Burkholderia strain-based coat-coated vetiver grass seeds, and preparation method and application thereof

Abstract

The invention belongs to the technical field of ecological environment protection, and particularly relates to vetiver grass seeds coated by burkholderia strains, a preparation method and application. The preparation method comprises the following steps: adding burkholderia into a sterilized LB culture medium according to the inoculum size of 1%, carrying out constant-temperature culture, then centrifuging to remove supernate, and carrying out sterile normal saline resuspension to obtain a resuspension; s2, dissolving polyvinyl alcohol in a solvent, stirring and cooling to obtain a polyvinyl alcohol solution, and mixing the polyvinyl alcohol solution with the resuspension in the S1 to obtain a coating agent; s3, adding the coating agent in the S2 into spraying equipment, adjusting the atomization pressure, the atomization particle size, the spraying rate and the seed transmission rate, and coating the surfaces of the seeds with the coating agent to obtain coated seeds. Through the preparation method, exogenous functional microorganisms can be successfully colonized in rhizosphere soil or plants, and the remediation efficiency can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of ecological environment protection, and specifically relates to a vetiver seed coated with Burkholderia seed coating, a preparation method and an application thereof. Background Art

[0002] Inorganic pollution in my country's cultivated soil is mainly cadmium (Cd), which is absorbed by crop roots and enters the plant body, and is transmitted through the food chain, eventually posing a serious threat to human health. Phytoremediation is a technology for remediating heavy metal pollution in cultivated soil that has been widely studied and has broad application prospects, but there are also some factors that limit the efficiency of phytoremediation.

[0003] Phytoremediation refers to the use of plants (such as vetiver, centipede grass, pokeweed, etc.) to reduce the concentration or toxic effects of pollutants in the environment (soil, water). It has the advantages of being economical, efficient, environmentally friendly, energy-saving and environmentally friendly. Among them, the plant extraction technology has the greatest development potential. However, the efficiency of plant extraction is affected by many factors, such as the total amount and effective content of heavy metals, soil physical and chemical properties, plant species and strains, plant biomass, etc. The existing technology research and development mainly focuses on the problems of small biomass of enriched plants, long growth cycle, low migration coefficient, low effective Cd content in soil, and low colonization efficiency of exogenous microorganisms, forming a series of technical methods to improve the efficiency of plant extraction, such as genetic modification and combined remediation technology (microorganism-plant combined remediation, physical / chemical method-plant combined remediation). Compared with microorganism-plant combined remediation, other remediation technologies have practical problems such as instability, easy to cause secondary environmental pollution, and high cost.

[0004] Existing "microbe-plant joint remediation" mostly uses exogenous addition of functional microbial agents to the rhizosphere / rhizosphere soil. For example, after sowing seeds or transplanting rice, the microbial agents will be cultured to a certain concentration (10 6 CFU / mL), and after centrifugation, the bacteria were resuspended and added to the target cultivated soil. Due to the competition and predation between exogenous strains and indigenous bacteria, the colonization rate of exogenous strains in the rhizosphere of restored plants is low, which makes it difficult to improve the efficiency of plant extraction.

[0005] Among the existing technologies, microbial-plant remediation technology is considered to be the most promising joint remediation technology due to its high efficiency, sustainability and ability to eradicate heavy metals. Although the existing technology can improve the efficiency of plant remediation to a certain extent, it has the following objective disadvantages: due to ecological relationships such as competition and predation, the functional microorganisms directly added to the rhizosphere soil have a low colonization rate in the rhizosphere microenvironment, so they cannot play a long-term promoting role, and thus it is difficult to give full play to the advantages of the "microbial-plant joint remediation" technology; exogenous functional microorganisms are limited by the amount used, and their functional metabolites or signal molecules are difficult to interact with plants in a long-term and effective manner; existing application technologies mostly use live bacterial liquids, which are difficult to store and are not suitable for long-distance and large-area application, and have operational defects. Summary of the invention

[0006] In view of the shortcomings and defects in the prior art, the present invention provides a method for preparing vetiver seeds coated with Burkholderia spp., comprising the following steps:

[0007] S1: Adding Burkholderia to a sterilized LB medium at a 1% inoculum volume, culturing at a constant temperature, centrifuging, removing the supernatant, and resuspending the cells in sterile physiological saline to obtain a resuspended liquid;

[0008] S2: dissolving polyvinyl alcohol in a solvent, stirring and cooling to obtain a polyvinyl alcohol solution, and mixing it with the resuspended solution in S1 to obtain a coating agent;

[0009] S3: Add the coating agent in S2 to the spray equipment, adjust the atomization pressure, atomization particle size, spraying rate, and seed transmission rate, and apply the coating agent on the surface of the seeds to obtain coated seeds.

[0010] Furthermore, the LB medium described in S1 includes 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, and a pH of 7.0 to 7.1.

[0011] Furthermore, the constant temperature culture described in S1 is 37° C., the speed is 120 rpm, and the culture time is 48 h.

[0012] Furthermore, the centrifugation speed in S1 is 4000 rpm and the centrifugation time is 5 min.

[0013] Furthermore, the degree of polymerization of the polyvinyl alcohol described in S2 is 500-2500.

[0014] Furthermore, the ratio of the polyvinyl alcohol solution and the resuspension described in S2 is 1:1.

[0015] Furthermore, the atomization pressure described in S3 is 1-1.5 bar, the atomization particle size is 80-150 μm, the spraying rate is 200-1000 mL / h, and the seed transmission rate is 0.5-1 m / s.

[0016] A Burkholderia-based seed coating for vetiver seeds.

[0017] An application of vetiveria seeds coated with Burkholderia seed coating, wherein the vetiveria seeds coated with Burkholderia seed coating are used in the preparation of plant seeds for treating Cd pollution.

[0018] Beneficial Effects

[0019] The preparation method of vetiver seeds coated with Burkholderia provided by the present invention, compared with the existing microorganism-plant joint remediation operation technology, the present invention makes the functional microorganism-Burkholderia into a coating agent, and improves the colonization efficiency of functional microorganisms in the rhizosphere and inside the plant in the form of seed coating on vetiver seeds; the existing microorganism-plant joint remediation technology is to directly add the functional microorganism liquid to the seed / plant rhizosphere soil. In the middle and late stages of the remediation stage, the survival rate and colonization efficiency of functional microorganisms in the rhizosphere soil of plants are almost 0%, while the method proposed by the present invention can increase the survival rate and colonization rate by more than 35%. Even at the end of the remediation period, a large number of Burkholderia can still be detected in the plants and rhizosphere soil.

[0020] Through the preparation method of vetiver seeds coated with Burkholderia provided by the present invention, the present invention successfully colonizes exogenous functional microorganisms in rhizosphere soil or plants, which helps to efficiently promote vetiver to extract heavy metal Cd in soil and improve the remediation efficiency; this is an effective measure for inoculation and improving the efficiency of Cd extraction by combined remediation technology for the existing method of adding exogenous microorganisms. In the contaminated soil, compared with the treatment group (Cd5, Cd10, Cd15) directly adding functional bacteria to the soil, the Cd content of the aboveground and underground parts of the seed coating treatment group (Cd5B, Cd10B, Cd15B) increased by 36.56% to 39.66% and 25.97% to 130.47%, respectively. In addition, the Cd accumulation capacity (BCF-shoots, BCF-roots) and migration capacity (TF) of Vetiver were also improved in the seed coating treatment group. The maximum values ​​of the three indicators appeared in Cd5B (2.74), Cd15B (21.59) and Cd5B (0.14) in the seed coating treatment group. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0022] Figure 1 The relative abundance change diagram of Burkholderia of the present invention in soil at 1d, 5d and 10d under different addition modes;

[0023] Figure 2 This is a graph showing the Cd content in different parts of Vetiveria zizyphus in different treatment groups of the present invention;

[0024] Figure 3 This is a diagram showing the Cd enrichment coefficients of different parts of Vetiveria zizanioides in different treatment groups of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with embodiments 1 to 5 of the present invention and the attached Figures 1 to 3 , the technical solution of the present invention is clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] S1: First, culture the microorganisms. Add 1% of the Burkholderia to 1000 mL of sterilized LB medium (10.0 g of tryptone, 5.0 g of yeast extract powder, and 10.0 g of sodium chloride per liter of medium, and adjust the pH to 7.0). Incubate the culture in an incubator at a constant temperature of 37°C, 120 rpm for 48 h. Centrifuge the culture at 4000 rpm for 5 min using a high-speed centrifuge. Remove the supernatant and resuspend the cells to 2*10 6 CFU / mL concentration level;

[0028] S2: Then prepare the coating agent, dissolve 100g of polyvinyl alcohol (PVA) with a molecular weight of 30000g / mol and a degree of polymerization of 500 in 1000ml of warm water at 50℃, and stir thoroughly until it is completely dissolved; after cooling the PVA solution to below 37℃, mix it thoroughly with 1000ml of the bacterial resuspension prepared in S1 at a ratio of 1:1 to obtain 5% PVA and 1*10 6 CFU / mL of the strain coating agent; stored in a closed and cool environment, try to prepare it before use;

[0029] S3: Add the mixed microbial coating agent prepared in S2 to the spray equipment, select an air atomization spray nozzle, and adjust the atomization pressure to 1 bar, ensure that the atomized particle size is within the range of 80 μm, control the spraying rate at 200 mL / h, and the seed transmission rate at 0.5 m / s, so that the coating agent is evenly coated on the clean seed surface; obtain a vetiver seed coated with Burkholderia seed coating, and store the coated seeds in a dry and cool place for future use.

[0030] Example 2

[0031] S1: First, culture the microorganisms. Add 1% of the Burkholderia to 1000 mL of sterilized LB medium (10.0 g of tryptone, 5.0 g of yeast extract powder, and 10.0 g of sodium chloride per liter of medium, and adjust the pH to 7.1). Incubate the culture in an incubator at a constant temperature of 37°C, 120 rpm for 48 h. Centrifuge the culture at 4000 rpm for 5 min using a high-speed centrifuge. Remove the supernatant and resuspend the cells to 2*10 6 CFU / mL concentration level;

[0032] S2: Then prepare the coating agent, dissolve 100 g of polyvinyl alcohol (PVA) with a molecular weight of 70000 g / mol and a degree of polymerization of 2500 in 1000 ml of warm water at 50°C, and stir thoroughly until completely dissolved; after cooling the PVA solution to below 37°C, mix it thoroughly with 1000 mL of the bacterial resuspension prepared in S1 at a ratio of 1:1 to obtain 5% PVA and 1*10 6 CFU / mL of the strain coating agent; stored in a closed and cool environment, try to prepare it before use;

[0033] S3: Add the mixed microbial coating agent prepared in S2 to the spray equipment, select an air atomization spray nozzle, and adjust the atomization pressure to 1.5 bar, ensure that the atomized particle size is within the range of 150 μm, control the spraying rate at 1000 mL / h, and the seed transmission rate at 1 m / s, so that the coating agent is evenly coated on the clean seed surface; obtain a vetiver seed coated with Burkholderia seed coating, and store the coated seeds in a dry and cool place for future use.

[0034] Example 3

[0035] S1: First, culture the microorganisms. Add 1% of the Burkholderia to 1000 mL of sterilized LB medium (10.0 g of tryptone, 5.0 g of yeast extract powder, and 10.0 g of sodium chloride per liter of medium, and adjust the pH to 7.1). Incubate the culture in an incubator at a constant temperature of 37°C, 120 rpm for 48 h. Centrifuge the culture at 4000 rpm for 5 min using a high-speed centrifuge. Remove the supernatant and resuspend the cells to 2*10 6 CFU / mL concentration level;

[0036] S2: Then prepare the coating agent, dissolve 100g of polyvinyl alcohol (PVA) with a molecular weight of 50000g / mol and a degree of polymerization of 1500 in 1000ml of warm water at 60℃, and stir thoroughly until completely dissolved; after cooling the PVA solution to below 37℃, mix it thoroughly with 1000ml of bacterial resuspension prepared in S1 at a ratio of 1:1 to obtain 5% PVA and 1*10 6 CFU / mL of the strain coating agent; stored in a closed and cool environment, try to prepare it before use;

[0037] S3: Add the mixed microbial coating agent prepared in S2 to the spray equipment, select an air atomization spray nozzle, and adjust the atomization pressure to 1.25 bar, ensure that the atomized particle size is within the range of 110 μm, control the spraying rate at 700 mL / h, and the seed transmission rate at 0.75 m / s, so that the coating agent is evenly coated on the clean seed surface; obtain a vetiver seed coated with Burkholderia seed coating, and store the coated seeds in a dry and cool place for future use.

[0038] Compared with the prior art, the present invention achieves a different method of adding exogenous microorganisms to improve the efficiency of the "microorganism-vetiver" joint restoration. The commonly used method of adding bacteria in this technical field is to add them directly to the soil or the rhizosphere soil of plants, while the present solution is to inoculate bacteria through seed coating. At the same time, the seed coating preparation process is different from the existing reported coating technology that is mainly used in agricultural production (insect prevention, disease prevention, and yield increase).

[0039] In the prior art, due to the ecological relationships such as competition and predation among microorganisms in the soil, the survival rate of exogenously added microorganisms is extremely low and the colonization rate is almost zero. However, through the preparation method of the present invention, the colonization rate of the functional microorganism - Burkholderia can reach 30% to 80%, truly achieving survival and functioning to improve the efficiency of plant extraction.

[0040] The present invention provides a method for exogenously adding functional microorganisms - coating vetiver seeds with microbial coating agents and a coating agent production process, and has a significant effect on improving the colonization rate of functional microorganisms and the effect of plant restoration. Through the coating process and technical methods described in the patent, Burkholderia can be colonized in the rhizosphere soil of plants. After colonization, it can change the physical and chemical properties of the soil through its own metabolic activities (such as lowering the soil pH, increasing the effective content of nutrients such as nitrogen and phosphorus), change the occurrence form of heavy metal cadmium in the soil, and increase the effective content to increase plant enrichment. In addition, after successful colonization, the microorganisms can change the plant biomass (increase plant height and fresh weight) by secreting plant auxin (IAA), and improve the plant's ability to transfer cadmium from roots to aboveground parts by secreting iron carriers. These are all due to the efficient colonization of Burkholderia in the soil. The relative abundance changes in the soil at 1d, 5d, and 10d under different addition methods are as follows: Figure 1 As shown;

[0041] In order to analyze the effects of seed coating with Burkholderia and direct addition of Burkholderia to the soil on plant absorption and migration of Cd. Contaminated soils with heavy metal cadmium contents of 5 mg / kg, 10 mg / kg and 15 mg / kg were selected for the experiment. Vetiver was harvested two months after planting at room temperature. We divided the aboveground and underground parts, dried them at 60°C to constant weight, and then digested them with a mixture of HNO3, HF and HCLO4. Then, the volume was fixed to 10 mL with 0.1% dilute nitric acid, and the Cd content of each part was analyzed using an inductively coupled plasma mass spectrometry (ICP-MS). In addition, the bioconcentration factor (BCF) of the aboveground and underground parts was used to analyze the extraction capacity of vetiver for Cd. The specific calculation formula is as follows

[0042] BCF- shoot = C shoot / C soil (1)

[0043] BCF- root = C root / C soil (2)

[0044] In the formula, C shoot : Cd content in aboveground parts; C root : Cd content in underground part; C soil : Cd content in soil, BCF is bioaccumulation factor.

[0045] The results of the effects of different inoculation methods on the extraction, migration and bioaccumulation efficiency of Cd in Vetiver are shown in Figure 2. Figure 2 and Figure 3As shown in Figure 2, with the increase of soil Cd concentration, the Cd content in different plant tissues also increased ( Figure 2 In addition, the results showed that the plant roots had a stronger ability to accumulate Cd than the aboveground parts. The Cd content in the roots of the vetiver grass in the direct addition group was 79.82 mg / kg. -1 (Cd5), 95.93mg kg -1 (Cd10) and 136.28 mg kg -1 (Cd15), while the Cd content in the aboveground part of vetiver was 5.96 mg kg -1 (Cd5), 7.38 mg kg -1 (Cd10) and 10.95 mg kg -1 (Cd15). At the same time, compared with the direct addition group, the Cd content in the aboveground and underground parts of the seed coating inoculation group increased by 36.56% to 39.66% and 25.97% to 130.47%, respectively. In addition, Figure 3 The results reflected the Cd accumulation capacity (BCF) of vetiver grass. The results showed that after inoculation with seed coating, the BCF- shoots and BCF- roots also improved, with the maximum values ​​appearing in Cd5B (2.74) and Cd15B (21.59) groups, respectively.

[0046] The preparation method of vetiver seeds coated with Burkholderia provided by the present invention improves the colonization efficiency of functional microorganisms in the rhizosphere and inside the plant, showing significant advantages over traditional microorganism-plant combined remediation technology. In contaminated soil, even at the end of remediation, high concentrations of Burkholderia can still be detected in the rhizosphere and plants, and the survival rate and colonization rate are increased by more than 35%. In addition, the Cd enrichment factor (BCF) and migration factor (TF) of vetiver are significantly improved in the seed coating treatment. This method effectively promotes the combined remediation efficiency of plants and functional microorganisms, and provides a new efficient and stable way for the remediation of contaminated soil.

Claims

1. A method for preparing vetiver seeds coated with Burkholderia spp., characterized in that: The following steps are involved: S1: Adding Burkholderia to a sterilized LB medium at a 1% inoculum volume, culturing at a constant temperature, centrifuging, removing the supernatant, and resuspending the cells in sterile physiological saline to obtain a resuspended liquid; S2: dissolving polyvinyl alcohol in a solvent, stirring and cooling to obtain a polyvinyl alcohol solution, and mixing it with the resuspended solution in S1 to obtain a coating agent; S3: Add the coating agent in S2 to the spray equipment, adjust the atomization pressure, atomization particle size, spraying rate, and seed transmission rate, and apply the coating agent on the surface of the seeds to obtain coated seeds.

2. The method for preparing vetiver seeds coated with Burkholderia seed coating according to claim 1, characterized in that: The LB medium described in S1 includes 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, and has a pH of 7.0 to 7.

1.

3. The method for preparing vetiver seeds coated with Burkholderia seed coating according to claim 1, characterized in that: The constant temperature culture described in S1 is 37°C, the speed is 120 rpm, and the culture time is 48 h.

4. The method for preparing vetiver seeds coated with Burkholderia seed coating according to claim 1, characterized in that: The centrifugation speed in S1 was 4000 rpm and the centrifugation time was 5 min.

5. The method for preparing vetiver seeds coated with Burkholderia seed coating according to claim 1, characterized in that: The degree of polymerization of the polyvinyl alcohol described in S2 is 500 to 2500.

6. The method for preparing vetiver seeds coated with Burkholderia seed coating according to claim 1, characterized in that: The ratio of the polyvinyl alcohol solution and the resuspension solution described in S2 is 1:

1.

7. The method for preparing vetiver seeds coated with Burkholderia seed coating according to claim 1, characterized in that: The atomization pressure described in S3 is 1-1.5 bar, the atomization particle size is 80-150 μm, the spraying rate is 200-1000 mL / h, and the seed transmission rate is 0.5-1 m / s.

8. Vetiveria seeds coated with Burkholderia seed coating obtained by the preparation method according to any one of claims 1 to 7.

9. The use of Burkholderia seed coating on vetiver seeds according to claim 8, characterized in that: The Burkholderia seed coating coated vetiver seeds is used in the preparation of plant seeds for treating Cd pollution.

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