected using a soil drill method. A 10cm diameter, 15cm high metal cylindrical drill bit was driven into the soil 5cm from the root base. Two soil cores were collected below the soil surface, i.e., soil samples at a depth of 0-30cm. The soil samples were passed through a 2mm sieve, and the roots were picked out with tweezers and washed with deionized water. Roots were scanned using an Epson Expression 1600pro root scanner, and the scanned images were analyzed using the 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. Specific root length (cm / g) was calculated by dividing the root length (cm) by the root dry weight (g).

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

[0061] Example 4: A greenhouse soil column experiment was conducted to verify the controversial effect of *Variovorax paradoxus* JKM1 on promoting maize growth under soil compaction stress.

[0062] 4.1 Soil column test setup

[0063] The experiment employed a soil column culture method. The test container was an assembled PVC pipe cut along its cross-sectional diameter, 15 cm in diameter and 35 cm high, with the bottom sealed using a 200-mesh nylon mesh during assembly. Sterilization was performed using radiation sterilization at a dose of 25000 Gy. Each container was filled with 6.9 kg of soil without compaction, resulting in a bulk density of 1.3 g·cm³. -3 For compaction, fill each pot with 8.5 kg of soil. For every 5 cm of soil added, place a flat, sealed metal post on the soil surface and tap it with a hammer to evenly compact the soil. The final soil density should be 1.6 g / cm³. -3A Rhizon sampler (Rhizo SMS) with a 1.5 μm pore size was placed 10 cm above the soil surface on a PVC soil column to collect gas and solution from the pores.

[0064] The maize variety selected was Zhengdan 958 (Zea mays L.cv ZD958). Seeds of similar shape and size were chosen for sowing. After disinfection by soaking in 10% H2O2 for 30 minutes, the seeds were rinsed thoroughly with deionized water and then placed in a saturated CaSO4 solution in the dark with ventilation for 12 hours to allow them to swell. The swollen seeds were then placed in trays containing moistened filter paper and placed in a plant culture room in the dark for 24 hours to germinate. During this period, deionized water was added every 4 hours to keep the filter paper moist. Once the seeds had sprouted to about 1 cm, seeds with relatively similar root lengths were selected. For each inoculated strain treatment, the seeds were soaked in the corresponding bacterial solution (OD600 0.5 ± 0.02) for 2 hours before sowing. For the control treatment, sterile water was used instead of the bacterial solution for soaking. Three seeds were sown per pot, and after emergence, thinning was performed, retaining two seedlings with relatively consistent growth characteristics with the rest. During the experiment, deionized water was added from the bottom of the soil column through infiltration to maintain the soil moisture content at 60-80% of field capacity. During the plant growth period, the inoculated strain treatment was irrigated with bacterial solution every 7 days after sowing, with 40 mL per pot. A total of 30 days were spent irrigating before harvest. The control treatment was irrigated with sterile water instead of the bacterial solution.

[0065] Ethylene was collected from the root zone 15 days after corn sowing using a 5 mL vacuum tube connected to a Rhizon sampler 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 Controversy over the preparation and inoculation method of Variovorus paradoxus JKM1 inoculum

[0067] The strain Variotrophus paradoxus JKM1 was activated and inoculated into LB liquid medium, incubated at 28°C and 160 rpm. -1 Incubate with shaking for 48 hours, then at 4℃ and 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. This bacterial agent was used for inoculation of greenhouse soil column tests.

[0068] 4.3 Measurement of ethylene content in root zone soil, IAA (indole-3-acetic acid) content and related synthetic genes in roots, and ABA (abscisic acid) content and related synthetic genes in roots.

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

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

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

[0072] IAA promotes root growth and root cell division; regulates root tropism, including hydrotropism and nutrient tropism; and promotes the growth of 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 root system's resistance to stress by regulating the physiological activities of the root system to enhance its drought resistance; maintain the ion balance of the plant root system under salt stress; and regulate the water absorption and transport of the root system.

[0074] The relative expression levels of the IAA-related synthetic gene ZmYUC4 and the ABA-related synthetic gene NCED1 in maize roots were determined by Nanjing Zhongding Biotechnology Co., Ltd., using real-time quantitative PCR with GADPH as an internal reference gene for relative quantitative detection.

[0075] The primer sequences used for real-time 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] The following will use 2 -△△Ct The method involves relatively quantitative analysis of the data:

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

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

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

[0086] The control group was a control treatment without compaction.

[0087] The determination of ABA and IAA content in maize roots was entrusted to Nanjing Ruiyuan Biotechnology Co., Ltd., using a double antibody sandwich method. The main materials included an enzyme-linked immunosorbent assay (ELISA) kit for abscisic acid (ABA) and an ELISA kit for plant growth regulators (IAA). The control group was also a control treatment under non-compacted conditions.

[0088] Controls were established for root soil ethylene content, IAA content and related gene expression, and ABA content and related gene expression. Results are as follows: Figure 3 As shown.

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

[0090] Thirty days after sowing, the plants were cut at the base of the stem with scissors, and the above-ground parts were placed in envelopes for sampling. The above-ground dry weight and total phosphorus content were to be determined, with the above-ground dry weight determined using the same method as in the field experiment. The above-ground dry samples were ground into powder, and the test solution was prepared first using the H2SO4-H2O2 digestion method. Then, the total phosphorus content of the above-ground parts was determined using the vanadium molybdenum yellow spectrophotometric method. After collecting the above-ground indicators, the assembled PVC pipe was disassembled, and the soil column was removed as a whole. The root system and soil column were placed on kraft paper, and the loose soil was gently shaken to separate the plant from the soil column. The rhizosphere soil adhering to the root surface was gently brushed off with a soft brush. The undamaged root samples were washed with deionized water and then dried with absorbent paper. The root samples were subsequently used to determine root morphology indicators, with the same measurement procedures as in the field experiment.

[0091] 4.4 Data Analysis Methods

[0092] Experimental data were analyzed using SPSS software, and bar charts were generated using GraphpadPrism 8 software, such as... Figure 3 and Figure 4 As shown in the figure. The data in the figure represent the mean (± standard error) of three replicates. A t-test was used to analyze the significance of differences between two groups, with * indicating significant differences. One-way ANOVA was performed at the 5% significance level using the LSD method to analyze differences between the three groups. Lowercase letters indicate significant differences between treatments.

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

[0094] Figure 4 The results showed that under soil compaction stress, after inoculation with Variovora 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%, respectively, the average root diameter decreased by 5.1%, and the phosphorus content in the aboveground parts also increased significantly.

[0095] Figure 5 This is a comparison chart of maize growth 30 days after inoculation with Variovorax paradoxus JKM1 under soil compaction stress in a greenhouse soil column experiment. As shown in the figure, maize with compacted soil had the worst growth, followed by maize with compacted soil but inoculated with JKM1. Maize with non-compacted soil had the best growth, but the growth of compacted soil inoculated with JKM1 was also significantly better than that of compacted soil without JKM1 inoculation.

[0096] Examples 3 and 4 of this invention compare plant growth and root conditions in non-compacted soil with those in single-factor (JKM1 bacteria only) conditions. The results show that although the growth is not as good as in non-compacted soil, it is better than that in compacted soil without JKM1 inoculation. This indicates that JKM1 does indeed promote plant growth in compacted soil. Based on this invention, physical measures such as deep plowing and application of organic fertilizer can be added to improve the soil structure of compacted soil and promote plant growth.

[0097] Example 4 of this invention further analyzes the mechanism by which the ACC deaminase bacterium Variovora paradoxus JKM1 regulates root morphology by influencing root hormone synthesis. It promotes plant growth in compacted soil by reducing ethylene content 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 within the protection scope of the present invention.

Claims

1. A controversial gluttonous bacterium producing ACC deaminase ( Variovorax paradoxus JKM1 was deposited on October 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 32218.

2. The ACC deaminase-producing Agrobacterium strain of claim 1, Variovorax paradoxus ) JKM1 for use in promoting plant growth in compacted soil.

3. The ACC deaminase-producing bacteriophage of claim 2, wherein the bacteriophage is a member of the genus Erwinia. Variovorax paradoxus ) Use of JKM1, characterized in that, The ACC deaminase-producing Agrobacterium tumefaciens (AT) strain Variovorax paradoxus JKM1 promotes plant growth by reducing ethylene content in plant roots under soil compaction stress.

4. The ACC deaminase-producing bacteriophage of claim 3, Variovorax paradoxus ) the use of JKM1, characterized in that, The reduction of the ethylene content of the plant root system under the stress of soil compaction is achieved by degrading the ethylene synthesis precursor ACC to inhibit the production of ethylene in the plant root in the compacted soil.

5. The ACC deaminase-producing bacteriophage of claim 2, wherein the bacteriophage is a member of the genus Erwinia. Variovorax paradoxus ) Use of JKM1, characterized in that, The ACC deaminase-producing Agrobacterium tumefaciens strain Variovorax paradoxus JKM1 promotes plant growth by increasing the content or synthesis of indole acetic acid and abscisic acid in plant roots.

6. The ACC deaminase-producing Agrobacterium strain of claim 2, Variovorax paradoxus ) Method of use of JKM1 in promoting plant growth in compacted soil, characterized in that, The debate surrounding inoculation of plants with ACC-producing deaminase-producing bacteria during the seedling stage ( Variovorax paradoxus JKM1, the activated bacterial strain was diluted in LB liquid medium to prepare OD. 600 Inoculation solution with a value of 0.5±0.02 was used for root drenching.

7. The ACC deaminase-producing bacteriophage of claim 6, wherein the bacteriophage is a member of the genus Erwinia. Variovorax paradoxus ) The use of JKM1 in promoting plant growth in compacted soil, characterized by, The inoculation by the irrigation method is to dig a hole deep to the root of the plant beside the plant with a small shovel, pour 50 mL of bacterial solution into the hole, cover the soil to fill the hole, and complete the inoculation.

8. The ACC deaminase-producing Agrobacterium strain of claim 2, Variovorax paradoxus ) Method of use of JKM1 in promoting plant growth in compacted soil, characterized in that, The plant seeds are germinated, and after the seeds grow to about 1 cm of sprouts, the seeds are soaked in OD 600 value of 0.5±0.02 before sowing; the OD 600 value of 0.5±0.02 is obtained by diluting the bacterial strain in sterile water.

9. A soil compacting conditioner, characterized in that, A bacteriophage comprising the ACC deaminase of claim 1 Variovorax paradoxus ) JKM1.

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