CN113699049B - Heavy metal tolerant fusarium X-2-5 and application thereof - Google Patents

Abstract

The application discloses heavy metal tolerant fusarium X-2-5 and application thereof, wherein the preservation number of the fusarium is CGMCC No.11609, and the ITS1-5.8S-ITS2 sequence is shown as SEQ ID NO 1; the fusarium X-2-5 and the bioremediation material containing the fusarium X-2-5 can be used for repairing heavy metal contaminated soil, and specifically, the fusarium X-2-5 or the bioremediation material is uniformly mixed with plant seeds and then sown, or the fusarium X-2-5 or the bioremediation material is directly placed in the soil where the plant seeds germinate or the plants grow. The fusarium X-2-5 provided by the application has strong tolerance to heavy metals, can promote plants to grow on barren, water-deficient and heavy metal polluted land and improve the heavy metal enrichment capacity, is safe and environment-friendly, and has a good application prospect.

Description

Heavy metal tolerant fusarium X-2-5 and application thereof

Technical Field

The invention relates to the technical field of environmental management, in particular to discovery and characteristics of heavy metal tolerant fusarium X-2-5 and application of the fusarium X-2-5 in bioremediation of heavy metal polluted areas.

Background

With the rapid development of mining and metal smelting industries, the problem of heavy metal pollution in soil and underground water in metal mining and smelting areas is more serious, great threat is caused to the production and life of surrounding residents, and the method is a key focus area for preventing and treating heavy metal pollution.

The heavy metal in the mining area is mainly migrated and diffused through the actions of leaching, metal-containing colloid, biology and the like, and the migration process is influenced by a plurality of factors such as the chemical characteristics of the heavy metal, the content of organic matters in the soil, the pH value of the soil and the like. Common pollutants in a non-ferrous metal smelting site mainly comprise cadmium, lead, arsenic, zinc, copper, chromium, mercury and the like, and the heavy metal pollution of the non-ferrous metal smelting site has the following characteristics: (1) multiple heavy metal composite pollution exists generally; (2) often involving co-contamination of deep soil and groundwater; (3) the ecological damage is serious, and the vegetation recovery difficulty is extremely high; (4) the migration path of the pollutant is many, the diffusion speed is fast, and the diffusion range is wide.

In recent years, scholars at home and abroad develop a great deal of research work aiming at the heavy metal pollution control of nonferrous smelting sites. In terms of repair materials and techniques, physical, chemical, biological or combination of the three methods can be used to transfer or remove heavy metals from soil and groundwater, or to reduce their bioavailability and mobility. However, how to realize long-acting stable restoration in a polluted area, how to realize synchronous restoration of multi-metal composite pollution, how to realize soil-underground water cooperative restoration and the like are common problems in the field of ecological restoration of domestic and foreign smelting sites.

At present, a plurality of mature repair technologies are applied, including chemical repair, physical repair, biological repair and the like. The chemical remediation method consumes a large amount of chemical agents and simultaneously generates secondary pollution, and is not suitable for large-area use; the physical repair needs special equipment, so that the cost is high and the effect is not ideal; bioremediation can satisfy ecosystem's healthy development because of the effect that relies on microorganism and plant are from life, can absorb accumulation or capture the solid-borne pollutant again, has resumeed the ecology promptly and can manage and control the pollutant diffusion again, has multiple advantages such as ecological, environmental protection, with low costs concurrently, receives student and trade company and favours.

Microbial remediation, which is a popular bioremediation technique in recent years, utilizes indigenous microorganisms or artificially domesticated microorganisms having specific functions to reduce the activity of harmful pollutants in the environment or degrade the harmful pollutants into harmless substances through the metabolic action of the microorganisms under suitable environmental conditions.

The endophytic bacteria serving as a new resource has ubiquitous property and functional diversity, is applied to soil heavy metal remediation, has the advantage of small negative influence on soil fertility and metabolic activity, and can avoid the influence on human health and environment caused by pollutant transfer. And based on the unique growing environment of the endophytic bacteria of the plants, the symbiotic relationship between the endophytic bacteria and the host plants is harmonious, the combined remediation with the plants can be realized, the poison of heavy metals to the plants is relieved, and the ecological restoration and the safe utilization of the land of the non-ferrous metal mining and metallurgy remaining site are further realized.

Therefore, the method for searching the endophytic bacteria with strong heavy metal tolerance from the plants in the heavy metal polluted area has strong purpose and feasibility, but the reports about the specific research results of the endophytic bacteria are less at present.

Disclosure of Invention

An object of this application is to provide an endogenetic bacterium of plant of tolerance heavy metal, it does not have the exogenous pollution destructiveness to the environment, and can realize safe, harmless, the quick restoration of heavy metal contaminated soil with the plant cooperation. The technical scheme of the application is as follows:

the application firstly provides heavy metal tolerant Fusarium X-2-5 which is classified and named as Fusarium nematophilum and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, the preservation time is 2015, 11 months and 27 days, and the preservation number is CGMCC No.11609.

Specifically, the ITS1-5.8S-ITS2 sequence is shown in SEQ ID NO. 1.

The application also provides a biological repair material which comprises the heavy metal tolerant fusarium X-2-5.

Further, the invention also provides application of the heavy metal tolerant fusarium X-2-5 or the bioremediation material in repairing heavy metal contaminated soil, wherein the heavy metal comprises at least one of manganese, cadmium, lead, zinc, arsenic, chromium and antimony.

The invention also provides a method for repairing heavy metal contaminated soil, which comprises the step of using the fusarium X-2-5 or the bioremediation material which can tolerate the heavy metal to cooperate with the plant to repair the soil, wherein the fusarium X-2-5 or the bioremediation material is uniformly mixed with the plant seeds and then sown, or the fusarium X-2-5 or the bioremediation material is directly placed in the soil where the plant seeds germinate or the plant grows.

In some embodiments, the bioremediation material is a liquid microbial inoculum for seed soaking treatment of plant seeds.

Specifically, the method comprises the following steps: selecting fresh potatoes, cleaning and peeling, weighing 200g of the potatoes, cutting the potatoes into small pieces, adding water, boiling for 20-30 minutes, boiling the potatoes, filtering the potatoes by using a plurality of layers of gauze, adding 5g of glucose into filtrate, uniformly stirring, fixing the volume to 1000mL, subpackaging the obtained mixture in conical bottles, plugging and binding, sterilizing the obtained product at the high temperature of 115 ℃ for about 20 minutes, cooling the obtained product to the room temperature, inoculating fusarium X-2-5, and culturing the obtained product at the room temperature for 20-30 days to obtain a liquid microbial inoculum of fusarium X-2-5. The cultured liquid microbial inoculum is stirred uniformly, diluted by 5 times, soaked in plant seeds for 2-3 days, and then broadcast.

In some specific embodiments, the bioremediation material is a solid microbial inoculum for dressing seeds or directly placing in soil to infect plant roots.

Specifically, the method comprises the following steps: the method comprises the steps of taking grass chaff and sawdust as culture mediums, ammonium nitrate as an exogenous nitrogen source, cane sugar as an exogenous carbon source and potassium dihydrogen phosphate as an exogenous potassium phosphate source, mixing and stirring uniformly, adjusting the water content to 65 percent and pH to 6.5, subpackaging by using polypropylene plastic bags, sealing by using lantern rings and films, sterilizing at 120 ℃ for about 90 minutes to obtain a solid culture medium, cooling to room temperature, inoculating fusarium X-2-5, culturing at room temperature for 30-35 days to obtain a solid microbial inoculum of fusarium X-2-5, mashing the cultured solid microbial inoculum into granules, mixing with seeds, and then spreading the granules in soil or directly placing the granules in the soil where plant seedlings grow.

When the microbial inoculum is used, the microbial inoculum is mixed with plant seeds and then is broadcast in soil, or is directly placed in soil where plant seedlings grow, so that the relative water content of the soil after the microbial inoculum is applied is maintained at about 65 percent for 10 days, and the smooth survival of the microbial inoculum and the seeds/seedlings in the soil is ensured.

In some specific embodiments, the plant is pokeweed.

The technical scheme provided by the application has at least the following beneficial effects:

1. the fusarium X-2-5 has obvious effects of promoting the field planting and growth of plants on the heavy metal contaminated soil and strengthening the enrichment of heavy metals, particularly manganese, cadmium, lead, zinc and the like, of the plants from the soil, is favorable for reducing the content of effective heavy metals in the soil and recovering the vegetation coverage rate, and has wide prospect in the aspect of realizing ecological recovery and reutilization of metal smelting fields by cooperating with the plants.

2. The fusarium X-2-5 has simple culture conditions, is easy for industrial production and has good market popularization value.

Drawings

In order to make the description of the embodiments or solutions of the prior art more clear, the drawings that are required for the description of the embodiments or solutions of the prior art are briefly described below, it being understood that the following drawings only show some embodiments of the present application and are therefore not to be considered limiting of scope, from which other drawings can be derived by a person skilled in the art without inventive effort.

FIG. 1 is a macroscopic morphological feature of Fusarium X-2-5 herein;

FIG. 2 is a microscopic morphological feature of Fusarium X-2-5 in this application.

Detailed Description

In order to facilitate understanding of the present application, the technical solutions in the present application will be described more fully and in detail with reference to the drawings and the preferred embodiments, but the scope of protection of the present application is not limited to the following specific embodiments, and all other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application.

The fusarium X-2-5 provided by the application is obtained by separating roots of a plant pokeweed with a good growth condition from electrolytic manganese smelting waste residues in a manganese ore area of Hunan Tan City in Hunan province in 2014, and researches show that the fusarium X-2-5 has a promoting effect on the growth of the pokeweed in barren soil, water-deficient soil and heavy metal polluted soil, and can obviously promote the planting, drought resistance and heavy metal enrichment of the pokeweed.

The morphological characteristics of Fusarium X-2-5 are shown in FIGS. 1 and 2. Observation according to morphological characteristics: the colony grows moderately on PDA culture medium (potato glucose agar culture medium), and is cultured at 25 deg.C for 7 days to generate colony with diameter of 3.5cm, and appearance of villiform-flocculent and white (see figure 1). Conidiophores stem is directly grown on hypha, and does not branch or simply branches; conidiophore sickle shape, usually 3 partitions, colorless, 25-40 μm × 4.5-8 μm (see FIG. 2).

The specific separation and extraction method of the strain X-2-5 comprises the following steps:

selecting good-growing pokeberry seedlings in a smelting waste residue storage yard, taking root samples of the pokeberry seedlings with the length of about 150mm and the diameter of about 5mm, bringing the pokeberry seedlings back to a laboratory, cleaning soil with tap water, cleaning the pokeberry seedlings with sterile water for three times, and placing the pokeberry seedlings on a super clean bench. Cutting the pokeweed roots into slices with the thickness of about 1-2mm by using a sterilized scalpel, soaking the root slices in 0.1% mercuric chloride solution for 1-2 minutes for sterilization, taking out the root slices, sucking water by using sterile absorbent paper, then inoculating the root slices on a sterilized PDA culture medium through sterile operation, sealing and wrapping, placing the root slices in a constant temperature cabinet, culturing at the constant temperature of 25 ℃, germinating white hyphae after 24 hours, transferring and purifying after three days, and after the surface of a culture medium is covered with the white hyphae, obtaining separated strains for identification and further culture and research. The ITS1-5.8S-ITS2 sequence of rRNA gene of strain X-2-5 is shown in SEQ ID NO 1 in the sequence table by the identification of institute of microbiology of Chinese academy of sciences.

Example 1

Preparing a potato liquid culture medium, placing the prepared culture medium in a 200mL conical flask, sealing, and sterilizing. Cooling, inoculating fusarium X-2-5 under aseptic condition, culturing at 25 deg.c for 20-30 days, diluting 2 times while stirring to soak American pokeberry seed, and germinating in culture dish after the seed absorbs water and expands. The control was inoculated with the same volume of pure potato broth that was not inoculated with Fusarium X-2-5.

Similarly, preparing Hoagland nutrient solution containing cadmium, manganese and zinc, wherein the cadmium is respectively 10 mu M and 50 mu M in two groups with different set concentrations, the manganese is respectively 100 mu M, 500 mu M and 1000 mu M in three groups with different set concentrations, and the zinc is respectively 50 mu M, 100 mu M and 500 mu M in three groups with different set concentrations; three replicates of each concentration were set up.

In the culture dish is evenly arranged in to the qualitative filter paper of foreshadowing in culture dish bottom, the seed after with the aforesaid water swelling that absorbs, corresponds to add each nutrient solution that disposes, and the volume of adding is based on just submerging half the seed, arranges the illumination incubator in after the culture dish covers the lid and cultivates in, and the cultivation condition is: the fluorescent lamp provides illumination with the intensity of 350 mu mol.m ^-2 s ^-1 The illumination time is 12 hours, the day-night circulation is simulated, the day temperature is 25 ℃, the night temperature is 20 ℃, and the relative humidity of air is 60-70% (RH). After 10 days, the germination rate of each group of seeds is counted, and the data are shown in table 1.

As can be seen from the table 1, the fusarium X-2-5 has a remarkable effect of promoting the germination of the pokeweed seeds, and the fusarium X-2-5 can improve the germination rate of the seeds by 5-15% even in the environment polluted by heavy metals.

TABLE 1 Effect of Fusarium X-2-5 on the germination percentage of Phytolacca americana in heavy metal-containing nutrient solutions

Example 2

Preparing a solid culture medium which takes grass chaff and wood dust as a culture medium, ammonium nitrate as an exogenous nitrogen source, cane sugar as an exogenous carbon source and potassium dihydrogen phosphate as an exogenous phosphorus and potassium source, adjusting the water content to 65 percent and the pH value to 6.5, subpackaging by using polypropylene plastic bags, sealing by using lantern rings and films, and sterilizing at the high temperature of 120 ℃ for 90 minutes. After cooling, fusarium X-2-5 is inoculated under aseptic condition and cultured for 30-35 days at 25 ℃ in indoor environment to prepare the solid microbial inoculum.

Selecting 2m multiplied by 2m land blocks respectively in a pollution-free wasteland, an electrolytic manganese waste residue storage yard and an electrolytic zinc waste residue storage yard (cadmium pollution and lead pollution exist) at the end of 3 months or in the beginning of 4 months, digging the land, loosening the soil, deeply applying a solid microbial inoculum, sprinkling water and preserving moisture (about 70 percent of water content), maintaining and balancing the soil, and covering a film and preserving moisture for 5 days. Meanwhile, selecting a plot with the same or similar conditions as a reference from a pollution-free wasteland, an electrolytic manganese waste residue storage yard and an electrolytic zinc waste residue storage yard, replacing the solid microbial inoculum with an equivalent amount of solid culture medium of non-inoculated fusarium X-2-5, and performing the same other operations.

While preparing land and preserving moisture, using common water to soak seeds for 3-5 days, then sowing, sowing the seeds of pokeberry and pennisetum seeds (about 30 g) with the same weight in each land, and adopting an intercropping mode, namely planting a row of pokeberry, then planting a row of pennisetum, then planting a row of pokeberry, and the like. After sowing, a sunshade net is covered above the land, and water is sprayed to preserve moisture. And after the plant seedlings grow to the height of about 15-20 cm, removing the sunshade net to enable the plant seedlings to naturally grow.

At the end of 7 months or at the beginning of 8 months (namely when vegetative growth is finished and buds begin to appear), randomly collecting overground parts of partial plants and counting the dry weight, wherein the data are shown in a table 2, and then, the content of heavy metals in different plant leaves is analyzed according to detection, and the data are shown in a table 3.

TABLE 2 Effect of Fusarium X-2-5 on the dry weight (g/strain) of the aerial parts of Phytolacca americana and Euphorbia fischeriana

TABLE 3 Effect of Fusarium X-2-5 on the enrichment of heavy metals in the leaves of Phytolacca americana and pennisetum alopecuroides

As can be seen from the table 2, fusarium X-2-5 has a remarkable effect of promoting the growth of pokeweed, and the fusarium X-2-5 also shows a growth promoting effect on pennisetum under the environment polluted by heavy metals, so that the applicant can reasonably speculate that the fusarium X-2-5 also has a certain growth promoting effect on other plants except the pokeweed, thereby being beneficial to the rapid recovery of the vegetation coverage rate of the polluted site and having a wide application range.

As can be seen from Table 3, the heavy metal enrichment capacity of the pokeweed with the solid microbial inoculum is significantly higher than that of the control group without the solid microbial inoculum, so that the fusarium X-2-5 has a significant effect on strengthening the heavy metal enrichment of plants from soil, and particularly has a prominent comprehensive effect on the synergistic restoration of the ecological environment of a metal smelting field with the pokeweed.

The above description is only a few examples of the present application and does not limit the scope of the claims of the present application, and it will be apparent to those skilled in the art that various modifications and variations can be made in the present application. Any improvement or equivalent replacement directly or indirectly applicable to other related technical fields within the spirit and principle of the present application by using the contents of the specification and the drawings of the present application shall be included in the protection scope of the present application.

Sequence listing

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<120> heavy metal tolerant fusarium X-2-5 and application thereof

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Claims (9)

1. The fusarium X-2-5 resistant to the heavy metal is characterized in that the fusarium X-2-5 is preserved in the China general microbiological culture Collection center (CGMCC) with the strain preservation number of CGMCC No.11609.

2. A bioremediation material comprising the heavy metal tolerant Fusarium X-2-5 of claim 1.

3. Use of a heavy metal tolerant fusarium X-2-5 according to claim 1 or a bioremediation material according to claim 2 for remediating heavy metal contaminated soil, wherein the heavy metal comprises at least one of manganese, cadmium, lead, zinc.

4. A method for remediating heavy metal contaminated soil comprising using the heavy metal tolerant fusarium X-2-5 of claim 1 or the bioremediation material of claim 2 in combination with phytolacca americana for soil remediation, wherein the fusarium X-2-5 or the bioremediation material is uniformly mixed with plant seeds and then sown, or the fusarium X-2-5 or the bioremediation material is directly placed in soil where the plant seeds germinate or the plants grow.

5. The method for remediating heavy metal contaminated soil as recited in claim 4, wherein the bioremediation material is a liquid microbial inoculum for seed soaking treatment of plant seeds.

6. The method for remediating heavy metal contaminated soil as recited in claim 5, wherein the preparation process of the liquid microbial inoculum comprises: cleaning peeled potato, boiling in water, filtering, adding carbon source, stirring, heating for sterilization, cooling to room temperature, inoculating Fusarium X-2-5, and culturing at room temperature.

7. The method for remediating heavy metal contaminated soil as recited in claim 4, wherein the bioremediation material is a solid microbial inoculum, and the solid microbial inoculum is crushed and used for dressing seeds or directly placed in soil to infect plant roots.

8. The method for remediating heavy metal contaminated soil as recited in claim 7, wherein the preparation process of the solid microbial inoculum comprises: adding grass chaff and sawdust as culture medium, adding nitrogen source, carbon source and phosphorus and potassium source, stirring, adjusting water content and pH value, heating for sterilization, cooling to room temperature, inoculating Fusarium X-2-5, and culturing at room temperature.

9. The method for remediating heavy metal contaminated soil as recited in claim 8, wherein the relative water content of the soil is controlled to be 60-70% after the microbial inoculum is applied.

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