CN105733958A - Fusarium oxysporum and application thereof to phytoremediation of heavy metal contaminated soil - Google Patents
Fusarium oxysporum and application thereof to phytoremediation of heavy metal contaminated soil Download PDFInfo
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- CN105733958A CN105733958A CN201610152355.1A CN201610152355A CN105733958A CN 105733958 A CN105733958 A CN 105733958A CN 201610152355 A CN201610152355 A CN 201610152355A CN 105733958 A CN105733958 A CN 105733958A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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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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Abstract
The invention provides fusarium oxysporum which is classified as fusarium. The fusarium oxysporum is preserved in the China General Microbiological Culture Collection Center on November 30, 2015, and a preservation number of the fusarium oxysporum is CGMCC 11607.Specifically, a ITS1-5 .8S-ITS2 sequence of the fusarium oxysporum is shown as SEQ ID NO:1. The invention further provides a bioremediation agent comprising the fusarium oxysporum and provides application of the fusarium oxysporum or the bioremediation agent to phytoremediation of heavy metal contaminated soil. The provided fusarium oxysporum which is a plant endophytic fungus is high in resistance to heavy metals and safe and environment friendly in remediation of the heavy metal contaminated soil.
Description
Technical field
The invention belongs to technical field of soil remediation, be specifically related to a kind of Fusarium oxysporum and the application in biological restoration heavy-metal contaminated soil thereof, particularly to being applied to the method that the heavy metals such as cadmium in heavy-metal contaminated soil are extracted in fortification of plants enrichment.
Background technology
Along with economic fast development, the production being not yet effectively addressed and sanitary wastewater, waste gas, waste residue and other garbage sharply increase, many harmful substances enter soil continuously and accumulate, the overall situation of soil pollution is severe, and ecological environment, agricultural safety and sustainable development are produced considerable influence.Wherein, mining and metallurgy activity is one of major reason of farmland and agricultural product heavy metals exceeding standard, and the exploitation of mineral resources cause the heavy metal pollution such as mining area and the arsenic of the soil of surrounding area, subsoil water and crop, lead and cadmium.Pollutant jeopardize residents ' health by drinking-water and food chain transmission, and heavy metal pollution event is situation occurred frequently.The whole nation there are about 1.2 ten thousand Tailings Dams, and wherein varehouse for dangerous goods, sick storehouse account for 12.4%, serious to ambient water and Soil Environmental Pollution, the most prominent to the health threat of resident.Although the soil pollution recovery technique that can enumerate is a lot, but actual practicality is little.Mostly the recovery technique that Application comparison is ripe at present is that dystopy is disposed, and multiple based technique for in-situ remediation is still in the research and development stage, including chemical redemption, biological restoration and agronomic measures etc..Chemical restoration also produces secondary pollution while consuming substantial amounts of chemical agent, be not suitable for the reparation in pollution in wide area farmland, biological restoration, because relying on microorganism and the spontaneous Ecology Action of plant, not only ecology but also environmental protection, low cost, does not produce secondary pollution and enjoys scholar and businessman's favor.Only utilizing the function of microorganism and plant, competence exertion ecosystem circulation digestive function, in conjunction with agronomic measures and physicochemical method, it is possible to achieve pollute the restoration of the ecosystem of degenerated soil.
But the work that this area is done in terms of microorganism and the plant cooperated reparation in field are repaired in heavy metal pollution is the most few.As Chinese patent CN 201110377736.7 relates to a kind of endophytic bacterium producing ACCD and soil remediation microbial inoculum thereof and application.Endophytic bacterium Jp3-3, Classification And Nomenclature is Pantoea agglomerans Jp3-3, is preserved in China typical culture collection center, and preservation date is on July 13rd, 2011, and culture presevation number is CCTCC NO:M2011250.Soil remediation microbial inoculum is directly used and can be promoted plant growing and absorb heavy metal, the efficiency of fortification of plants restoration of soil polluted by heavy metal (the especially soil of copper staining).
And Chinese patent application CN201510708559.4 provides a kind of by plant and degradation bacteria combine d bioremediation cadmium and the method for oxytetracycline combined contamination soil, described method includes using pot experiment, every basin loads cadmium, oxytetracycline combined contamination soil plantation Herba Tagetis Patulae or Mirabilis jalapa seed, after germination, oxytetracycline efficient degrading bacteria bacterium solution is inoculated in plant rhizosphere soil pouring root, use greenhouse production, plant entirety from contaminated soil is removed by plant growing to during the period of maturation, the most repeatedly plant this plant, until the pollutant in soil reach safety of soil environment standard.The method rehabilitation plant Herba Tagetis Patulae or Mirabilis jalapa, plant strain growth is strong, has good ornamental value, and has good accumulation ability to cadmium；Also the feature can beautified the environment the while of having low cost, easy operation, non-secondary pollution, has most important theories and realistic meaning for soil antibiotic, the preventing and treating of heavy metal pollution and crop production safety.
But for the microorganism field with plant cooperated restoration of soil polluted by heavy metal, also it is worth developing more effective and practical restorative procedure.
Summary of the invention
Present invention firstly provides a kind of Fusarium oxysporum, its Classification And Nomenclature is Fusarium Fusarium oxysporum (Fusarium oxysporum), bacterial strain number is J-2-1-2, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is CGMCC No.11607, preservation date is on November 30th, 2015, is preservation Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Specifically, its ITS1-5.8S-ITS2 sequence is as shown in SEQ ID NO:1.
The present invention also provides for a kind of bioremediation agents, including Fusarium oxysporum described above.
The present invention also provides for a kind of Fusarium oxysporum described above or bioremediation agents application in Phytoremediation of Soils Contaminated by Heavy Metals.
In a kind of specific embodiment, described plant is phytolacca american, and described heavy metal includes one or more in cadmium, lead, zinc and manganese.
The present invention also provides for a kind of method of restoration of soil polluted by heavy metal, soil remediation is carried out including using described Fusarium oxysporum or bioremediation agents to work in coordination with plant, wherein, described Fusarium oxysporum or bioremediation agents are mixed thoroughly with plant seed and broadcasts sowing, or described Fusarium oxysporum or bioremediation agents are placed directly within the soil of plant seed germination or plant growing, such as will state Fusarium oxysporum or bioremediation agents is placed directly within the soil of plant seedling growth.
In a kind of specific embodiment, described bioremediation agents is solid fungicide, also comprises one or more in wood flour, grass bran, cotton seed hulls and bog moss in described solid fungicide.
Specifically: this Fusarium oxysporum (Fusarium oxysporum) J-2-1-2 is prepared as solid fungicide, smashs into graininess to pieces, for broadcasting sowing in soil with seed mixing, or it is placed directly within the soil of plant seedling growth.After using solid fungicide, soil relative water content maintains about 65% 10 day, to guarantee that mycelia and seed or seedling survive in soil.
Wherein, described solid fungicide is prepared as follows obtaining: add cotton seed hulls as culture matrix with wood flour (or grass bran), ammonium nitrate is exogenous nitrogen source, sucrose is external source carbon source, potassium dihydrogen phosphate is external source phosphorus potassium source, mixing is mixed thoroughly and is adjusted the water content to 65%, pH6.5, polypropylene plastics pocket subpackage, with the collar and film seal, 120 DEG C of high temperature sterilizes 90 minutes, obtain solid medium, inoculation Fusarium oxysporum (Fusarium oxysporum) J-2-1-2, cultivate 30～35 days under the conditions of 25 DEG C, obtain point spore reaping hook (Fusarium oxysporum) J-2-1-2 solid fungicide, smash into cultured microbial inoculum to pieces graininess, for dressing seed or being placed directly within soil, it is allowed to infection plant's root system.
The plant endogenesis epiphyte Fusarium oxysporum heavy metal patience that the present invention provides is strong, and the reparation to heavy metal pollution of soil is safe and environmentally friendly.
The present invention at least has the advantage that and good effect:
1) Fusarium oxysporum (Fusarium oxysporum) J-2-1-2 effect to promoting plant absorption and accumulation heavy metal is the most notable, the effect that fortification of plants extracts heavy metal in soil is more notable, has remarkable result to reducing soil exchangeable species content of beary metal.
2) Fusarium oxysporum (Fusarium oxysporum) J-2-1-2 condition of culture is simple, it is easy to industrialized production, has good development prospect.
Accompanying drawing explanation
Fig. 1 is the morphological characteristic (both macro and micro) of Fusarium oxysporum in the present invention (Fusarium oxysporum) J-2-1-2,
Fig. 2 is that Fusarium oxysporum in the present invention (Fusarium oxysporum) is at phytolacca american root (Fig. 2 A) and stem (figure
Infection form in 2B).
Biological deposits
The Fusarium oxysporum that the present invention provides is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and deposit number is CGMCC 11607, and preservation date is on November 30th, 2015.
Detailed description of the invention
The present invention is further described below in conjunction with specific embodiment.But these embodiments are only exemplary, the scope of the present invention is not constituted any restriction.It will be understood by those skilled in the art that and technical scheme and detail view can be modified or replace without departing from the spirit and scope of the present invention, but these amendments and replacement each fall within the scope of protection of the invention.
Above-mentioned bacterial strains J-2-1-2 is that the plant phytolacca american root that upgrowth situation was good on the Pb-Zn ore district electrolytic zinc melting waste slag of Xiangxi Autonomous Prefecture of Hunan Province Huayuan County in 2013 separates acquisition, research finds that this bacterium has facilitation in barren, hydropenia, heavy metal pollution compared with the waste aerial of heavy soil to phytolacca american, can be obviously promoted phytolacca american field planting, drought resisting and enriching heavy metal.
The colony characteristics of Fusarium oxysporum (Fusarium oxysporum) J-2-1-2 is as follows:
Morphological observation shows: bacterium colony is very fast in the upper growth of PDA culture medium (potato dextrose agar), 25 DEG C, cultivates 7 days, diameter 8cm.Fine hair shape-cotton-shaped, white-lilac (see Fig. 1).Conidiophore is directly born in mycelia, not branch or simple branch.Macroconidium sickleshaped, microconidia oblong, kidney shape, cylindricality, 0-3 every, colourless, 7～20 μ m 2.5～4 μm.
The concrete separating and extracting process of bacterial strain J-2-1-2:
Choose well-grown phytolacca american seedling in melting waste slag stockyard, take its root samples, be about 150mm, diameter about 5mm, take back laboratory, first clean earth with tap water, then use sterile water wash three times, be placed in superclean bench.With the dissecting knife after sterilizing, phytolacca american root is cut into the section of thick about 1-2mm, root section is placed in 0.1% mercuric chloride solution immersion sterilization in 1-2 minute, taking-up root is cut into slices, with aseptic absorbent paper suck dry moisture, then above alcohol burner flame, pass through sterile working, root section is inoculated on the PDA nutrient chemical of sterilizing, after sealing wrapping, is placed in calorstat, 25 DEG C of constant temperature culture, after 24 hours, white hypha is sprouted, and after three days, switching purifies, and treats that media surface is covered with white hypha, obtain separated strain, for identifying and cultivating further and research.Identifying through Institute of Microorganism, Academia Sinica, the ITS1-5.8S-ITS2 sequence of the rRNA gene of bacterial strain J-2-1-2 is as shown in SEQ ID NO:1 in sequence table.
Embodiment 1
Configuration is containing cadmium, lead, the PDA fluid medium of manganese, one group of cadmium, manganese and zinc concentration are respectively 10,100,100 μMs, another group cadmium, manganese and zinc concentration are respectively 50,500,500 μMs, one group of PDA fluid medium being not added with any heavy metal is also had to compare, every kind of concentration for the treatment of repeats three, the culture medium prepared is placed in 200ml conical flask, sealing, sterilizing.After cooling, aseptically inoculate Fusarium oxysporum (Fusarium oxysporum) J-2-1-2,25 DEG C, cultivate 20 days, treat that mycelium is covered with fluid medium, with strainer filtering, pure water cleans several times, absorbent paper suck dry moisture, obtain Fusarium oxysporum (Fusarium oxysporum) J-2-1-2 mycelium, drying to constant weight, analyze cadmium, lead, manganese and Zn content for 80 DEG C, data are shown in Table 1.
Equally, configuration is containing cadmium, lead, the Gram nutritional solution suddenly of manganese, one group of cadmium, manganese and zinc concentration are respectively 10, 100, 100 μMs, another organizes cadmium, manganese and zinc concentration are respectively 50, 500, 500 μMs, one group of PDA fluid medium being not added with any heavy metal is also had to compare, every kind of concentration for the treatment of repeats three, the nutritional solution configured is placed in nutritive cube, combine sponge with cystosepiment punching to support fixing method and be supported on liquid nutritional alms bowl by phytolacca american seedling and cultivate, change one time of nutrition liquid every other day, plant roots is taken respectively after 20 days, stem, leaf sample, after pure water cleans, dry to constant weight for 80 DEG C, pulverize respectively with agate mortar again, for analyzing cadmium, lead, manganese and Zn content, data are shown in Table 1.
As can be seen from Table 1, Fusarium oxysporum (Fusarium oxysporum) J-2-1-2 mycelium enriching heavy metal very capable, exceeds well over the super enriching plant phytolacca american root under the conditions of same concentrations water planting, stem, the accumulation ability of leaf each position heavy metal.
Table 1 Fusarium oxysporum J-2-1-2 and the comparison of super enriching plant phytolacca american heavy metal accumulation ability
Embodiment 2
The solid medium that configuration adds the culture matrix of cotton seed hulls based on wood flour (or grass bran), ammonium nitrate is exogenous nitrogen source, sucrose is external source carbon source, potassium dihydrogen phosphate is external source phosphorus potassium source, moisture is adjusted to 65%, pH6.5, polypropylene plastics pocket subpackage, with the collar and film seal, 120 DEG C of high temperature sterilizes 90 minutes.After cooling, under aseptic condition, Fusarium oxysporum (Fusarium oxysporum) J-2-1-2 is inoculated in this solid medium, 25 DEG C, indoor cultivation 30～35 days, it is prepared as solid fungicide.
Take smeltery's debris dump periphery contaminated soil, air-dry, smash to pieces, cross 20 mesh sieves, be sufficiently mixed uniformly, be placed in polypropylene plastics pocket, every bag of 2kg soil, be placed in high-pressure sterilizing pot, 120 DEG C of high temperature sterilizes 90 minutes, standby after cooling.
Soil in plastic bag is placed in the plastic tub alms bowl (2kg/ basin) of sterilization.Smash into solid fungicide to pieces graininess, with phytolacca american seed mixes, then broadcast sowing in the basin alms bowl soil prepared, soil relative water content maintains about 65%, to guarantee that mycelia survives and seed germination, directly sow cultivated plant and compare being not added with solid fungicide, do 6 repetitions respectively.Treating seed germination, observe root system infection conditions, then carry out thinning, every basin stays 5 strains, plant growing 20 days, takes plant shoot sample for the first time, takes back laboratory, cleans, and dries, for analysis heavy metal content；After 25 days, again take plant regeneration Seedling aerial parts sample, take plant three lives Seedling overground part sample after 30 days third times, same analysis plant content of beary metal, the results are shown in Table 2.
From table 2 it can be seen that the phytolacca american overground part example enrichment heavy metal ability using solid fungicide is significantly higher than the plant not using solid fungicide.Additionally, after three times in same soil plantation phytolacca american, although total cadmium in soil, total lead, total manganese, total zinc changes of contents not quite, but the content of exchangeable species heavy metal significantly reduces.
Table 2 is inoculated Fusarium oxysporum J-2-1-2 and super enriching plant phytolacca american is absorbed the impact of heavy metal
Additionally, what those skilled in the art knew, in current soil, total heavy metal is difficult to be extracted efficiently out, even if the method that dystopy is disposed also is difficult to heavy metal be proposed and the most do not damage other composition of soil.Thus the technology generally advocated at present and praise highly is to use the heavy metal in plant extract soil, then it is distributed into the smelting of ore deposit enterprise to realize its efficient recovery and utilization by gained ash after being burned by heavy metal plant.Because the method using phytoremediation heavy metal soil has the effect of ecological, environmental protective, the first can be with the ecosystem of restoration of soil polluted by heavy metal by the plantation of special plant, promote the ecological diversity of this area's soil, and prevent heavy metal part as airborne dust in groundwater infiltration and entrance air；It two is can progressively the heavy metal in soil to be guided in plant by the plantation of special plant, can be as mining and metallurgy raw material after burning.
Although after in above-mentioned table 2 scheme, the phytolacca american seedling of microbial inoculum is used in plantation for 1～3 time, in soil, the changes of contents of total heavy metal is inconspicuous.But a reason is in laboratory conditions, and phytolacca american only grow to plantlet stage, thus between its plant dry weight and soil gross weight, gap is obvious.Second reason is in laboratory conditions, heavy metal in soil is slower to the rate of transformation of free state from stable state, thus after the phytolacca american seedling of microbial inoculum is used in continuous three plantations, can the bioaccumulation efficiency of heavy metal in soil declined phytolacca american as seen from Table 2.But after this strain being applied in the plantation of outdoor phytolacca american, on the one hand diminish with the difference of soil dry weight because of the plant gross dry weight (the most repeatedly after plantation) of phytolacca american, the most outdoor ecosystem contributes to stationary state heavy metal in soil and changes to free state (exchangeable species) heavy metal, thus generally, can be it is contemplated that the phytolacca american of microbial inoculum be used in outdoor continuous plantation, it will be conducive to the essence of total heavy metal content in soil to improve.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (7)
1. a Fusarium oxysporum, its Classification And Nomenclature is Fusarium Fusarium oxysporum (Fusarium oxysporum), its
Deposit number is CGMCC 11607.
Fusarium oxysporum the most according to claim 1, it is characterised in that its ITS1-5.8S-ITS2 sequence such as SEQ ID
Shown in NO:1.
3. a bioremediation agents, including Fusarium oxysporum as claimed in claim 1 or 2.
4. Fusarium oxysporum as claimed in claim 1 or 2 or bioremediation agents as claimed in claim 3 are at phytoremediation weight
Application in metallic pollution soil.
Apply the most according to claim 4, it is characterised in that described plant is phytolacca american, the most described heavy metal
Including one or more in cadmium, lead, zinc and manganese.
6. a method for restoration of soil polluted by heavy metal, including using Fusarium oxysporum as claimed in claim 1 or 2
Or as claimed in claim 3 bioremediation agents is worked in coordination with plant and is carried out soil remediation, wherein, by described Fusarium oxysporum or biology
Renovation agent is mixed thoroughly with plant seed and is broadcasted sowing, or described Fusarium oxysporum or bioremediation agents are placed directly within plant seed germination or
In the soil of plant growing.
Method the most according to claim 6, it is characterised in that described bioremediation agents is solid fungicide, described solid
Microbial inoculum also comprises one or more in wood flour, grass bran, cotton seed hulls and bog moss.
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| CN106995786A (en) * | 2016-10-25 | 2017-08-01 | 广州大学 | A kind of reaping hook bacteria strain and its application |
| CN109182227A (en) * | 2018-10-30 | 2019-01-11 | 知合环境（北京）有限责任公司 | A kind of complex micro organism fungicide and its preparation method and application |
| CN110560469A (en) * | 2019-04-19 | 2019-12-13 | 南华大学 | Method for restoring uranium-cadmium combined polluted soil by using uranium-cadmium-resistant fungi enhanced plants |
| CN113699049A (en) * | 2021-08-30 | 2021-11-26 | 湖南省环境保护科学研究院 | Heavy metal tolerant fusarium X-2-5 and application thereof |
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| CN113699049A (en) * | 2021-08-30 | 2021-11-26 | 湖南省环境保护科学研究院 | Heavy metal tolerant fusarium X-2-5 and application thereof |
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| CN105733958B (en) | 2018-03-13 |
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