Nano-material complex microbial inoculant and application thereof

Abstract

The invention discloses a nano-material complex microbial inoculant and application thereof, and relates to the technical field of microorganisms. The invention discloses application of a nano-material complex microbial inoculant in promoting growth of overground parts of plants, and results show that compared with a treatment group singly inoculated with YH-18, the plant height, leaf area, ground diameter, fresh weight, dry weight and chlorophyll content of tomato seedlings subjected to complex treatment are all remarkably increased. The invention discloses application of a nano-material complex microbial inoculant in promoting plant root growth, and results show that the root length, the root surface area and the root tip number are respectively increased by 35.76%, 13.19% and 57.84% compared with those of single inoculation of YH-18. After compounding and inoculating, the content increase of rapidly available potassium, rapidly available phosphorus and hydrolytic nitrogen in the soil is effectively promoted, the content of organic matters in the soil is remarkably increased, and the nutrient structure of the soil is improved; pseudomonas is significantly enriched through composite inoculation, and the abundance of TRA3-20 is significantly reduced.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a nanomaterial composite bacterium agent and its application. Background Art

[0002] Bacillus velezensis YH-18, as a pleiotropic Gram-positive bacterium with functions of biocontrol, growth promotion and pollutant degradation, has shown significant application value in the fields of agricultural biological control and soil remediation. Its core advantage lies in its ability to secrete lipopeptide antibiotics (such as surfactin, iturin), hydrolases (protease, cellulase, etc.) and siderophores and other active metabolites, and to achieve disease prevention and control and soil ecological function restoration by directly antagonizing plant pathogenic bacteria, degrading organic pollutants (such as pesticide residues, petroleum hydrocarbons) or inducing plant systemic resistance. However, in complex environments (such as extreme pH, ultraviolet radiation, indigenous microbial competition), free microorganisms generally face bottleneck problems such as low colonization efficiency, unstable metabolic activity, and inhibited functional expression, which seriously restrict their large-scale application efficiency. For example, in arid or saline-alkali soils, the spore germination rate of YH-18 may decrease by more than 30% due to water stress; in heavy metal polluted environments, the extracellular polymeric substance (EPS) synthesis pathway of YH-18 is easily interfered by the toxicity of metal ions, resulting in hindered biofilm formation, and thus weakening the adsorption-degradation ability of pollutants. Therefore, how to improve the environmental tolerance and functional persistence of microorganisms through technological innovation has become a key direction to break through the existing technical barriers.

[0003] Carbon-based nanomaterials (such as graphene oxide, carbon nanotubes, carbon quantum dots and biomass-derived carbon materials) provide a new idea for the development of microbial enhancement technology due to their unique physical and chemical properties (high specific surface area, tunable surface functional groups, excellent mechanical strength and electron transfer ability). Research shows that carbon-based materials can form functional complementarity with microorganisms through multiple mechanisms: on the one hand, their porous structure and surface active sites can serve as physical carriers for microbial loading, and fix the bacterial cells by means of π-π stacking, electrostatic adsorption or covalent bonding, reducing cell loss caused by environmental stress; on the other hand, the catalytic properties (such as peroxidase-like activity) or conductivity of the materials themselves can mediate the electron exchange between microorganisms and the external environment, thereby activating specific metabolic pathways.

[0004] However, the compounding of microorganisms and carbon-based nanomaterials is not a simple physical mixture, and its interfacial interaction mechanism, long-term ecological safety, and engineering application feasibility still need to be systematically studied. For example, the surface properties of carbon-based materials (such as functional group type, Zeta potential, hydrophobicity) may have significant differences in the effects on microbial adhesion behavior and metabolic activity: positively charged amino-functionalized carbon nanotubes may disrupt the cell membrane integrity of YH-18 through electrostatic interaction, resulting in the leakage of intracellular substances; while hydroxylated carbon materials are more easily wrapped by bacterial biofilms due to enhanced hydrophilicity, forming a stable symbiotic interface. Therefore, it is particularly important to select appropriate carbon-based nanomaterials and appropriate concentrations for compounding with microbial agents. In addition, the biotoxicity and environmental fate of nanomaterials cannot be ignored: some studies have shown that unmodified carbon nanotubes may damage the non-target soil microbial community structure through physical puncture or oxidative stress, while small-sized carbon quantum dots have the risk of bioaccumulation through the food chain. While paying attention to the functional role of the compound microbial agent, it is still necessary to pay attention to its impact on the soil environment.

[0005] Therefore, to develop a compound microbial agent system with high efficiency, stability, and ecological safety, it is necessary to analyze the ternary interaction mechanism of "material-microorganism-environment" from multiple scales of molecule-cell-community. In this way, the growth rate, colonization success rate, and plant growth promotion efficiency of the compound microbial agent in the environment can be improved, providing a theoretical innovation and research basis for the green production and sustainable development of agriculture and forestry. Summary of the Invention

[0006] Aiming at the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide a nanomaterial compound microbial agent. Another technical problem to be solved by the present invention is to provide an application of the nanomaterial compound microbial agent for realizing plant disease resistance and growth promotion.

[0007] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A nanomaterial compound microbial agent is composed of the compounding of Bacillus velezensis YH-18 and a novel nano-fertilizer absorption promoter X7.

[0009] The preparation method of the nanomaterial compound microbial agent is to mix the novel nano-fertilizer absorption promoter X7 and the bacterial suspension of Bacillus velezensis YH-18 according to a volume ratio of 1:2000 to obtain the nanomaterial compound microbial agent.

[0010] The application of the nanomaterial compound microbial agent in regulating the growth of the above-ground part of plants.

[0011] The application of the nanomaterial compound microbial agent in regulating the growth of plant roots.

[0012] The application of the nanomaterial compound microbial agent in regulating the chlorophyll content of plants.

[0013] Application of nanomaterial composite microbial agent in regulating soil organic matter content.

[0014] Application of nanomaterial composite microbial agent in regulating available potassium, available phosphorus and hydrolyzable nitrogen contents in soil.

[0015] Application of nanomaterial composite microbial agent in regulating bacterial diversity and richness in soil.

[0016] Application of nanomaterial composite microbial agent in enriching soil microorganisms, wherein the soil microorganisms are Pseudoxanthomonas and Pseudomonas.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1) The nanomaterial composite microbial agent prepared in this application was inoculated into tomato seedlings, significantly promoting the growth of the above-ground part of tomato seedlings. The results show that compared with the treatment group inoculated with YH-18 alone, the plant height and leaf area of tomato seedlings increased by 11.84% and 28.3% respectively after the compound treatment, the ground diameter increased by 21.33%, and the fresh weight and dry weight increased by 38.75% and 33.33% respectively; the SPAD value reflecting the chlorophyll content increased by 10.66% compared with the control group.

[0019] 2) The nanomaterial composite microbial agent prepared in this application was inoculated into tomato seedlings, significantly promoting the root growth of tomato seedlings. The results show that the compound treatment X7+YH-18 has the best growth-promoting effect on tomato roots, and the root length, root surface area and root tip number increased by 35.76%, 13.19% and 57.84% respectively compared with the treatment of inoculating YH-18 alone.

[0020] 3) The nanomaterial composite microbial agent prepared in this application significantly promoted the increase in the content of organic matter in the soil. The results show that after the compound inoculation, it effectively promoted the increase in the contents of available potassium, available phosphorus and hydrolyzable nitrogen in the soil, and significantly increased the content of organic matter in the soil, improving the soil nutrients; the compound inoculation significantly enriched Pseudomonas and significantly reduced the abundance of the TRA3-20 genus. Description of the Drawings

[0021] Figure 1 It is the TEM morphology diagram of the novel nano-fertilizer absorption promoter X7;

[0022] Figure 2 It is the diagram of the effect of different concentrations of the novel nano-fertilizer absorption promoter X7 on the growth of YH-18;

[0023] Figure 3 It is the diagram of the effect of different concentrations of the novel nano-fertilizer absorption promoter X7 on the biofilm formation of YH-18;

[0024] Figure 4 Effect diagram of different inoculation treatments on the growth of tomato seedlings (A is plant height; B is ground diameter; C is fresh weight; D is dry weight; E is SPAD value; F is leaf area);

[0025] Figure 5 Morphological diagram of tomato seedlings under different inoculation treatments;

[0026] Figure 6 Effect diagram of different inoculation treatments on soil nutrients (A is organic matter; B is available potassium; C is hydrolyzable nitrogen; D is available phosphorus);

[0027] Figure 7 Diagram of the top 15 bacterial taxa with the largest number of bacteria at the phylum level (A is a stacked column chart of the top 15 bacterial taxa with the largest number of bacteria at the phylum level; B is the relative abundance of Bdellovibrionota in different treatments; C is Desulfobacterota in different treatments);

[0028] Figure 8 Diagram of the top 15 bacterial taxa with the largest number of bacteria at the genus level (A is a stacked column chart of the top 15 bacterial taxa with the largest number of bacteria at the genus level; B is the relative abundance of Pseudoxanthomonas in different treatments; C is the relative abundance of Pseudomonas in different treatments; D is the relative abundance of the TRA3-20 genus in different treatments);

[0029] Figure 9 Principal coordinate analysis diagram of the total bacterial 16S rRNA gene based on the weighted similarity index at 97% identity (operational taxonomic unit level) (A, PC1 and PC2 explain 11.53% and 9.18% of the variance respectively) and Venn diagram between different treatments (B). Specific implementation manner

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well-known to those skilled in the art.

[0031] The Bacillus velezensis strain YH-18 used in this application was previously isolated and screened from the branches of cherry blossoms by the Forest Protection Laboratory of Nanjing Forestry University (Jiang Mingming, 2016; Wei Danping, 2020).

[0032] The novel nano-fertilizer absorption promoter X7 used in this application (provided by Shenzhen Yuewang Energy Saving Technology Service Co., Ltd.) is a molecular hydrated solution of derivatives based on the framework of brown humic acid / fulvic acid, and is supported on a nano-carrier. Compared with traditional humic acid molecules, this derivative molecule removes the parts that are easily degraded in the environment, such as the original sugars and amino acid residues, and replaces them with a polycyclic polyphenol framework with good biological activity as the main body, greatly extending the half-life of this molecule in the environment. The novel nano-fertilizer absorption promoter X7 is a dark brown liquid, odorless, with a pH of 6 - 6.5, C>95.85%, H<1%, N<1%, S<1%, K: (0.75 - 2.32%), Fe<0.05%, Mn<0.01%, Ni<0.01%, and it is a carbon-based nano-material.

[0033] Example 1

[0034] 1. The effect of the novel nano-fertilizer absorption promoter X7 on the growth of YH-18

[0035] After culturing the YH-18 strain in an artificial shaking incubator at 28°C and 200 rpm for 24 h, the culture solution was centrifuged at 4°C and 10000 r / min for 10 min to collect the bacterial cells, washed twice with 0.85% normal saline, and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL seed solution for standby. Subsequently, the YH-18 seed solution was inoculated into LB medium supplemented with different concentrations of the novel nano-fertilizer absorption promoter X7 at 0.2%, and the growth of the YH-18 strain within 48 h was observed using a fully automatic microbial growth curve analyzer. The liquid novel nano-fertilizer absorption promoter X7 was characterized by TEM.

[0036] The results are as Figure 1 shown. The liquid novel nano-fertilizer absorption promoter X7 was characterized by TEM. The particle size was about 10 nm, and it was basically spherical. The Zata potential was -50 mV. The average particle size of its aggregated particles was measured to be 1052 nm by a BT-90 nano laser particle size distribution analyzer.

[0037] The results are as Figure 2 shown. After the novel nano-fertilizer absorption promoter X7 at different concentrations was compounded with YH-18, the high-concentration (1 / 10 addition amount) novel nano-fertilizer absorption promoter X7 significantly inhibited the growth of YH-18. The medium-concentration (1 / 1300 - 1 / 50 addition amount) novel nano-fertilizer absorption promoter X7 slightly reduced the growth of the strain, but the impact was not significant. The low-concentration (1 / 1700 addition amount) novel nano-fertilizer absorption promoter X7 significantly promoted the growth of the strain in the later stage of strain growth and delayed the decline period of YH-18.

[0038] 2. Effects of the new nano-fertilizer absorption promoter X7 on the YH-18 biofilm

[0039] Inoculate the YH-18 seed solution into a sterile 24-well plate containing LB medium with different concentrations of the new nano-fertilizer absorption promoter X7 at an inoculation amount of 1%. After culturing in a shaker at 28 °C and 200 r / min for 24 hours, transfer it to a constant temperature medium at 28 °C for static culture for two days. Then remove the planktonic bacteria, gently wash with PBS to remove the non-adherent bacteria, then add methanol to fix the biofilm, stain with 0.1% crystal violet for 15 minutes. Wash off the excess dye, dissolve the stained biofilm with 33% acetic acid, and measure the OD 590 Absorbance.

[0040] The results are as Figure 3 shown. The new nano-fertilizer absorption promoter X7 at higher concentrations (1 / 1000 - 1 / 100 addition amount) inhibits the biofilm formation of YH-18, and the new nano-fertilizer absorption promoter X7 at lower concentrations (1 / 2000 - 1 / 1500 addition amount) is more suitable for compounding with the YH-18 strain.

[0041] Example 2

[0042] Centrifuge the culture solution of the YH-18 strain after shaking culture in an artificial shaking incubator at 28 °C and 200 rpm for 24 h at 4 °C and 10000 r / min for 10 min to collect the bacterial cells. Select plump tomato seeds without pests and diseases. After rinsing with clear water, soak the tomato seeds in a 5% NaClO solution for 5 minutes. After soaking, wash the tomato seeds with sterile water 3 - 5 times. Sow the disinfected tomato seeds into pots filled with soil, sowing several seeds in each pot. After the seeds germinate and grow stably, thin the seedlings according to the situation, and keep 1 seedling in each pot. For the CK treatment, inoculate 30 mL of sterile water into the rhizosphere of each tomato seedling. For the YH-18 treatment, inoculate 30 mL of YH-18 suspension (1×10 8 CFU / mL) into the rhizosphere of each tomato seedling. For the X7 treatment, inoculate 30 mL of the new nano-fertilizer absorption promoter X7 solution diluted 2000 times into the rhizosphere of each tomato seedling. The compound treatment of X7 + YH-18 is to inoculate 30 mL of the X7 and YH-18 complex bacterial agent solution (the volume ratio of the new nano-fertilizer absorption promoter X7 to the YH-18 bacterial suspension is 1:2000) (bacterial suspension concentration: 1×10 8 CFU / mL). Each treatment includes 16 pots and 4 replicates.

[0043] Fifteen days after inoculation, the plant height of tomato seedlings was measured with a tape measure, and the plant ground diameter was measured with a vernier caliper. Three leaves without pests, diseases, and mechanical damage and at the same position around the fruits were selected from each seedling, and the chlorophyll content in the leaves (expressed by SPAD value) was measured with a portable chlorophyll meter SPAD-502. The dry weight and fresh weight were weighed with an electronic balance. A small square of 1 cm×1 cm was drawn on a white cardboard, and the leaf and the small square were placed in the same plane for taking pictures. The pixels of the small square and the leaf were read using PS software, and the leaf area = leaf pixels / square pixels × 1 cm 2 The roots of tomato seedlings in each treatment after 15 days were rinsed with water, dried with filter paper, and the roots of tomato seedlings were scanned with a root scanner to obtain the total root length, root volume, root surface area, number of forks, and number of root tips.

[0044] 1. Effects on the growth of tomato seedlings

[0045] The results are as Figure 4 shown. Compared with the control, after inoculation with YH-18 and the new nano-fertilizer absorption promoter X7, the plant height and leaf area of tomato seedlings were significantly increased. The plant height increased by 20.63% and 16.67% compared with the control, and the leaf area increased by 102.96% and 59.38% compared with the control. The compound treatment had the best effect on increasing the plant height and leaf area, which was significantly higher than the other three treatments, and was also increased by 11.84% and 28.3% compared with the treatment group inoculated with YH-18 alone. After inoculation with the new nano-fertilizer absorption promoter X7, there was no effect on the growth of the plant ground diameter. After inoculation with YH-18, although there was a certain promoting effect, the promoting effect on the ground diameter was not significant. After the compound treatment, the plant ground diameter could be significantly increased, which was increased by 21.33% compared with the control. After inoculation with the new nano-fertilizer absorption promoter X7, although there was a certain increase in the fresh weight and dry weight of tomato seedlings, the promoting effect was not significant. After inoculation with YH-18, the promotion effect was significant, which increased by 66.67% and 50% compared with the control. The compound treatment still had the best growth-promoting effect, which was increased by 38.75% and 33.33% compared with the treatment of applying YH-18 alone. The SPAD value reflecting the chlorophyll content also increased after several different inoculation treatments. The treatment of inoculating X7 had an increasing effect but not significant. The treatments of inoculating YH-18 and the compound treatment both significantly increased the SPAD value of tomato leaves, which increased by 13.07% and 10.66% compared with the control respectively.

[0046] 2. Effects on the root growth of tomato seedlings

[0047] The results are as Figure 5As shown in Table 1, compared with the control, inoculation with the new nano-fertilizer absorption promoter X7 had a certain promoting effect on the tomato roots. Specifically, it increased the root length and the number of root forks of tomatoes to a certain extent, but the promotion effect was not significant. The promotion effects on root surface area and the number of root tips were significant, increasing by 65.99% and 256.09% respectively compared with the control. After inoculation with the YH-18 microbial inoculant, the promotion effects on the root length, root surface area, the number of root tips and the number of root forks of tomato roots were all significant, increasing by 140.8%, 76.91%, 218.13% and 106.71% respectively compared with the control, and the treatment effect of inoculating YH-18 was better than that of inoculating X7. The compound treatment X7+YH-18 had the best growth-promoting effect on tomato roots. On the basis of the treatment of inoculating YH-18, it also significantly increased the root length, root surface area and the number of root tips, increasing by 35.76%, 13.19% and 57.84% respectively compared with inoculating YH-18 alone.

[0048] Table 1 Effects of different inoculation treatments on the root growth of tomato seedlings

[0049]

[0050]

[0051] Example 3

[0052] On the basis of the treatment in Example 2, 15 days after inoculation, the surface soil of the plants was removed, and the rhizosphere soil was collected. One sample was mixed from every 4 pots, and 4 replicates were taken for each treatment. The soil samples were stored in sterilized and sealed polyethylene bags. A part of the samples was freeze-dried for soil property determination. Another part of the rhizosphere soil was stored at -80 °C for DNA extraction and high-throughput sequencing analysis.

[0053] After air-drying the soil, the hydrolyzable nitrogen content of the soil was determined by the alkali-hydrolysis diffusion method, the available potassium content was determined by the ammonium acetate extraction method, the available phosphorus was determined by the double-acid extraction-molybdenum antimony anti-colorimetry method, and the soil organic matter content was determined by the potassium dichromate oxidation-external heating method.

[0054] Take 0.5 g of fresh soil and extract total soil DNA using the Power Soil DNA Isolation Kit (MOBIO Laboratories Inc, Carlsbad, CA, USA). Determine the concentration and purity of the DNA using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific., Waltham, MA, USA), and detect the integrity of the DNA using 1% (m / v) agarose gel electrophoresis. Then store the extracted DNA at -20 °C for future analysis. Use an equal amount of DNA extracted from each sample as the amplification template. Use 343F (5'-TACGRAGGCAGCAG-3') and 798R (5'-AGGGTATCTAATCCT-3') to amplify the bacterial 16S rRNA gene targeting the V3-V4 variable region. Detect the PCR products using electrophoresis, and purify them using Agencourt AMPure XP beads (Beckman Coulter Co, USA) after detection. Quantify using the Qubit dsDNA Assay Kit. Then, after adjusting the concentration, perform 16S rRNA sequencing using the Illumina Miseq platform (Illumina Inc., San Diego, CA; OE Biotech Company; Shanghai, China).

[0055] Evaluate the microbial community diversity using the Shannon and Simpson indices, and estimate the microbial community richness at the OTU level using Chao1 and Ace. Calculate the Unifrac distance matrix using QIIME software, and perform unweighted Unifrac principal coordinate analysis (PCoA) and phylogenetic tree construction. Use canonical principal coordinates analysis (CAP) and PERMANOVA to visually detect significant differences in the community structure and functional structure.

[0056] 1. Effects on soil nutrients

[0057] The results are as Figure 6 shown. Compared with the control, the available phosphorus and hydrolyzable nitrogen in the soil increased significantly after inoculating with the new nano-fertilizer absorption promoter X7. Inoculating the YH-18 microbial inoculant alone had a certain promoting effect on the available potassium, available phosphorus, and hydrolyzable nitrogen in the soil. After compound inoculation, it effectively promoted the increase in the contents of available potassium, available phosphorus, and hydrolyzable nitrogen in the soil, and significantly increased the content of organic matter in the soil, improving the soil nutrients.

[0058] 2. Effects on soil flora

[0059] The results are shown in Table 2. Different inoculation treatments had no significant effect on the bacterial diversity and richness of plant roots, indicating that the inoculation treatment had little effect on the rhizosphere microecology of plants.

[0060] Table 2 Effects of different inoculation treatments on the α-diversity of rhizosphere soil bacterial communities

[0061]

[0062] The results are as Figure 7 shown. Among the dominant phyla, the single inoculation of the new nano-fertilizer absorption promoter X7 and the combined inoculation both significantly reduced Bdellovibrionota. The single inoculation of YH-18 significantly enriched Desulfobacterota compared with the single inoculation of the new nano-fertilizer absorption promoter X7.

[0063] The results are as Figure 8 shown. Among these dominant genera, the single inoculation of the new nano-fertilizer absorption promoter X7 significantly enriched Pseudoxanthomonas and reduced the abundance of the TRA3-20 genus to a certain extent. The single inoculation of YH-18 treatment extremely significantly enriched Pseudomonas, enriched Pseudoxanthomonas to a certain extent, and reduced the abundance of the TRA3-20 genus. The combined inoculation significantly enriched Pseudomonas and significantly reduced the abundance of the TRA3-20 genus.

[0064] 3. Community composition at the OTU level

[0065] The results are as Figure 9 shown. The PERM ANOVA pairwise test confirmed that there was a significant difference between the treatment of single inoculation of the new nano-fertilizer absorption promoter X7 and the control treatment, while there was no significant difference between other treatments (p < 0.05). The Venn diagram showed the similarity and difference in the bacterial community composition among different treatments. After 15 days of inoculation, the four treatments shared OTUs (2814 OTUs). The CK treatment had the fewest unique OTUs (325 OTUs), and the YH-18 treatment had a high number of unique OTUs (606 OTUs). The single inoculation of X7 and the combined inoculation had a medium number of unique OTUs (435 OTUs and 490 OTUs).

[0066] In summary, the novel nano-fertilizer absorption promoter X7 at low concentration delays the growth decline period of Bacillus velezensis YH-18 and promotes the formation of biofilms by the strain. After the combined use of the novel nano-fertilizer absorption promoter X7 at low concentration and the strain, it promotes plant growth and improves soil nutrient conditions. Moreover, the effect of the composite microbial agent is significantly better than that of applying only one microbial agent alone.

[0067] The above description is illustrative rather than restrictive to the present invention. Those of ordinary skill in the art understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A nanomaterial composite microbial agent, characterized in that, It is composed of the compounding of Bacillus velezensis YH-18 and the novel nano-fertilizer absorption promoter X7.

2. The preparation method of the nano-material composite bacterial agent according to claim 1, characterized in that, The novel nano-fertilizer absorption promoter X7 and the bacterial suspension of Bacillus velezensis YH-18 are mixed according to a volume ratio of 1:2000 to prepare a nano-material compound microbial agent.

3. Application of the nano-material compound microbial agent described in claim 1 in regulating the growth of the above-ground part of plants.

4. Application of the nano-material compound microbial agent described in claim 1 in regulating the growth of plant roots.

5. Application of the nano-material compound microbial agent described in claim 1 in regulating the chlorophyll content of plants.

6. Application of the nano-material compound microbial agent described in claim 1 in regulating the content of soil organic matter.

7. Application of the nano-material compound microbial agent described in claim 1 in regulating the contents of available potassium, available phosphorus and hydrolyzable nitrogen in soil.

8. Application of the nano-material compound microbial agent described in claim 1 in regulating the bacterial diversity and richness in soil.

9. Application of the nano-material compound microbial agent described in claim 1 in enriching soil microorganisms.

10. The application according to claim 9, characterized in that, The soil microorganisms are Pseudoxanthomonas and Pseudomonas.

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