Application of graphene in improvement of species diversity of rhizosphere microflora of sorghum in saline-alkali soil

Abstract

The invention discloses application of graphene to improvement of species diversity of saline-alkali soil sorghum rhizosphere microflora, and belongs to the technical field of crop ecological carbon sink. The graphene sol fertilizer is used for treating sorghum crops in saline-alkali soil, it is accidentally found that graphene can remarkably promote abundance and diversity of rhizosphere microbial community species of sorghum, and some potential plant probiotic communities are recruited in root systems. According to the method, the condition of rhizosphere microflora of sorghum is classified and counted by utilizing an amplicon sequencing method, and it is proved that graphene promotes growth of saline-alkali soil sorghum to depend on change of a rhizosphere microflora structure, so that the application range of graphene is widened, and a theoretical basis is laid for wide application of graphene in agriculture.

Description

Technical Field

[0001] This invention belongs to the field of ecological carbon sequestration technology and relates to the application of graphene in improving the species diversity of rhizosphere microbial communities in saline-alkali land sorghum. Background Technology

[0002] Sorghum (Sorghum bicolor) is an important cereal crop, widely cultivated in tropical and subtropical regions worldwide. Sorghum is drought-tolerant, tolerant of poor soil, and highly resilient, making it an important source of food and feed in areas where climatic conditions are unsuitable for other crops. Saline-alkali soils, due to their high content of soluble salts and alkalis, suffer from decreased soil organic matter content and a lack of microbial diversity, hindering plant growth. Planting alkali-tolerant crops and using drip irrigation to apply water-soluble fertilizers can improve soil physical and chemical properties, thereby promoting soil health and increasing crop productivity (such as sorghum). Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides an application of graphene in improving the species diversity of crop rhizosphere microbial communities.

[0004] Graphene is a nanomaterial with many unique properties, including a unique two-dimensional structure, an extremely large specific surface area, tunable surface chemistry, and excellent electrical, thermal, optical, and mechanical properties. Soil microorganisms are an important component of the soil ecosystem, often referred to as the 'second genome' of plants, participating in numerous ecological processes such as the decomposition of organic matter and nutrient transformation and cycling in the soil. This invention utilizes graphene to improve the diversity of plant rhizosphere microbial communities, effectively promoting soil health and increasing crop (such as sorghum) productivity.

[0005] According to an embodiment of the present invention, the crop is preferably a saline-alkali soil crop. For example, the crop is sorghum.

[0006] According to an embodiment of the present invention, the graphene is used to improve the species diversity of the rhizosphere microbial community of crops in saline-alkali land; specifically, it is used to improve the species diversity of the rhizosphere microbial community of sorghum crops in saline-alkali land.

[0007] According to an embodiment of the present invention, the graphene is graphene oxide.

[0008] According to an embodiment of the present invention, graphene is applied to saline-alkali land in the form of graphene sol fertilizer. For example, graphene is dispersed in water to obtain graphene sol fertilizer, which is then applied to the soil.

[0009] This invention addresses the composition and differences of rhizosphere microbial community species in sorghum grown in saline-alkali soil. By treating sorghum crops in saline-alkali soil with graphene sol fertilizer, and using amplicon sequencing to classify and statistically analyze the rhizosphere microbial community status of sorghum, this invention improves the microbial diversity of sorghum crops in saline-alkali soil in northern Shanxi Province, and clarifies the application of graphene in improving the rhizosphere microbial diversity of sorghum in saline-alkali soil.

[0010] According to an embodiment of the present invention, the concentration of graphene in the graphene sol fertilizer is 5 to 20 mg / L, with exemplary values ​​of 5 mg / L, 8 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L.

[0011] According to an embodiment of the present invention, the graphene is prepared by a two-step electrochemical method using flexible graphite paper and platinum foil as the cathode and anode, respectively.

[0012] In one embodiment of the present invention, in the two-step electrochemical method, the initial voltage of the first step of the electrochemical method is, for example, 1.5 to 2.0 V, and exemplarily 1.60 V.

[0013] In one embodiment of the present invention, in the two-step electrochemical method, the initial voltage of the second electrochemical step is, for example, 2.0 to 3.0 V, and exemplarily 2.5 V.

[0014] In one embodiment of the present invention, in the two-step electrochemical method, the electrolyte in the second step of the electrochemical method is a sulfuric acid solution. For example, the concentration of the sulfuric acid solution is 40–60 wt.%, exemplarily 50 wt.%.

[0015] In one embodiment of the present invention, the second electrochemical step in the two-step electrochemical method includes fixing the blue intermediate product obtained in the first electrochemical step with a platinum foil as a cathode.

[0016] According to embodiments of the present invention, the microbial community species include at least one of bacteria and fungi. For example, it includes at least one of Pseudomonas, Bacillus, Mortierella, and Trichoderma.

[0017] According to an embodiment of the present invention, the species diversity includes at least one of the following: species abundance (e.g., species dilution curves, Venn diagrams based on rhizosphere microbial OTUs, average number of OTUs), Alpha diversity indices (Chao1 and Shannon), principal coordinate analysis, composition and abundance of potential plant probiotics, and taxonomic analysis based on phylum and genus levels.

[0018] Furthermore, the graphene improves the dilution curve of rhizosphere microbial samples of crops (such as sorghum) in saline-alkali land.

[0019] Furthermore, the graphene promotes the total number and average number of OTUs of rhizosphere-specific fungi and bacteria in crops (such as sorghum) in saline-alkali soils.

[0020] Furthermore, the graphene promotes the alpha diversity index (Chao1 and Shannon) of rhizosphere microbial communities in crops (such as sorghum) in saline-alkali land.

[0021] Furthermore, the principal coordinate analysis revealed significant differences in β-diversity between the rhizosphere microbial communities of sorghum in graphene-treated and control-treated saline-alkali land.

[0022] Furthermore, the graphene promotes the abundance of potential beneficial plant microbial communities such as Pseudomonas, Bacillus, Mortierella, and Trichoderma in the rhizosphere microbial community of crops (e.g., sorghum).

[0023] The present invention also provides the application of the above-mentioned graphene in the preparation of crop growth fertilizer.

[0024] According to an embodiment of the present invention, the fertilizer is a water-soluble fertilizer. Preferably, the concentration of graphene in the fertilizer is 5-20 mg / L, exemplarily 5 mg / L, 8 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L.

[0025] The present invention also provides a fertilizer comprising the above-mentioned graphene.

[0026] According to an embodiment of the present invention, the fertilizer is a water-soluble fertilizer. Preferably, the concentration of graphene in the fertilizer is 5-20 mg / L, exemplarily 5 mg / L, 8 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L.

[0027] The present invention also provides the application of the fertilizer in improving the species diversity of the rhizosphere microbial community of crops and / or promoting crop growth. Preferably, it is used in improving the species diversity of the rhizosphere microbial community of crops in saline-alkali soils and / or promoting crop growth in saline-alkali soils.

[0028] The present invention also provides a method for improving the species diversity of crop rhizosphere microbial communities, comprising applying the above-mentioned graphene and / or fertilizer to the crop growing soil.

[0029] The present invention also provides a method for promoting crop growth, comprising applying the above-mentioned graphene and / or fertilizer to the soil in which the crop grows.

[0030] The beneficial effects of this invention are:

[0031] The inventors unexpectedly discovered through extensive experiments that applying graphene sol to saline-alkali land improved the stress resistance of sorghum to saline-alkali conditions, thereby increasing sorghum yield. However, whether graphene affects microbial diversity in saline-alkali land remains unclear. Therefore, the inventors studied the impact of graphene on microbial diversity, providing a scientific basis for assessing its environmental safety and contributing to understanding its potential impacts and benefits in agricultural and environmental applications. This has significant theoretical and practical implications for promoting the safe application of nanomaterials, developing new agricultural technologies, and maintaining the health of ecosystems. Specifically:

[0032] This invention provides the application of graphene in improving the diversity of rhizosphere microorganisms in sorghum in saline-alkali land, thus broadening the application scope of graphene. By applying water-soluble fertilizer containing graphene to saline-alkali land, this invention helps improve the community structure and diversity of rhizosphere microorganisms in sorghum, thereby increasing agricultural productivity in saline-alkali land. Simultaneously, it provides a good technical approach for utilizing graphene to enhance crop growth and stress resistance through microorganisms. Attached Figure Description

[0033] Figure 1 Characterization diagram of graphene enrichment in the roots of sorghum crops after graphene treatment; where: Figure 1 Image A is a phenotypic photograph of the entire sorghum plant; Figure 1 Image B is a characterization image of graphene enrichment in roots taken using a scanning electron microscope. Figure 1 C in the figure represents the Raman spectrum of graphene.

[0034] Figure 2 A dilution curve of rhizosphere microbial species based on OTU level after graphene treatment of sorghum crops; where: Figure 2 Figure A shows the bacterial dilution curve of the sorghum rhizosphere. Figure 2 Figure B shows the fungal dilution curve of the sorghum rhizosphere; the control groups in all figures are water treatment.

[0035] Figure 3 Venn diagram results of rhizosphere microbial species based on OTU level after graphene treatment of sorghum crops; where: Figure 3 A represents the Venn diagram of rhizosphere bacteria in sorghum. Figure 3 B represents the Venn diagram of rhizosphere fungi in sorghum.

[0036] Figure 4 A bar chart showing the average number of OTUs in the rhizosphere microorganisms after graphene treatment of sorghum crops; where: Figure 4 A is a bar chart showing the average number of OTUs in the rhizosphere of sorghum; Figure 4 B is a bar chart showing the average number of OTUs in the rhizosphere of sorghum.

[0037] Figure 5 Box plot of Alpha diversity indices (Chao1 and Shannon) for rhizosphere microorganisms after graphene treatment of sorghum crops; where: Figure 5 Figure A shows the Chao1 index statistics of rhizosphere bacteria in sorghum. Figure 5 Figure B shows the Shannon index statistics of rhizosphere bacteria in sorghum. Figure 5 C represents the Chao1 index statistics of rhizosphere fungi in sorghum; Figure 5 The middle section (D) shows the Shannon index statistics of rhizosphere fungi in sorghum.

[0038] Figure 6 Principal Coordinates Analysis (PCoA) plot for rhizosphere microorganisms after graphene treatment of sorghum crops; Figure 6 Figure A shows the PCoA analysis of rhizosphere bacteria in sorghum. Figure 6 Figure B shows the PCoA analysis of rhizosphere fungi in sorghum.

[0039] Figure 7 Bar chart of species composition analysis of rhizosphere microorganisms at the phylum and genus levels after graphene treatment of sorghum crops; Figure 7 A is a bar chart showing the species composition analysis of sorghum rhizosphere bacteria at the phylum level; Figure 7 Bar chart B is a species composition analysis of sorghum rhizosphere bacteria at the genus level; Figure 7 C is a bar chart representing the species composition analysis of sorghum rhizosphere fungi at the phylum level; Figure 7 The bar chart in section D represents the species composition analysis of sorghum rhizosphere fungi at the genus level. Detailed Implementation

[0040] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0041] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0042] Example 1

[0043] This embodiment provides a method for preparing graphene-enhancing liquid and its application in the field.

[0044] Flexible graphite paper and platinum foil were used as the cathode and anode, respectively. An initial voltage of 1.60V was set for the intercalation reaction. The current gradually decreased as the conductivity of graphite decreased. During this process, the color of the flexible graphite paper changed from silver-gray to blue. Since the blue intermediate product was light, soft, and easily broken, it was fixed with platinum foil as the cathode for oxidation exfoliation. Platinum foil was also used as the anode. The electrolyte, sulfuric acid solution, was controlled at a concentration of 50 wt.% and the voltage at 2.5V to obtain graphene oxide. Subsequently, after filtration, repeated washing with distilled water, and freeze-drying, graphene oxide powder was obtained. The graphene oxide powder was then dispersed in sterile water to obtain a graphene mother liquor with a concentration of 5 mg / L (0.5%).

[0045] The aforementioned 0.5% graphene mother liquor (Engineering Research Center of Coal-based Ecological Carbon Sequestration Technology, Ministry of Education, Datong University, Shanxi Province) was diluted with water to a final concentration of 10 mg / L to obtain a graphene synergistic solution. This solution was then applied via drip irrigation to saline-alkali land planted with sorghum (Jinza 22) (experimental field of Shanxi Nongquan Technology Co., Ltd., Dabaideng Town, Yanggao County, Datong City, Shanxi Province). Each irrigation consisted of 3000 L of 10 mg / L graphene synergistic solution per acre, with an interval of 20 days between irrigations, for a total of four irrigations. During this period, field management measures such as fertilization, pesticide application, weeding, and thinning were consistent with the control group (water irrigation). Phenotypic images of sorghum seedlings at 3 months of age (after four irrigations) were taken using a camera (results are shown in the figure). Figure 1 As shown in Figure A), the results showed that graphene promotes the growth of sorghum in saline-alkali land.

[0046] Example 2

[0047] This embodiment provides a method for characterizing the graphene composition enriched in sorghum roots using scanning electron microscopy.

[0048] Sorghum root tissue treated with graphene-enhancing solution four times in Example 1 was sliced ​​into 1 mm thick cross sections using a microtome. After fixation with 2.5% glutaraldehyde and washing, the tissue was dehydrated using gradient ethanol solutions of 30%, 50%, 70%, 90%, and 100% concentrations. Natural drying followed by gold sputtering and graphene surface morphology observation were performed using a scanning electron microscope (SEM, TESCAN MAIA 3LMH). The morphology of graphene oxide enriched in sorghum roots is shown below. Figure 1 As shown in Figure B, the sorghum root system contains numerous relatively transparent, wrinkled, veil-like sheet structures with a large surface area similar to graphene oxide.

[0049] In addition, the graphene Raman spectra of sorghum roots treated with graphene-enhancing solution four times in Example 1 were measured using a Qontor Raman spectrometer (Renishaw, UK). The results are as follows: Figure 1 As shown in Figure C, the graphene in sorghum roots exhibits the typical characteristic peaks of graphene oxide, with a peak at 1500 cm⁻¹. -1 and 1750cm -1 The peaks between them represent the sp of carbon atoms. 2 Vibrations of a heterogeneous two-dimensional hexagonal lattice, at 1250 cm⁻¹. -1 and 1500cm -1 The peaks between are disordered SP 2 The vibration of carbon atoms.

[0050] Example 3

[0051] This embodiment shows the changes in the number, abundance, and diversity of rhizosphere microbial communities of sorghum crops in saline-alkali land after four treatments using graphene-enhanced liquid, as described in Example 1.

[0052] Three-month-old sorghum seedlings were collected, and rhizosphere soil samples were collected from the control group (water-irrigated) and the group treated with graphene synergist solution (as described in Example 1) after four irrigations using a destructive sampling method. Five samples were taken from each treatment group for replication. Soil microbial DNA was then extracted. The extraction, amplification, and sequencing process mainly included five steps: sample DNA extraction and testing, PCR amplification and purification, PCR product quantification and homogenization, sequencing library construction, and sequencing. The specific method is as follows: a soil DNA extraction kit (SPINeasy) was used. TMTotal microbial DNA was extracted from soil samples (SKU: 116530050). For bacteria, the V3-V4 variable region of the 16S rRNA gene was amplified by PCR using the forward primer 338F5′-ACTC-CTACGGGAGGCAGCAG-3′ and the reverse primer 806R 5′-GGACTACHVGGGTWTCTAAT-3′. For fungi, ITS1 was amplified by PCR using the forward primer ITS5F 5′-GGAAGTAAAAGTCGTAACAAGG-3′ and the reverse primer ITS2R5′-GCTGCGTTCTTCATCGATGC-3′. Sequencing library construction was performed by Beijing Biomarker Biotechnology Co., Ltd. For data processing, Trimmomatic v0.33 software was first used to filter the raw reads obtained from sequencing. Then, cutadapt 1.9.1 software was used to identify and remove primer sequences, resulting in clean reads free of primer sequences. The dada2 method in QIIME2 2020.6 was used for noise reduction, pairwise sequence splicing, and removal of chimeric sequences to obtain the final valid data. The dilution curve was constructed by randomly selecting a certain number of sequences from the sample, counting the number of species represented by these sequences, and plotting the sequence number against the number of species. This curve was used to verify whether the amount of sequencing data was sufficient to reflect the species diversity in the sample and indirectly reflect the species richness of the sample.

[0053] In Example 1, the dilution curves of rhizosphere bacteria and fungi based on OTU levels after irrigating sorghum crops four times with graphene-enhanced solution are shown below. Figure 2 As shown in Figures A and B, it can be seen that within a certain range, as the number of sequencing reads increases, the curve shows a sharp rise followed by a flattening, indicating that the species in this environment do not increase significantly with the increase in the number of sequencing reads. This indicates that the sample sequences meet the analytical requirements and can be used for data analysis.

[0054] In Example 1, the Venn diagram results of rhizosphere bacteria and fungi based on OTU levels after irrigating sorghum crops four times with graphene-enhanced solution are as follows: Figure 3 As shown in Figures A and B, the control group (water irrigation) had 8043 OTUs of bacteria after four irrigations of sorghum, while the group treated with graphene-enhanced solution in Example 1 had 11063 OTUs. Similarly, the control group (water irrigation) had 915 OTUs of fungi after four irrigations of sorghum, while the group treated with graphene-enhanced solution in Example 1 had 1253 OTUs. This indicates that the number of OTUs in the rhizosphere microorganisms of sorghum increased after irrigation with graphene-enhanced solution, further suggesting that this treatment promotes the species number and abundance of rhizosphere microorganisms in sorghum.

[0055] The bar charts showing the average OTU counts of rhizosphere bacteria and fungi after four applications of graphene-enhanced solution to sorghum crops in Example 1 are shown in Figures 4A and 4B, respectively. The results also indicate that the average OTU count of rhizosphere microorganisms in sorghum significantly increased after treatment with the graphene-enhanced solution. This further demonstrates that graphene treatment promotes the species number and abundance of rhizosphere microorganisms in sorghum.

[0056] Example 4

[0057] This embodiment provides the Alpha diversity index and Beta diversity (principal coordinate analysis) of the rhizosphere microbial community of sorghum crops treated with graphene-enhanced solution four times in Example 1.

[0058] Microbial diversity indices, abundance, principal coordinate analysis, and species composition analysis at different taxonomic levels were all performed on the Biocloud platform (https: / / www.biocloud.net / ) and R4.2.3 software. First, the Alpha diversity index was calculated using Mothur software (http: / / www.mothur.org / wiki / Calculators), and the inter-group differences in Alpha diversity were analyzed using the Wilcoxon rank-sum test. The Chao1 algorithm was used to estimate the number of OTUs in the community, reflecting species richness; the Shannon-Wiener index is used to estimate the diversity of microorganisms in the sample. The similarity of microbial community structure was tested using principal coordinate analysis (PCoA) with the Bray-Curtis distance algorithm, and the significance analysis used the PERMANOVA nonparametric test.

[0059] The results of the box plots of the Alpha diversity indices (Chao1 and Shannon) of rhizosphere bacteria and fungi after four applications of graphene-enhanced liquid to sorghum crops in Example 1 are as follows: Figure 5 As shown in Figures A and B, the results in the figure show that the Chao1 index and Shannon index of the rhizosphere microbial community of sorghum were significantly increased after the graphene-enhanced liquid was applied.

[0060] The PcoA results of rhizosphere bacteria and fungi after irrigating sorghum crops four times with graphene-enhanced solution are as follows: Figure 6As shown in Figures A and B, the results indicate a significant difference in microorganisms between the control group (water irrigation) and the groups treated with graphene-enhanced solution after four irrigations (bacteria P = 0.045; fungi P = 0.001). These results suggest that graphene influences the structure and diversity of the rhizosphere microbial community in sorghum.

[0061] Furthermore, an analysis of the changes in species composition based on the top 10 abundances at the phylum and genus levels was conducted. The bar chart results of the species composition analysis of rhizosphere microorganisms at the phylum and genus levels after sorghum crop irrigation with graphene-enhanced liquid are shown below. Figure 7 As shown in the figure, the results indicate that irrigation with graphene-enhanced solution can promote the abundance of potential beneficial bacteria in the rhizosphere microbial community of sorghum, including Pseudomonas, Bacillus, Mortierella, and Trichoderma. This further suggests that the promotion of sorghum growth in saline-alkali soil by graphene-enhanced solution depends on changes in the rhizosphere microbial community structure.

[0062] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of graphene in improving species diversity of crop rhizosphere microbial communities.

2. The application as described in claim 1, characterized in that, The graphene is graphene oxide. Preferably, graphene is applied to the soil in the form of graphene sol fertilizer. Preferably, the graphene concentration in the graphene sol fertilizer is 5–20 mg / L.

3. The application as described in claim 1 or 2, characterized in that, The graphene was prepared by a two-step electrochemical method using flexible graphite paper and platinum foil as the cathode and anode, respectively.

4. Application of graphene in the preparation of crop growth fertilizers.

5. The application as described in claim 4, characterized in that, The concentration of graphene in the fertilizer is 5–20 mg / L.

6. A fertilizer, characterized in that, The fertilizer comprises graphene as described in any one of claims 1-3.

7. The fertilizer as described in claim 6, characterized in that, The concentration of graphene in the fertilizer is 5–20 mg / L.

8. The use of the fertilizer of claim 6 or 7 in improving the species diversity of crop rhizosphere microbial communities and / or promoting crop growth.

9. A method for improving species diversity in crop rhizosphere microbial communities, characterized in that, This includes applying the graphene according to any one of claims 1-3 and / or the fertilizer according to claim 6 or 7 to the soil in which crops grow.

10. A method for promoting crop growth, characterized in that, This includes applying the graphene according to any one of claims 1-3 and / or the fertilizer according to claim 6 or 7 to the soil in which crops grow.

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