CN111657304B - Method for repairing heavy metal contaminated soil by using Aspergillus flavus TL-F3 enhanced ryegrass - Google Patents
Method for repairing heavy metal contaminated soil by using Aspergillus flavus TL-F3 enhanced ryegrass Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/34—Aspergillus
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/002—Solid waste from mechanical processing of material, e.g. seed coats, olive pits, almond shells, fruit residue, rice hulls
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses a method for repairing heavy metal contaminated soil by Aspergillus flavus TL-F3 reinforced ryegrass, which is used for repairing heavy metal contaminated soil by combining Aspergillus flavus TL-F3 with ryegrass. The invention discovers for the first time that the aspergillus flavus TL-F3 has a growth promoting effect on plants, and the aspergillus flavus TL-F3 still has good growth promoting effects when the concentrations of Pb (II), Zn (II), Cu (II) and Cd (II) are respectively 50, 200, 300 and 5 mg/L. The inoculated aspergillus flavus TL-F3 obviously promotes the growth of ryegrass, improves the enrichment efficiency of ryegrass on Pb (II), Zn (II), Cu (II) and Cd (II), and reduces the concentration of heavy metals in soil. The invention provides a new means for restoring the heavy metal combined contaminated soil.
Description
Technical Field
The invention relates to the technical field of heavy metal contaminated soil remediation, in particular to a method for repairing heavy metal contaminated soil by Aspergillus flavus TL-F3 reinforced ryegrass.
Background
In the soil ecosystem, the sources of heavy metals are very wide, and two ways of natural sources and artificial sources are mainly available. Among natural factors, natural disasters such as earthquake, debris flow, volcanic eruption and the like have great influence on the heavy metal content of soil. Among human factors, the heavy metal content of soil caused by industrial, agricultural and traffic sources is high in proportion, and is a main source of heavy metal pollution of soil. Heavy metal pollution in the environment is not caused by single heavy metal, but is accompanied by combined pollution of multiple heavy metals. Due to the characteristics of concealment, complexity, nondegradable property and the like of heavy metals, the heavy metals are difficult to be thoroughly treated. Long-term heavy metal pollution not only can affect the crop yield and cause economic loss, but also enters human bodies through food chains and harms human health.
At present, the applied heavy metal soil pollution remediation technologies comprise physical remediation, chemical remediation, phytoremediation, microbial remediation, combined remediation and the like. The traditional physical remediation modes (electric remediation, electric heating remediation, soil leaching and the like) not only have high engineering quantity and high cost, but also are difficult to apply to large-area soil remediation. Chemical remediation (chemical precipitation, ion exchange, electrolysis, etc.) is complex to operate, can damage the soil structure, causes the soil fertility to be reduced, and can cause secondary pollution. The phytoremediation has low cost and no secondary pollution, and is a green, environment-friendly and sustainable soil heavy metal pollution remediation technology. But due to the defects of small biomass, long growth cycle and the like, the ideal effect is difficult to achieve by single plant restoration.
Soil microorganisms almost participate in all biological and biochemical reactions in soil, play an important role directly or indirectly in soil functions and soil development processes, and are considered to be the most sensitive index for evaluating soil environmental quality standards. Soil microorganisms can promote plant growth by a variety of mechanisms, such as secretion of indoleacetic acid (IAA) and 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase, phosphorus solubilization, siderophore production, nitrogen fixation, etc., or relieve environmental stress by increasing the rate of uptake of soil nutrients by host plants. Indoleacetic acid (IAA) is a ubiquitous natural auxin in plants and promotes the formation of plant shoots or buds. 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase can degrade the precursor ACC of ethylene, thereby reducing the ethylene level during plant growth and contributing to plant growth. Most of phosphorus in the soil is insoluble and can not be directly utilized by plants, and microorganisms can convert the phosphorus into soluble phosphorus, so that the utilization efficiency of the phosphorus is greatly improved. A siderophore is a low molecular weight substance that can bind ferric ions and donate to microbial cells. In addition, the microorganism also has the effects of potassium removal, nitrogen fixation, disease resistance, harm prevention and the like. The rhizosphere secretion of the plant can provide nutrition and energy sources for the growth of microorganisms. Therefore, the combination of microorganisms and plants for repairing the heavy metal pollution of the soil is a repair technology with great potential.
Disclosure of Invention
The invention aims to provide a method for strengthening Aspergillus flavus TL-F3 to repair heavy metal contaminated soil by ryegrass.
Another object of the invention is to provide the application of Aspergillus flavus TL-F3 in plant growth promotion.
In order to achieve the object of the present invention, in a first aspect, the present invention provides the use of Aspergillus flavus TL-F3 in plant growth promotion.
The Aspergillus flavus TL-F3 of the invention is shown in ZL 201410639891.5, and the preservation number is CGMCC No. 8146.
In a second aspect, the invention provides the use of Aspergillus flavus TL-F3 in plant growth promotion under heavy metal stress, wherein the heavy metal is at least one selected from Pb (II), Zn (II), Cu (II) and Cd (II).
The application comprises the following steps: aspergillus flavus TL-F3, metabolite thereof, fermentation liquor, bacterial suspension, microbial inoculum and/or spore suspension are mixed for seed dressing, or used as base fertilizer or used as top dressing to be applied to a plant growing area.
Such plants include, but are not limited to, ryegrass.
In a third aspect, the invention provides any one of the following uses of Aspergillus flavus TL-F3:
1) for promoting plant growth and development and increasing crop yield;
2) dissolving phosphorus;
3) the method is used for preparing agricultural fertilizers;
4) used for preparing a phosphorus activator;
5) is used for preparing plant growth promoting agent.
In a fourth aspect, the invention provides application of aspergillus flavus TL-F3 in dissolving refractory phosphorus under heavy metal stress, wherein the heavy metal is at least one selected from Pb (II), Zn (II), Cu (II) and Cd (II).
The heavy metal stress is selected from at least one of the following materials:
the concentration of Pb (II) is less than or equal to 50 mg/L;
concentration of Zn (II) is less than or equal to 200 mg/L;
concentration of Cu (II) is less than or equal to 300 mg/L;
the concentration of Cd (II) is less than or equal to 5 mg/L.
The insoluble phosphorus is insoluble organic phosphorus and/or inorganic phosphorus.
Preferably, the poorly soluble phosphorus is calcium phosphate.
In a fifth aspect, the present invention provides materials secreted from Aspergillus flavus TL-F3, including but not limited to IAA, ACC deaminase, and siderophore.
In a sixth aspect, the invention provides a method for repairing heavy metal contaminated soil by Aspergillus flavus TL-F3 reinforced ryegrass, which is to repair heavy metal contaminated soil by combining Aspergillus flavus TL-F3 with ryegrass. The method comprises the following steps: the Aspergillus flavus TL-F3 microbial inoculum is applied to heavy metal contaminated soil to promote the growth of ryegrass and improve the heavy metal enrichment capacity of the ryegrass.
Wherein the heavy metal is selected from at least one of Pb (II), Zn (II), Cu (II) and Cd (II).
The concentration of Pb (II) in the heavy metal polluted soil is less than or equal to 50 mg/L;
the concentration of Zn (II) is less than or equal to 200 mg/L;
the concentration of Cu (II) is less than or equal to 300 mg/L;
the concentration of Cd (II) is less than or equal to 5 mg/L.
Preferably, the microbial inoculum is added into the soil according to the mass ratio of the Aspergillus flavus TL-F3 microbial inoculum to the soil of 3g:2 kg-5 g:2kg (preferably 4 g:2 kg); wherein the concentration of the Aspergillus flavus TL-F3 microbial inoculum is 108～1010Spors/mL (preferably 10)9Spores/mL)。
Experiments show that (1) the spore liquid of the aspergillus flavus TL-F3 is inoculated into a liquid culture medium containing 0.05 percent of L-tryptophan or a liquid culture medium containing 0.05 percent of L-tryptophan, 50mg/L Pb (II), 200mg/L Zn (II), 300mg/L Cu (II) and 5mg/L Cd (II), and the liquid culture medium is subjected to shake culture at 28 ℃ and 150r/min for 7 d. The bacterium has the ability to secrete IAA.
(2) Inoculating the spore liquid of Aspergillus flavus TL-F3 into a liquid culture medium or a liquid culture medium containing 50mg/L Pb (II), 200mg/L Zn (II), 300mg/L Cu (II) and 5mg/L Cd (II), and carrying out shake culture at 28 ℃ and 150r/min for 7 d. The bacterium has the capability of secreting ACC deaminase.
(3) Inoculating the spore liquid of the aspergillus flavus TL-F3 into an inorganic phosphorus liquid culture medium or the inorganic phosphorus liquid culture medium containing 50mg/L Pb (II), 200mg/L Zn (II), 300mg/L Cu (II) and 5mg/L Cd (II), and carrying out shake culture at 28 ℃ and 150r/min for 7 d. The bacterium has the ability to convert inorganic phosphorus into soluble phosphorus.
(4) Inoculating the spore liquid of Aspergillus flavus TL-F3 into SA liquid culture medium or SA liquid culture medium containing 50mg/L Pb (II), 200mg/L Zn (II), 300mg/L Cu (II) and 5mg/L Cd (II), and shake-culturing at 28 deg.C and 150r/min for 7 d. The bacterium has siderophore production ability.
Wherein the liquid isThe components of the culture medium are as follows: 20g of glucose, 10g of peptone, 0.2g of NaCl and CaCl2 0.1g、KCl 0.1g、K2HPO4 0.5g、NaHCO3 0.05g、MgSO4 0.25g、Fe(SO4)2·7H2O0.005 g and distilled water to a constant volume of 1L, natural pH, and sterilizing at 121 deg.C for 30 min.
The inorganic phosphorus liquid culture medium comprises the following components: sucrose 10g, Ca3(P04)25g、(NH4)2SO4 0.5g、NaCl 0.1g、KCl 0.2g、MgSO4·7H2O 0.1g、FeSO4 0.03g、MnSO4·H2O0.03 g and distilled water to a constant volume of 1L, natural pH, and sterilizing at 121 deg.C for 30 min.
The SA liquid culture medium comprises the following components: sucrose 20.0g, L-asparagine 2.0g, K2HPO4 0.5g、MgSO4.7H2O0.5g, distilled water to constant volume of 1L, natural pH, 121 ℃ sterilization for 30 min.
Further, the invention provides a method for repairing Pb (II), Zn (II), Cu (II) and Cd (II) combined contaminated soil by using Aspergillus flavus TL-F3(A. flavus TL-F3) reinforced ryegrass, which comprises the following specific steps:
1) selecting a proper amount of A.flavus TL-F3 inclined plane and flat plate, adding sterile normal saline (0.85% NaCl solution) to fully wash the spores, filtering the washing solution by using sterile absorbent cotton, transferring the washing solution to a sterile triangular flask, and oscillating the washing solution for 2 hours at the temperature of 28 ℃ and the rotating speed of 150r/min to prepare the spore solution. Counting with a hemocytometer and diluting the concentration to 108Spors/mL. Storing in a refrigerator at 4 deg.C for use.
2) Loading water, testa Tritici and testa oryzae into 1L beaker with matrix of 6 mL: 3g: 2g (V/W/W), sealing with kraft paper, autoclaving at 121 deg.C for 30min, cooling, and air drying. Then, the spore solution and the matrix were mixed in a sterile plastic box at a ratio of 1mL to 10g (V/W). Culturing at 28 deg.C for 7 days, and counting with spread plate to obtain 10-concentration bacterial agent9Spors/mL. Storing in a refrigerator at 4 deg.C for use.
3) Selecting rye grass seeds with uniform size and full granules, soaking in 10% sodium hypochlorite, and sterilizing for 8min to avoid adding non-indigenous microorganisms into the system. Then, the seeds were rinsed thoroughly three times with pure water and soaked overnight. The seeds were spread on a tray attached with absorbent cotton, supplemented with appropriate amount of water daily, and cultured in an incubator at 25 ℃ for 2 weeks.
4) A. flavus TL-F3 microbial inoculum is prepared according to the weight ratio of 4 g:2kg of the fertilizer is evenly mixed into the soil, so that the field water capacity is kept at 60 percent and is stabilized for 3 days. Transplanting the ryegrass seedlings into rhizosphere bags filled with 400g of soil (microbial inoculum soil mixture), and then sequentially filling pots (microbial inoculum soil mixture), wherein 5 plants are planted in each pot. The soil culture rhizosphere bag experiment is carried out in an experiment shed of a university agriculture extraction garden of Anhui agriculture, the daytime mode is 30 ℃, the nighttime mode is 20 ℃, the time duration is set to be 12 hours respectively for white light and dark, and the illumination is set to be 1400-1600 mu mol/m in illumination intensity under natural conditions2/s1Watering every 2 days to keep 60% of the field water capacity.
Ryegrass was harvested after 30 days and its biomass, plant and soil heavy metal content were determined. The results show that compared with the missed bacteria, the inoculated bacteria agent promotes the growth of the root and the overground part of the rye grass, and increases the dry weight of the plant; the enrichment efficiency of ryegrass on Pb (II), Zn (II) and Cu (II) is improved, and the toxic effect of Cd (II) on ryegrass is relieved. The invention provides a new means for restoring the heavy metal combined contaminated soil.
Drawings
FIG. 1 is a graph showing the effect of different treatments on rye grass biomass in example 1 of the present invention.
FIG. 2 is a graph showing the effect of different treatments on Pb (II) enrichment of rye grass in example 1.
FIG. 3 is a graph showing the effect of different treatments on Zn (II) enrichment of rye grass in example 1 of the present invention.
FIG. 4 is a graph showing the effect of different treatments on Cu (II) enrichment of rye grass in example 1 of the present invention.
FIG. 5 is a graph showing the effect of different treatments on the enrichment of Cd (II) in rye grass in example 1.
FIG. 6 is a graph showing the effect of different treatments on the Pb (II) content of the soil in example 1 of the present invention.
FIG. 7 is a graph showing the effect of different treatments on the Zn (II) content of the soil in example 1 of the present invention.
FIG. 8 is a graph showing the effect of different treatments on the Cu (II) content of the soil in example 1 of the present invention.
FIG. 9 is a graph showing the effect of different treatments on the Cd (II) content of the soil in example 1 of the present invention.
FIG. 10 shows the effect of different concentrations of Pb (II) on the growth of A.flavus TL-F3 cells in example 2 of the present invention.
FIG. 11 shows the effect of different concentrations of Cd (II) on the growth of A.fTavus TL-F3 cells in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 Aspergillus flavus TL-F3 method for strengthening ryegrass to repair heavy metal contaminated soil
1. Experimental Material
Tetraploid extra-high annual ryegrass (70 in winter) is purchased from Kaiyuan industry Co., Ltd in Jilin province.
The strain is Aspergillus flavus TL-F3 with the preservation number of CGMCC No. 8146.
The soil is collected from the vicinity of a certain mining area in the city of holy, Anhui province, and the physicochemical properties of the soil are as follows: the pH value is 6.5, the total nitrogen is 1.21g/kg, the total phosphorus is 0.14g/kg, the total potassium is 0.16g/kg, the organic matter is 8.74g/kg, and the EC is 250.00 mS/cm. The initial concentrations of the soil heavy metals Pb (II), Zn (II), Cu (II) and Cd (II) were 16.17, 171.74, 330.91 and 0.30mg/kg, respectively.
Selecting a proper amount of A.flavus TL-F3 inclined plane and flat plate, adding sterile normal saline (0.85% NaCl solution) to fully wash the spores, filtering the washing solution by using sterile absorbent cotton, transferring the washing solution to a sterile triangular flask, and shaking for 2 hours at the temperature of 28 ℃ and the rotating speed of 150r/min to prepare the spore solution. Counting with a hemocytometer and diluting the concentration to 108Spors/mL. Storing in a refrigerator at 4 deg.C for use.
Loading water, testa Tritici and testa oryzae into 1L beaker with matrix of 6 mL: 3g: 2g (V/W/W), sealing with kraft paper, and filteringSterilizing at 121 deg.C under high pressure for 30min, cooling, and air drying. Then, the spore solution and the matrix were mixed in a sterile plastic box at a ratio of 1mL to 10g (V/W). Culturing at 28 deg.C for 7 days, and counting with spread plate to obtain 10-concentration bacterial agent9Spores/mL。
2. Design of experiments
Selecting rye grass seeds with uniform size and full granules, soaking in 10% sodium hypochlorite, and sterilizing for 8min to avoid adding non-indigenous microorganisms into the system. Then, the seeds were rinsed thoroughly three times with pure water and soaked overnight. The seeds were spread on a tray attached with absorbent cotton, supplemented with appropriate amount of water daily, and cultured in an incubator at 25 ℃ for 2 weeks.
3. Process group setup
The growth promotion test is provided with 5 treatment groups in total: a.flavus TL-F3 spore liquid was inoculated into liquid media containing Pb (II), Zn (II), Cu (II) and Cd (II) at concentrations of 50, 200, 300 and 5mg/L, respectively, denoted as Pb50, Zn200, Cu300 and Cd5, respectively. Meanwhile, a blank group without any heavy metal added to the medium was designated as CK. Each treatment was repeated three times.
The pot experiment was set up with 4 treatment groups in total: a is blank (CK), b is only added bacterium (A), c is only seed ryegrass (R), d is added bacterium and is planted with ryegrass (R + A). Each treatment was repeated three times. The microbial inoculum is evenly mixed into the soil according to the proportion of 4g to 2kg, so that the field water holding capacity is kept at 60 percent and is stabilized for 3 days. The seedlings of ryegrass were transplanted into rhizosphere bags containing 400g of soil and then potted, 5 plants per pot. The soil culture rhizosphere bag experiment is carried out in a large-school agriculture and forestry garden experiment shed of Anhui agriculture, the daytime mode is 30 ℃, the nighttime mode is 20 ℃, the time duration is set to be 12 hours respectively for white light and dark, and the illumination is set to be 1400-year-old 1600 mu mol/m in the natural condition2/s1Adding proper amount of water every two days, culturing for 30 days, harvesting, and measuring.
4. Measurement method
(1) Measurement of the secretion of IAA by Flavus TL-F3
0.5mL of spore solution was aspirated and inoculated into a liquid medium containing 0.05% L-tryptophan and CK, Pb50, Zn200, Cu300 and Cd5, and shake-cultured at 28 ℃ and 150r/min for 7 d. The culture solution was centrifuged at 6000r/min at 4 ℃ for 10 min. Mixing 2mL of supernatant with 4mL of Salkowski reagent, reacting at 25 deg.C in the dark for 30min, and measuring absorbance at 530 nm.
(2) Assay for the secretion of ACC deaminase by flavus TL-F3
0.5mL of spore solution is sucked and inoculated into CK, Pb50, Zn200, Cu300 and Cd5 liquid culture medium, and shaking culture is carried out at 28 ℃ and 150r/min for 7 d. Centrifuging for 10min (4 deg.C, 8000rpm), collecting thallus, washing thallus with DF culture solution, centrifuging twice, and suspending thallus in solution containing 0.5g/L (NH)4)2S04The ADF culture solution of (1) was further cultured for 7 days, and the cells were collected by centrifugation (4 ℃, 8000rpm, 10min), and then treated with 10mL of Tris-HCl buffer (0.1 mol. multidot.L)-1pH7.5) was washed twice and resuspended in 600. mu. L of a Tris-HCl buffer (0.1 mol. multidot.L)-1pH8.5), 30. mu.L of toluene was added, and the cells were disrupted by ultrasonication for 30 seconds to obtain a crude enzyme solution.
Immediately taking 200. mu.L of crude enzyme solution and 20uL of 0.5 mol/L-1ACC was mixed well in a 1.5mL centrifuge tube, reacted in a water bath (30 ℃ C., 15min), and 1.0mL HCl (0.56 mol. L) was added-1) The reaction was stopped and two controls were set up, without addition of crude enzyme solution and without addition of ACC.
Centrifuging the culture solution at 4 ℃ under 12000r/min for 5min, taking 1mL of supernatant and 0.15mL of 2, 4-dinitrophenylhydrazine in a 10mL centrifuge tube, carrying out water bath reaction (30 ℃ for 30min), taking out, adding 2mL of NaOH, uniformly mixing, carrying out the same reaction by using Tris-HCI buffer solution as blank zero adjustment, and measuring the absorbance at the wavelength of 540nm by using alpha-ketobutyric acid as standard solution.
Total protein content was determined by Coomassie Brilliant blue. 100 mu L of toluidized bacteria suspension and 5mL of Coomassie brilliant blue G250 staining solution are mixed uniformly, placed for 5min at room temperature, and subjected to color comparison at 595nm to calculate the protein content (mg). 1 unit of ACC deaminase activity (U) is the activity to form 1. mu. mol of alpha-ketobutyrate per mg of protein per minute.
(3) Measurement of phosphorus solubilizing ability of flavus TL-F3
0.5mL of spore solution is sucked and inoculated into CK, Pb50, Zn200, Cu300 and Cd5 inorganic phosphorus liquid culture medium, and shaking culture is carried out at 28 ℃ and 150r/min for 7 d. Centrifuging the culture solution at 4 ℃ under 10000r/min for 15min, taking 1mL of supernatant into a 50mL volumetric flask, diluting with water to about 3/5 of the total volume, adding 2-3 drops of 2, 4-dinitrophenol, adjusting with 10% sodium hydroxide solution or 10% sulfuric acid solution until the solution is just yellowish, adding 5mL of molybdenum-antimony anti-reagent, shaking up, adding water to fix the volume, standing at room temperature for 30min, and carrying out color comparison at 720 nm.
(4) Determination of Flavus TL-F3 siderophore production Capacity
0.5mL of spore solution is absorbed and inoculated into SA liquid culture medium of CK, Pb50, Zn200, Cu300 and Cd5, shaking table culture is carried out for 7d at 28 ℃ and 150r/min, the culture solution is centrifuged for 15min at 4 ℃ and 10000g, 3mL of supernatant and 3mLCAS detection solution are uniformly mixed, the mixture is placed for 60min at room temperature, and color comparison is carried out at 630 nm. The absorbance of the supernatant of the uninoculated SA liquid medium was used as a reference value (Ar). The siderophore active unit (SU) ═ Ar-a/Ar ] × 100%.
(5) Determination of rye grass biomass
The harvested ryegrass is taken back to a laboratory to be repeatedly cleaned by distilled water, put into an oven at 105 ℃ to be de-enzymed for 30min, then the temperature is adjusted to 75 ℃ to be continuously dried until the weight is constant, the ryegrass is divided into roots and overground parts by scissors, and the weight is weighed and recorded.
(6) Determination of Pb (II), Zn (II), Cu (II) and Cd (II) content in ryegrass
Weighing 0.1000g of dried plant samples of roots and overground parts respectively, shearing into pieces, putting into a graphite digestion tank, and adding 1mL of HClO4And 9mL concentrated HNO3And standing overnight (10-12 h). The solution was then digested continuously at 150 ℃ until clear, transferred and made up to a 25mL volumetric flask, filtered through a 0.22 μm filter head and the filtrate was used for the assay.
(7) Determination of Pb (II), Zn (II), Cu (II) and Cd (II) contents in soils
And (3) taking the soil back to a laboratory for natural air drying, and weighing 0.1000g of ground and sieved soil sample in a graphite digestion tank. Adding HNO3And HCl 4mL each, left overnight (10-12 h). Heating at 150 deg.C for 90min, cooling for 30min, and adding 1mL HClO4Heating at 150 deg.C for 40min, and adding 1mL HNO3Finally, adding appropriate amount of distilled water, heating continuously until the solution is clear, transferring the solution and fixing the volume to a 25mL volumetric flask, filtering with a 0.22 μm filter head, filtering the filtrateFor the determination.
5. Results and analysis
Effect of different treatments on the growth promoting effect of a. flavus TL-F3: favus TL-F3 growth promoting properties are shown in Table 1.
TABLE 1A. Flavus TL-F3 growth promoting Properties
Under the culture condition without adding any heavy metal, the IAA, the ACC deaminase, the phosphorus and the siderophore secreted by the A.flavus TL-F3 are respectively 18.65 +/-2.175 mg/L, 1.70 +/-0.008U/mg, 89.16 +/-4.60 mg/L and 21.48 percent. Under the culture conditions of Pb (II), Zn (II), Cu (II) and Cd (II), the A.flavus TL-F3 still has good growth promotion effect. The Cd5 treatment increased the IAA secretion of A.flavus TL-F3 by 67.56% and decreased the Pb50, Zn200 and Cu300 by 23.75%, 43.59% and 73.99%, respectively. Pb50 increased the secretion of ACC deaminase from A.flavus TL-F3 by 83.6%, and decreased Zn200, Cu300 and Cd5 by 48.92%, 78.49% and 66.13%, respectively. The Pb50, Zn200, Cu300 and Cd5 treatments reduced the phosphorus dissolving capacity by 10.13%, 40.06%, 63.97% and 90.13%. The Pb50 and Zn200 treatment improves the siderophore production capacity by 36.08 percent and 81.01 percent, and reduces Cu300 and Cd5 by 71.42 percent and 40.18 percent.
Effect of flavus TL-F3 on biomass of ryegrass: the effect of different treatments on ryegrass biomass is shown in figure 1. The biomass of the R-treated black wheat grass root and the overground part were 0.11 g/plant and 0.48 g/plant, respectively. R + A treatment promoted ryegrass growth, increasing root and overground biomass to 0.12 g/plant and 0.64 g/plant by 14.67% and 33.38%, respectively.
Effect of flavus TL-F3 on extraction of heavy metals from ryegrass: the ryegrass has the potential of enriching heavy metals and repairing heavy metal pollution of soil. As shown in fig. 2-5, the roots of ryegrass are more enriched with heavy metals than the overground parts. The root part is enriched with Pb (II), Zn (II), Cu (II) and Cd (II) in the content of 13.16, 226.43, 73.02 and 2.54mg/kg, the overground part is enriched with heavy metals in the content of 2.79, 59.67, 16.11 and 1.22mg/kg, and the root part is enriched with heavy metals in the content of 4.71, 3.79, 4.53 and 2.08 times of the overground part. The R + A treated group extracted Zn (II) and Cu (II) more effectively than the R treated group, as shown in FIGS. 3 and 4. The R + A treatment increases the Zn (II) enriched content of the ryegrass roots from 226.43mg/kg to 227.10mg/kg, the overground part content from 59.67mg/kg to 59.75mg/kg, and the roots and the overground part are respectively increased by 0.30% and 0.13%; the content of Cu (II) enriched in ryegrass roots is increased from 73.02mg/kg to 74.40mg/kg, the content of overground parts is increased from 16.11mg/kg to 17.77mg/kg, and the content of roots and overground parts are respectively increased by 1.89% and 10.27%. However, the content of Cd (II) enriched in ryegrass roots is reduced from 2.54mg/kg to 2.20mg/kg by the R + A treatment, the content of overground parts is reduced from 1.22mg/kg to 0.85mg/kg, the content of roots and overground parts is respectively reduced by 13.36 percent and 30.43 percent, and A.flavus TL-F3 can relieve the toxic effect of Cd (II) on ryegrass. The content of Pb (II) enriched in ryegrass roots is reduced from 13.16mg/kg to 11.71mg/kg by 10.99% through the R + A treatment, and the content of Pb (II) in the overground part is increased from 2.79mg/kg to 3.15mg/kg by 12.73%.
Influence of different treatments on the heavy metal content of the soil: as shown in FIGS. 6 to 9, the contents of Pb (II), Zn (II), Cu (II) and Cd (II) in the CK-treated groups were 15.66, 162.73, 310.40 and 0.75mg/kg, respectively. The heavy metal content in treatment group A was 15.69, 171.30, 325.00 and 0.60mg/kg, respectively. Compared with CK, Pb (II), Zn (II) and Cu (II) in the soil are respectively increased by 0.16%, 5.26% and 4.71%. The content of Cd (II) in the soil is reduced by 19.94 percent. Probably because a. flavus TL-F3 is able to alter the morphology of heavy metals and the physicochemical properties of soil. The contents of Pb (II), Zn (II), Cu (II) and Cd (II) in the R-treated groups were 14.90, 159.52, 287.25 and 0.60mg/kg, respectively, and the heavy metal contents in the soil were reduced by 4.83%, 1.97%, 7.46% and 19.40% compared with CK, respectively. The contents of Pb (II), Zn (II), Cu (II) and Cd (II) in the R + A treatment groups were 12.85, 129.57, 221.68 and 0.59mg/kg, respectively, and the heavy metal contents in the soil were reduced by 13.78%, 18.78%, 22.83% and 2.22% compared with the R treatment groups, respectively.
The results show that the A.flavus TL-F3 still has good growth promotion effect at the concentrations of Pb (II), Zn (II), Cu (II) and Cd (II) of 50, 200, 300 and 5mg/I respectively. The inoculation of the A.flavus TL-F3 obviously promotes the growth of ryegrass, improves the enrichment efficiency of the ryegrass on Pb (II), Zn (II), Cu (II) and Cd (II), and reduces the concentration of heavy metals in the soil.
Example 2 resistance test of Aflavus TL-F3 to Pb (II) and Cd (II)
The experimental method comprises the following steps: 1mL of the solution was added to a concentration of 107-108Spores/mL A. flavus TL-F3 spore liquid is respectively added into liquid culture media containing Pb (II) and Cd (II) with different concentrations, and shake-cultured for 7d at 30 ℃ and 130 r/min. Pb (II) concentrations of 0, 50, 100, 250, 500, 700, 900 and 1000mg/L, respectively; cd (II) concentrations were 0, 10, 20, 40, 80 and 100mg/L, respectively. Filtering the mycelium pellets obtained by culturing, cleaning the mycelium pellets by using deionized water, drying the mycelium pellets in an oven at 60 ℃ to constant weight, weighing the growth amount of the mycelium according to a gravimetric method (figure 10 and figure 11), and determining the minimum inhibitory concentrations of Pb (II) and Cd (II) to the growth of the A.flavus TL-F3 mycelium as 1000mg/L and 100mg/L respectively.
Liquid medium for growth of cells: 20g of glucose, 10g of peptone, 0.2g of NaCl, CaCl2 0.1g、KCl 0.1g、K2HPO4 0.5g、NaHCO3 0.05g、MgSO4 0.25g、FeSO4·7H2O0.005 g, pH5.0, sterilizing at 121 deg.C for 30min, cooling, and adding sterilized Pb (II) and Cd (II) with different concentrations.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (5)
1. The application of the aspergillus flavus TL-F3 in plant growth promotion under heavy metal stress is disclosed, wherein the heavy metal is Pb (II), and the plant is ryegrass.
2. The application according to claim 1, wherein the application comprises: aspergillus flavus TL-F3, metabolite thereof, fermentation liquor, bacterial suspension, microbial inoculum and/or spore suspension are subjected to seed dressing, or used as base fertilizer or used as top dressing to be applied to a ryegrass growing area.
3. The method for repairing heavy metal contaminated soil by using Aspergillus flavus TL-F3 reinforced ryegrass is characterized by comprising the following steps: applying the Aspergillus flavus TL-F3 microbial inoculum to heavy metal contaminated soil to promote the growth of ryegrass and improve the heavy metal enrichment capacity of the ryegrass;
wherein the heavy metal is Pb (II).
4. The method as claimed in claim 3, wherein the concentration of Pb (II) in the heavy metal contaminated soil is 50mg/L or less.
5. The method as claimed in claim 3 or 4, characterized in that the microbial inoculum is added into the soil according to the mass ratio of the Aspergillus flavus TL-F3 microbial inoculum to the soil being 3g:2 kg-5 g:2 kg; wherein the concentration of the Aspergillus flavus TL-F3 microbial inoculum is 108～1010Spores/mL。
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