Application of plant metabolites in promoting microbial rhizosphere colonization and enhancing the growth of inoculants

Abstract

This invention discloses the application of plant metabolites in promoting microbial rhizosphere colonization and enhancing the growth-promoting effect of inoculants. The plant metabolites include purine and / or malurin. The combined use of these metabolites and inoculants significantly enhances the growth-promoting effect of synthetic inoculants on crops under drought stress. The metabolites purine and malurin increase the colonization rate of the inoculant. The concentration of purine is 10 μM, and the concentration of malurin is 500 μM. The application time for the metabolites and microbial community is the two-leaf-one-heart stage of wheat. The combined use of metabolites and compound inoculants significantly improves the growth (biomass accumulation) of crops under drought conditions. Rhizosphere microbial biomass measurements show that the metabolites purine and malurin increase the rhizosphere colonization rate of the inoculant.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to the application of plant metabolites in promoting microbial rhizosphere colonization and enhancing the growth of inoculants. Background Technology

[0002] Microbial inoculants are environmentally friendly agricultural products that show promise in promoting soil biodiversity and improving crop productivity. However, current research indicates that methods such as inoculating rhizosphere growth-promoting bacteria alone or adding synthetic communities are often hindered by the native community, leading to the rapid disappearance or inactivation of introduced strains. This is because these microbial inoculants do not consider the plasticity of plant metabolites, microbial interactions, and the importance of microbial diversity in promoting soil function, and therefore often fail to achieve their intended functions under field conditions. Root exudates, as mediators of plant-microbe interactions, can act as nutrients or screening agents to reshape the rhizosphere microbial community.

[0003] Drought stress inhibits wheat root development, photosynthetic efficiency, and nutrient absorption, leading to reduced yields and seriously threatening food security. Rhizosphere microorganisms (such as growth-promoting bacteria) and plant root metabolites, as key regulatory factors in the soil-plant system, have become a research hotspot in the field of agricultural stress resistance because they can enhance stress resistance by improving the soil microenvironment and regulating plant physiological metabolism. Currently, research on "microbial growth promotion" and "metabolite regulation" has confirmed that some rhizosphere bacteria can promote crop growth and certain root metabolites can regulate microbial community structure. However, how to combine the two to cope with drought stress has not yet formed a mature technical system, and related research is still in the exploratory stage.

[0004] Therefore, existing technologies have shortcomings and need to be improved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of plant metabolites in promoting microbial rhizosphere colonization and enhancing the growth of inoculants, which is an improvement over the shortcomings of the prior art.

[0006] The technical solution of the present invention is as follows: The application of plant metabolites in promoting microbial rhizosphere colonization and enhancing the growth of microbial agents, wherein the plant metabolites include purine (CAS: 120-73-0) and / or malurin (CAS: 519-34-6).

[0007] In the aforementioned application, the combined use of the metabolites and microbial agents can significantly enhance the growth-promoting effect of synthetic microbial agents on crops under drought stress.

[0008] In the aforementioned application, the metabolites Purine and Maclurin can increase the colony density of the bacterial agent.

[0009] In the aforementioned applications, Purine is 10 μM and Maclurin is 500 μM.

[0010] The application of metabolites and microbial communities is scheduled for the wheat at the two-leaf-one-heart stage.

[0011] The combined use of metabolites and compound microbial agents can significantly improve the growth of crops under drought conditions (biomass accumulation). The addition of metabolites Purine and Maclurin can increase the colonization rate of the microbial agents. Attached Figure Description

[0012] Figure 1 The effects of combined use of metabolites and microbial agents on wheat growth under drought conditions; different letters indicate significant differences between different treatments (Tukey test, HSD, p < 0.05), and error bars represent standard errors.

[0013] Figure 2 Effects of different metabolites on wheat rhizosphere microbial colonization; different letters indicate significant differences between different treatments (Tukey test, HSD, p < 0.05), and error bars represent standard errors. Detailed Implementation

[0014] The present invention will be described in detail below with reference to specific embodiments.

[0015] I. Sample Collection and Metabolite Screening 1. Sample collection The experiment was conducted in Dingxi City, Gansu Province (35°34′N, 104°37′E), a hilly and gully region in the central Loess Plateau. This area has a typical temperate semi-arid continental monsoon climate, with an average annual precipitation of approximately 400 mm, mainly concentrated in July to September; the average annual temperature is approximately 6.5℃, and the frost-free period is about 150 days. The soil parent material in this area is loess, with a predominantly loam texture. The soil layer is deep but has a low organic matter content and poor water and fertilizer retention capacity. The soil pH in Dingxi is 7.91, the organic carbon content is 6.42 g kg⁻¹, and the total nitrogen content is 0.615 g kg⁻¹.

[0016] The field experiment began in March 2024, employing a completely randomized design. Treatments included two spring wheat varieties: Longchun 35 (LC35) and Longchun 43 (LC43); and two rainfall treatments: normal rainfall and drought (40% reduction in rainfall). For the drought treatment, multiple U-shaped transparent plexiglass panels tilted at approximately 10° were used above each experimental plot, suspended by metal supports. The transparent plexiglass covered 40% of the soil surface area; during rainfall, water trapped by the U-shaped plexiglass flowed out along the slope. Two nitrogen fertilizer treatments were also included: 0 kg / ha of nitrogen fertilizer. -1 and 120 kg ha-1 Each treatment was repeated four times. Plots were 5 m × 4 m in size, separated by 0.25 m wide ridges, with 1.0 m protective rows. The wheat seeding rate was 140 kg ha. -1 .

[0017] Samples were collected from wheat at the jointing stage (May 2024). A five-point sampling method was used within each plot, with sampling depths ranging from 0 to 20 cm. Rhizosphere soil was collected from the surface of fine roots with a diameter < 2 mm. Before sampling, large clumps of soil were gently removed from the roots, and the soil adhering to the roots was gently brushed off with a sterilized small brush. A layer of aluminum foil was placed underneath to collect the rhizosphere soil. The collected rhizosphere soil was then placed into sterilized cryovials, labeled, and used for metabolite detection.

[0018] 2. Metabolite determination Rhizosphere metabolite profiling was performed on freeze-dried soil samples using LC-MS-based metabolomics analysis. One gram of soil sample was placed in a centrifuge tube, and 10 mL of pre-chilled extraction buffer (methanol:acetonitrile = 3:1, pre-chilled to -40°C) was added. Two steel balls were added, and the mixture was ground and incubated at 4°C for 1 hour. Subsequently, the sample was centrifuged at 13400 g at 4°C for 15 minutes, and the supernatant was collected and concentrated to dryness under vacuum. To dissolve the sample, 200 μL of 50% methanol-water solution (methanol:water = 1:1, v / v) was added, and the mixture was vortexed for 3 minutes (4°C, 2200 g), followed by centrifugation at 13400 g at 4°C for 15 minutes. The supernatant was collected for injection analysis. Metabolite annotation was performed using the Kyoto Encyclopedia of Genetics and Genomes (KEGG) and the Human Metabolite Database (HMDB).

[0019] 3. Key metabolites and microbial selection: Based on the co-occurrence network and differential analysis of metabolites and microorganisms under different treatments, four metabolites potentially associated with microbial colonization were screened: adenosine (CAS: 58-61-7), pyruvaldehyde (CAS: 78-98-8), purine (CAS: 120-73-0), and malurin (CAS: 519-34-6). Six strains associated with changes in metabolites were also screened: Agrobacterium tumefaciens, Bacillus stercoris, Pseudomonas glycinae, Achromobacter marplatensis, Microbacterium phyllosphaerae, and Olivibactersoli.

[0020] 4. Determination of metabolite concentration: Six commercially available bacterial strains were purchased. A co-culture experiment with metabolites was conducted to determine the OD values ​​of the strains after 24 hours of growth under different concentrations of metabolites, assessing the effect of metabolites on bacterial growth. Specific steps: The strains were cultured in liquid LB medium (Luria-Bertani Medium) until the OD600 value reached 0.6, then diluted with sterile water to a suitable concentration with an initial OD600 value of 0.05-0.1. Solutions of adenosine, pyruvaldehyde, purine, and malurin were prepared using sterile water at concentrations of 10 μM, 100 μM, 500 μM, and 2000 μM. Adenosine, pyruvaldehyde, and purine were purchased from Shanghai Haohong Biomedical Technology Co., Ltd., and malurin was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. Bacterial growth assays were performed in 96-well microplates. Each well contained 200 μL of liquid LB medium, 20 μL of the test chemical solution (the blank control group used an equal volume of sterile water), and 5 μL of bacterial suspension. Each treatment was repeated in quadruplicate. OD600 values ​​were measured hourly using a microplate reader to monitor the growth curves of the strains in real time. The effects of different metabolite concentrations on bacterial growth were then analyzed.

[0021] Based on the growth curves of the strains, the concentrations of metabolites that promote the growth of most strains were screened. The optimal concentrations for adenosine, pyruvaldehyde, and purine were 10 μM, and the optimal concentration for malurin was 500 μM.

[0022] 5. Pot experiment on the combined use of metabolites and synthetic community inoculants: (1) The synthetic community combinations are: Combination 1: Agrobacterium tumefaciens, Bacillusstercoris, Pseudomonas glycinae; Combination 2: Achromobacter marplatensis, Microbacterium phyllosphaerae, Olivibacter soli.

[0023] (2) Preparation of synthetic community inoculum: The selected strains of the two synthetic communities were activated. The previously stored glycerol cryovials were taken out from the -80°C freezer. A small amount of bacterial solution was dipped into the LB solid medium plate with a sterile inoculation loop and streaked onto the plate. The plate was inverted and placed in a 30°C incubator for incubation. After 1-2 days of incubation, a single colony was picked and cultured in LB liquid medium by shaking at a temperature of 180 r·min. -1 At 28°C, when the OD600 value was measured to be around 0.8, equal volumes of the culture were mixed to prepare the synthetic bacterial solution.

[0024] (2) Metabolite preparation: Prepare the four metabolites to the target concentration; (3) Experimental treatment: sterile, added synthetic community inoculant, added synthetic community inoculant and four metabolites; wheat variety was Longchun 35, drought level was moderate drought: 50% field water holding capacity.

[0025] (4) Specific steps: After removing debris and stones from the long-term unfertilized field soil, it was sieved through a 2 mm sieve and then mixed with vermiculite at a 1:1 ratio as a potting substrate. The soil was then subjected to high-pressure sterilization (121°C for 1 hour, repeated twice) to ensure that the soil was completely sterile.

[0026] For wheat seeds, use Longchun 35. First, select plump wheat seeds and soak them in sodium hypochlorite for 30-45 seconds. During the soaking process, gently shake the conical flask to ensure that the seeds are fully in contact with the disinfectant. Then discard the disinfectant and rinse thoroughly with sterile water to obtain seeds that have been surface disinfected for later use.

[0027] Sterilized wheat seeds were sown in soil for pot cultivation, with 8 seeds in each pot. The soil was kept at 70% field capacity by weighing. After 6-7 days of cultivation, when most of the wheat seeds had grown roots of about 5cm, the seedlings were thinned out, leaving 4 strong and uniform seedlings in each pot. The soil was kept at 70% field capacity by weighing.

[0028] When wheat reached the two-leaf-one-heart stage, 10 mL of the corresponding synthetic community inoculant suspension (the control group received an equal volume of sterile water) was added to the soil surface using a syringe, with only one inoculation. 5 mL of the corresponding metabolite was then added (the control group received an equal volume of sterile water), and the metabolite was replenished every 4 days thereafter. Drought treatment: Once the soil moisture content dropped to 50% of field capacity, this moisture content was maintained by weighing (water was added every 2 days) until the end of the experiment. Four weeks after treatment, the drought-treated sterile wheat showed significant growth restriction, and samples were collected. The plants were carefully removed, keeping the root system as intact as possible. The non-rhizosphere soil adhering to the roots was gently shaken or brushed off, and the soil adhering closely to the roots (rhizosphere soil) was collected, sieved through a 2mm sieve, and stored at -80°C for microbiological analysis. The roots were then systematically rinsed with sterile water to remove surface soil. The wheat plant height and root length were measured using a ruler. The plants were then dried in a 65°C oven to constant weight, and the dry weight was recorded.

[0029] 6. Determination of the absolute amount of rhizosphere microbial colonization Accurately weigh 0.5g of rhizosphere soil sample and extract total genomic DNA using the FastDNA® SPIN Kit for Soil (MPBiomedicals, Santa Ana, CA). DNA integrity is assessed by agarose gel electrophoresis. DNA concentration and purity are measured using a Nanodrop 2000 and Qubit 3.0 apparatus to ensure the sample concentration meets subsequent experimental requirements (typically adjusted to 10 ng / μL, with a total volume exceeding 500 ng). Sample quality is recorded during DNA extraction, and the target gene copy number per unit sample is calculated based on the amount of sequencing template DNA. A synthetically produced spike-in internal standard sequence is added to the extracted DNA. The internal standard contains the same conserved region as the natural 16S rRNA gene. It is added in a known gradient copy number configuration to construct a standard curve. PCR amplification is performed using specific primers (341F / 805R) targeting the V3-V4 or V4-V5 region of the 16S rRNA gene. The amplification product contains the target genomic region of the sample and the internal standard sequence. After amplification, sample-specific tag sequences were introduced via high-fidelity PCR using primers with index sequences to construct a library. Finally, after purification and quantification, the library was sent to the Illumina NovaSeq 6000 platform for sequencing, employing a 2×250 bp paired-end sequencing strategy to obtain high-quality data.

[0030] The raw reads obtained from sequencing were processed in QIIME2 software. First, the adapter and primer sequences were removed using the cutadapt plugin; then, the raw data was denoised, spliced, and dechimeric processed using the DADA2 plugin to generate a high-quality ASV (amplicon sequence variants) feature table and representative sequences. Based on the RDP (version 11.5) database, a pre-trained Naïve Bayes classifier was used for taxonomic annotation of ASVs (16S rRNA gene confidence threshold of 0.8, ITS region confidence threshold of 0.6). Simultaneously, spike-in sequences were identified and their read counts were counted for plotting a standard curve. Combining the sample DNA extraction amount, sequencing template DNA amount, and sample mass, the absolute copy number of each ASV per unit sample was calculated (formula: ASV absolute copy number copies / g = [ASV copy number × DNA extraction amount] / [template DNA amount × sample mass]), or absolute quantification was performed per unit DNA amount. By comparing the representative ASV sequences with the full-length 16S sequences of the strains, the added strains were identified, and their absolute copy numbers were calculated.

[0031] 7. Experimental Results refer to Figure 1 -A, the metabolites themselves do not promote plant growth, but when used in combination with compound microbial agents, they can significantly improve the effect of the agents on crop growth (biomass accumulation) under drought conditions. Figure 1 -B, Figure 1 -C).

[0032] refer to Figure 2 Absolute quantitative determination of target microorganisms in wheat rhizosphere showed that the metabolites Purine and Maclurin significantly increased the rhizosphere colonization of the target microbial agent.

[0033] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The application of plant metabolites in promoting microbial rhizosphere colonization and enhancing the growth-promoting effects of inoculants, characterized in that, The plant metabolites include purine and / or malurin.

2. The application according to claim 1, characterized in that, The combined use of the metabolites and microbial agents can significantly enhance the growth-promoting effect of synthetic microbial agents on crops under drought stress.

3. The application according to claim 1, characterized in that, Metabolites Purine and Maclurin can increase the rhizosphere colonization of the inoculant.

4. The application according to claim 1, characterized in that, Purine is 10 μM and Maclurin is 500 μM.

5. The application according to claim 1, characterized in that, The application period for metabolites and microbial flora is during the two-leaf-one-heart stage of wheat.

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