CN102747002A - Agrobacteriumsp. having free-living nitrogen fixing ability, and applications thereof - Google Patents
Agrobacteriumsp. having free-living nitrogen fixing ability, and applications thereof Download PDFInfo
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Abstract
The present invention discloses Agrobacteriumsp. having free-living nitrogen fixing ability, wherein a classification name of the Agrobacteriumsp. is Agrobacteriumsp. Ymu6-2, the Agrobacteriumsp. is preserved in the China General Microbiological Culture Collection Center (CGMCC), and a preservation number is CGMCCNo.5007. The Agrobacteriumsp. having free-living nitrogen fixing ability in the present invention has the following advantages that: high nitrogen fixing ability is provided; high concentration Cd growth can be resisted; indole-3-acetic acid can be secreted to produce iron atoms; with the prepared microbial agent, over-accumulated plant growth can be significantly promoted, biomass of over-accumulated plants can be increased, a heavy metal enrichment coefficient and a heavy metal transfer coefficient of over-accumulated plants can be substantially increased, soil ecology environment can be improved, and advantages of environment-friendliness and low cost are provided.
Description
Technical field
The present invention relates to the edaphic bacillus that a strain has spontaneous nitrogen fixing capacity, this edaphic bacillus obtains with ultraviolet ray-plasma body complex mutation, has the ability of preventing from heavy metal growth, can be used for the heavy metal pollution of soil environmental improvement, belongs to technical field of microbe application.
Background technology
Heavy metal contamination is meant the environmental pollution that is caused by heavy metal or its compound, mainly by due to the human factors such as mining, exhaust gas emission, sewage irrigation.In recent years, a large amount of uses of fast development, chemical fertilizer and agricultural chemicals etc. along with Increase of population, industry make heavy metal pollution of soil increasingly sharpen.
Cd is the very big heavy metal of a kind of toxicity, and world-shaking Japan " itai-itai " causes because of cadmium pollution.Cadmium can replace the calcium in the bone, makes skeleton softening, and bone is broken into pieces; Cadmium also can cause gastrointestinal dysfunction, disturbs the enzyme system of zinc in human body and the organism, causes hypertension.Cadmium is many-sided to the murder by poisoning of tissue and organ, and treatment is very difficult.Therefore, various countries have all made extremely strict regulation to the cadmium in the industrial discharge " three wastes ".Japan's regulation rice contains cadmium and is " cadmium rice " above 1 milligram/kilogram, forbids eating.The Japan regulation 0.3ppm of Environmental Agency is the highest normal contents of cadmium concentration in the rice.
At present, the technology that is used for the heavy metal pollution of soil reparation mainly contains: physics reparation, chemistry are repaired and the biological prosthetic method.Physics repairing method treatment effect is better, but quantities is big, and cost is high.The chemistry repairing method mainly comprises methods such as chemical modifying, surfactant washing and organic improvement.Its advantage is simple to operate, and cost is lower, and shortcoming is fundamentally not remove heavy metal, and the modifying agent that adds has the risk of secondary pollution.Biological prosthetic comprises animal reparation, phytoremediation, mikrobe reparation and mikrobe and plant combined reparation etc.Plant extract is to study maximum restorative procedures at present; This method is the heavy metal ion of utilizing in the plant absorbing soil that surpasses the accumulation heavy metal; Heavy metal ion is enriched in the over-ground part of plant in a large number, reduces and remove heavy metal-polluted soil through harvesting or the over-ground part of pulling out plant.The ultra accumulation plant of having reported has penny cress genus, Herba avenae fatuae, Indian mustard, rape, alfalfa, false indigo etc., is mainly used in the Pb that removes in the contaminated soil, Cd etc. at present.The advantage of this method is to repair thoroughly, does not have secondary pollution, and shortcoming is shortcomings such as there is poor growth in most of ultra accumulation plant, living weight is little, the heavy metal semi-invariant is not high, and repair time is long.The plant-microorganism united restoration method is to study more a kind of new restorative procedure in recent years both at home and abroad, and this method is to utilize the efficient of the ultra accumulation of microbial augmentation phytoremediation, thoroughly removes the heavy metal in the contaminated soil.In soil-mikrobe-plant symbiosis environment; Mikrobe can be converted into the secretory product of soil with organic matter and root system of plant small molecules and supply self to utilize; Simultaneously, the heavy metal of organic acids such as the siderophore that mikrobe produces in self metabolic process, indolylacetic acid (IAA), oxalic acid in can activating soil increases the available state of heavy metal; And mikrobe also has very strong redox ability; Can reduce to iron, Mn oxide, discharge heavy metal, thereby improve of enrichment and the transhipment of ultra accumulation plant heavy metal.In addition, mikrobe also can be synthesized IAA, siderophore, and effect such as acc deaminase and fixed nitrogen, phosphorus decomposing and potassium decomposing promotes plant-growth, increases the plant-growth amount, thereby quickens the removal of heavy metal.Like people such as Whiting blue colza of holding back of surface sterilization is inserted in the soil that contains heavy metal Zn of sterilization; Insert bacterium again with Zn resistance; It is 2 times of plant that do not connect bacterium that the plant shoot that the result inserts bacterium divides the amount of accumulation heavy metal Zn; The total Zn of cumulative amount in over-ground part and the root, insert bacterium for not inserting 4 times of schizomycete semi-invariant.
Nitrogen is one of macroelement essential in the crop growth process, is 40%-50% to the contribution of crop ultimate capacity.The heavy metal of high density can make plant forfeiture root nodule tubercle ability, thereby suppresses the symbiotic nitrogen fixation process in the soil fully, is prone to cause the plant nitrogen stress.If rely on nitrogen fertilizer application merely, not only uneconomical, and cause soil compaction easily, nitrogenous fertilizer excessive used the pollution that can cause the underground water nitrite and the eutrophication of rivers and lakes.The nitrogen of azotobacter agent in can fixed air is transformed into ammonium nitrogen with nitrogen, and to compare use range wider with root nodule bacterium, produce and use all comparatively convenient.But most of wild vinelandii all exist poor growth, preventing from heavy metal growth and the lower shortcoming of nitrogen fixing capacity, can not satisfy the needs that contaminated soil is repaired.For this reason; Adopt various methodologies to break the homergy of bacterial classification, make it to produce needed target meta-bolites (like proteolytic enzyme), improve its resistance capacity etc., reach this purpose; Major measure is exactly to carry out the seed selection of bacterial classification, as carries out physics and chemomorphosis etc.
Summary of the invention
One of the object of the invention is to obtain the edaphic bacillus that a strain can be used for spontaneous nitrogen fixing capacity of having of heavy metal pollution of soil environmental improvement and preventing from heavy metal energy for growth through ultraviolet ray-plasma body complex mutation.
Two of the object of the invention provides microbiobacterial agent that contains above-mentioned edaphic bacillus and preparation method thereof;
Three of the object of the invention provides the application of above-mentioned edaphic bacillus in heavy metal pollution of soil environmental improvement and soil ecology reparation.
Implementation procedure of the present invention is following:
A kind of edaphic bacillus with spontaneous nitrogen fixing capacity; Its classification called after edaphic bacillus (Agrobacterium sp.) Ymu6-2; (address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.5007 to be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 29th, 2011.
Ymu 6-2 bacterium colony on solid medium A is creamy white, circular protrusions, and neat in edge, periphery of bacterial colonies has transparent circle, G
-, aerobic or amphimicrobian, it is shaft-like that the ESEM demonstration is tubbiness, 28 ~ 32 ℃ of the righttest growth temperatures, the righttest pH value is 6.0 ~ 8.0, its nitrogen fixing capacity is at 0.56 ~ 0.73nmol10
7Cfu
-1H
-1, separating the inorganic phosphorus ability is 264.5mg/l, and its ability of producing indolylacetic acid (IAA) is 15.25 ~ 30.6 μ g/ml, and the ability (A/Ar) of producing siderophore is 0.806 ~ 0.402; Said culture medium A consists of: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2O 0.005-0.01g, Na
2MoO
42H
2O 0.0025-0.005 g, agar powder 15-20g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2.
The mutagenic breeding method of high-efficiency nitrogen-fixing microorganism bacterial strain provided by the invention may further comprise the steps:
1) with the laboratory from the edaphic bacillus YSGD06 of plant rhizosphere screening as starting strain;
2) mutagenic and breeding
(1) single cell suspension of preparation starting strain YSGD06
Starting strain YSGD06 is inoculated among the liquid nutrient medium A, and 28-32 ℃, 120-150rpm cultivates 12-16hrs, and is centrifugal, with the SPSS washing, places in the triangular flask that granulated glass sphere is housed, and vibration makes it be dispersed into single celled bacteria suspension;
Described culture medium A consists of: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2O 0.005-0.01g, Na
2MoO
42H
2O 0.0025-0.005 g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2.
(2) ultraviolet mutagenesis
The bacteria suspension of step (1) gained is regulated concentration to 10 respectively
5-10
7Individual/ml, get the 0.1ml separate application in containing 400-800mg/L Cd
2+On the no nitrogen solid medium B, carry out ultraviolet mutagenesis, the frequency of ultraviolet mutagenesis is 10-18W, and irradiation distance is 25-50cm, irradiation time 5-10min; 28-32 ℃ leaves standstill cultivation 5-7days; Respectively select 20-30 bigger single bacterium colony, shake the multiple sieve of bottle, measure the nitrogenase activity of each bacterial strain with the acetylene reduction method; Select the highest bacterial strain of 5 strain nitrogenase activities; Shake the multiple sieve of bottle more respectively, select the bacterial strain YSGD06-Z of a strain nitrogenase activity height and good stability, process the mutagenesis that bacteria suspension is used for next step plasma body;
Described no nitrogen solid medium B component is: glucose 10-20g, KH
2PO
40.2g, MgSO
47H
2O 0.1-0.2g, NaCl 0.2g, CaSO
42H
2O 0.2g, CaCO
35g, CdCl
20.080-0.32g, agar 15-18g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2;
The step that described shake flask fermentation sieves again is: individual being inoculated among the above-mentioned liquid nutrient medium A of 100ml of 20-30 that at first above-mentioned separation is obtained, cultivate 8-12hrs.Respectively get in the Erlenmeyer flask that 5ml bacterium liquid is inoculated into the 250ml that 100ml liquid nutrient medium C is housed, 28 ℃, behind the 150rpm shaking table shaking culture 48h; Change tampon into rubber plug; Seal film and drip the wax sealing, syringe extracts the air of 10-15%, injects the acetylene gas of 10-15%; Continue reaction, measure nitrogenase activity with the acetylene reduction method;
The contained component of described liquid nutrient medium C is: glucose 10-20 g, KH
2PO
40.2-0.41 g, K
2HPO
40.3-0.52 g, CaCl
20.1-0.2 g, MgSO
47H
2O 0.1-0.2 g, FeSO
47H
2O 0.005-0.01g, Na
2MoO
42H
2O 0.0025-0.005 g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2.
(3) plasma body mutagenesis
With the YSGD06-Z bacterial strain of step (2) gained, process 10
5-10
7The bacteria suspension of individual/ml is got 0.1-0.2ml and is evenly coated respectively in the sterile petri dish, and petridish is put on the electrode below the plasma; Regulate the position of top electrode, make that the distance between the upper/lower electrode is controlled at about 3-8mm, regulating voltage is 3-5V; Electric current is 0.5-0.8A; Make air or argon gas discharging, obtain uniform air or argon medium barrier discharge plasma, be 2-7min discharge time.Immediately with SPSS or phosphoric acid salt wash-out, separate application is in containing 400-800mg/L Cd after the mutagenesis
2+On the no nitrogen solid medium B, shake the multiple sieve of bottle again, choose the highest strain bacterium YSGD06-Z2 of a strain nitrogenase activity, process bacteria suspension and be used for next step mutagenesis; The described same step of method (2) of shaking the multiple sieve of bottle.
(4) the YSGD06-Z2 bacteria suspension viable count with step (3) gained transfers to 10
5-10
7Individual/ml, cycle repeats ultraviolet mutagenesis → plasma body mutagenesis 1-2 time obtains strain tolerance heavy metal Cd and the highest bacterial strain Ymu6-2 of nitrogenase activity at last.
Mutagenic fungi Ymu 6-2 and edaphic bacillus have the homology of height, and the result of strain identification is following:
CCGGGGGCTGCTTACCATGCAGTCGAGCGGAGTGATGGTGCTTGCACTATCACTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGGAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTGAAGTTAATACCTTCAGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTAGCTAGAGTGTGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAACACTGACGCTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCCTTACCTGGGCCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGAATGTTGGTTAAGTCCCGCAACGAGCGCAACCATTTTCCTTATTTGCCAGCGAGTTATGTCGGGACTTTAAGATACTGCAGTGAAAAACTGGAGGAGCGGACGACGTCAAGTCATCATGGCCTACGCAAGGCTAACAACACGTGCTACATGGTCGGTACAATGTGCTACTAAGGAATGGAAGCCTAACCTCCAAAAAAGGGCG
The present invention also provides a kind of high-efficiency nitrogen-fixing microbial inoculum, can be liquid bacterial agent or solid fungicide according to nutrition carrier difference.
The preparation of microbial inoculum may further comprise the steps:
1) strain fermentation
1. one-level triangular flask liquid culture
With frozen Ymu6-2 quick-thawing under 37 ℃ of conditions, be equipped with in the triangular flask of liquid culture A according to the inoculum size access of 0.5-1%, 28-32 ℃, shaking culture 15-20hrs;
2. second order fermentation jar fermentation
The one-level nutrient solution of Ymu6-2 is equipped with in the fermentor tank of substratum E according to the inoculum size access of 5-10%, carries out fermentation culture, warm 28-32 ℃ in jar is cultivated pH7-8, aerobic culture 20-30 hrs;
Described substratum E consists of: sucrose 10-15g, starch 2-3g, dregs of beans 20-30g, KH
2PO
40.2-0.41 g, K
2HPO
40.3-0.52 g, CaCl
20.1-0.2 g, MgSO
47H
2O 0.1-0.2 g, Na
2MoO
42H
2O 0.0025-0.005 g, water 1000ml, p H 7.0 ~ 7.2.
2) straw powder, turfy soil, rice chaff are pulverized with high speed disintegrator; Cross 60 mesh sieves; According to mass percent is the ratio uniform mixing of 50-70:20-30:10-20; Also contain certain density Sodium orthomolybdate and ferrous sulfate in this matrix, wherein Sodium orthomolybdate is 0.2 ~ 0.5g/Kg, and ferrous sulfate is 0.05 ~ 0.08g/Kg;
3) the Ymu6-2 fermented liquid that obtains in the step 1) is directly squeezed into container for storing liquid; Promptly get liquid high-efficiency nitrogen-fixing microbial inoculum; With dosage and the step 2 of the fermented liquid that obtains according to 50-100ml/kg) in the solid state substrate mixing of gained, 28-32 ℃ of fermentation 5-7days obtains solid-state high-efficiency nitrogen-fixing microbial inoculum.
The method of use of microbial inoculum provided by the invention; It is characterized in that; Liquid bacterial agent soaks when root, nursery stage or vegetative period irritating root when can be used for the seed soaking of ultra accumulation plant, transplanting and adds in proportion; When plant is soaked seed, soak seed or plant root 2-5hrs with 50-100 diluent doubly, irritate the dosage pouring diluent that root adopts 100-500 diluent 20-40ml/Kg soil doubly in nursery stage or vegetative period, the whole growth phase is irritated root 1-3 time; Solid-state microbial inoculum can be used as base manure and topdresses use, and using dosage is a 5-20g/Kg soil, and the clearance of Cd has increased 31.6-92.5% than control group in the soil.
Advantage of the present invention and beneficial effect:
1) using plasma mutagenesis and the ultraviolet mutagenesis method of circular treatment has repeatedly guaranteed the stability of mutant strain;
2) azotobacter that provides has high nitrogen fixing capacity, can resist the Cd growth of higher concentration, can secrete indolylacetic acid, produce iron atom, has ACC desaminase activity etc., and stabilization characteristics of genetics goes down to posterity 14 times.Can promote the growth of ultra accumulation plant, improve the coefficient of concentration and transhipment coefficient of ultra accumulation plant;
3) the big production of mass-producing is lacked, can be carried out to simple, the easy cultivation of microbial inoculum nutritional requirement of the present invention, growth cycle;
4) microbial inoculum of the present invention can obviously promote the growth of ultra accumulation plant, increases the living weight of ultra tired massive planting thing, greatly improves the heavy metal accumulation coefficient and transhipment coefficient of ultra accumulation plant, can improve ecological environment of soil again, environmental friendliness and with low cost;
5) the quick removal for heavy metal in the short living bacterium reinforced soil of development and use plant rhizosphere provides new way, and the improvement and the restoration of the ecosystem of Heavy-metal Polluted Environment had great importance.
Description of drawings
Fig. 1 is a Ymu 6-2 stereoscan photograph;
Fig. 2 produces iron ion qualitative experiment photo for Ymu 6-2;
Fig. 3 is a Ymu 6-2 mitotic stability;
Fig. 4 is containing Cd for Ymu 6-2
2+Growing state among the liquid nutrient medium A;
Fig. 5 is the living weight (dry weight) of Indian mustard in containing Cd soil;
Fig. 6 is the content of Indian mustard over-ground part accumulation Cd;
Fig. 7 is the clearance of Indian mustard to Cd;
Fig. 8 is the living weight of alfalfa in containing Cd soil;
Fig. 9 is the content of alfalfa over-ground part accumulation Cd;
Figure 10 is the clearance of alfalfa to Cd;
Figure 11 is the living weight of rape in containing Cd soil;
Figure 12 is the content of rape over-ground part accumulation Cd;
Figure 13 is the clearance of rape to Cd.
Embodiment
According to the screening method of azotobacter strain provided by the invention, seed selection can tolerate the high azotobacter strain of nitrogenase activity of heavy metal cadmium, and the step of preparation is following:
1) with the laboratory from the edaphic bacillus YSGD06 of plant rhizosphere screening and preservation as starting strain;
2) mutagenic and breeding
(1) single cell suspension of preparation starting strain YSGD06
Starting strain YSGD06 is inoculated among the liquid nutrient medium A, and 28 ℃, 150rpm cultivates 16hrs, and is centrifugal, with the SPSS washing, places in the triangular flask that granulated glass sphere is housed, and vibration makes it be dispersed into single celled bacteria suspension;
Described culture medium A consists of: yeast powder 10g, NaCl 5g, peptone 10g, FeSO
47H
2O 0.005g, Na
2MoO
42H
2O 0.0025g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2.
(2) ultraviolet mutagenesis
The bacteria suspension of step (1) gained is regulated concentration to 10
5Between individual/ml, get 0.1ml and coat and contain 600mg/L Cd
2+On the no nitrogen solid medium B, carry out ultraviolet mutagenesis, the frequency of ultraviolet mutagenesis is 18W, and irradiation distance is 25cm, irradiation time 5min; 28 ℃ leave standstill cultivation 5days; Respectively select 25 bigger bacterium colonies, shake the multiple sieve of bottle, measure the nitrogenase activity of each bacterial strain with the acetylene reduction method; Select the bacterial strain of 5 strain nitrogenase activity height and good stability altogether, process the mutagenesis that bacteria suspension is used for next step plasma body;
The contained component of described no nitrogen solid medium B is: glucose 20g, KH
2PO
40.41g, MgSO
47H
2O 0.2g, NaCl 0.2g, CaSO
42H
2O 0.2g, CaCO
35g, CdCl
20.32g, agar 18g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2;
The step that described shake flask fermentation sieves again is: the 50 strain azotobacters that at first above-mentioned separation obtained are inoculated among the above-mentioned liquid nutrient medium A of 100ml, cultivate 12hrs.Respectively get in the Erlenmeyer flask that 5ml bacterium liquid is inoculated into the 250ml that 100ml liquid nutrient medium C is housed, 28 ℃, behind the 150rpm shaking table shaking culture 48hrs; Change tampon into rubber plug; Seal film and drip the wax sealing, syringe extracts 10 air, injects 10 acetylene gas; Continue reaction, measure nitrogenase activity with the acetylene reduction method;
The contained component of described liquid nutrient medium C is: glucose 20 g, KH
2PO
40.41 g, K
2HPO
40.52g, CaCl
20.2 g, MgSO
47H
2O 0.1 g, FeSO
47H
2O 0.005, Na
2MoO
42H
2O 0.0025 g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2.
(3) plasma body mutagenesis
With the 5 strain nitrogenase activity height of above-mentioned selection and the bacterial strain of good stability, process 10 respectively
5The bacteria suspension of individual/ml is respectively got 0.1ml and is evenly coated in the sterile petri dish, and petridish is put on the electrode below the plasma; The distance of regulating between the upper/lower electrode is 3mm; Voltage is 3V, and electric current is 0.5A, utilizes atmospherical discharges; Obtain uniform air dielectric barrier discharge plasma, be 3min discharge time.Immediately with SPSS or phosphoric acid salt wash-out, coat and contain 600mg/L Cd after the mutagenesis
2+On the no nitrogen solid medium B, shake the multiple sieve of bottle again, obtain the bacterial strain of 2 strain nitrogenase activity height and good stability, process bacteria suspension and be used for next step mutagenesis.
(4) cycle repeats ultraviolet mutagenesis and plasma body mutagenesis are 1-2 time, obtain strain tolerance heavy metal Cd and the high bacterial strain YMU6-2 of nitrogenase activity at last.
Fig. 1 is the Ymu6-2 stereoscan photograph, and Fig. 2 produces iron ion qualitative experiment photo for Ymu6-2, and Fig. 3 is the Ymu6-2 mitotic stability, and table 1 is wild azotobacter and the contrast of mutagenic strain nitrogenase activity.
According to the method for preparing azotogen provided by the invention, preparation can tolerate the high azotogen of nitrogenase activity of heavy metal cadmium, and the step of preparation is following:
The preparation of microbial inoculum may further comprise the steps:
1, strain fermentation
1. one-level triangular flask liquid culture
With frozen Ymu6-2 quick-thawing under 37 ℃ of conditions, the inoculum size according to 0.5% inserts 28 ℃ of 150rpm shaking culture 18hrs is housed in the triangular flask of liquid culture A;
Culture medium A consists of: yeast powder 10g, NaCl 5g, peptone 10g, FeSO
47H
2O 0.01g, Na
2MoO
42H
2O 0.005 g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2;
2. second order fermentation jar fermentation
1) the one-level nutrient solution with Ymu6-2 is equipped with in the fermentor tank of liquid nutrient medium E according to 5% inoculum size access, carries out fermentation culture, warm 28-32 ℃ in jar, pH7-8 aerobic culture 28 hrs;
Described substratum E consists of: sucrose 15g, starch 3g, dregs of beans 30g, KH
2PO
40.41 g, K
2HPO
40.3 g, CaCl
20.2 g, MgSO
47H
2O 0.2 g, Na
2MoO
42H
2O 0.005 g, water 1000ml, p H 7.0 ~ 7.2;
2) straw powder, turfy soil, rice chaff are pulverized with high speed disintegrator; Cross 60 mesh sieves; According to mass percent is the ratio uniform mixing of 50:30:20, is 0.3g/Kg with Sodium orthomolybdate and ferrous sulfate according to Sodium orthomolybdate, and ferrous sulfate is that the dosage of 0.05g/Kg adds and abundant mixing;
3) the Ymu6-2 fermented liquid that obtains in the step 1) is directly squeezed into container for storing liquid and promptly get the liquid microbial microbial inoculum; With dosage and the step 2 of the fermented liquid that obtains according to 100ml/kg) in the solid state substrate mixing of gained; 28 ℃ of fermentation 5days, colony counting method counts that Ymu6-2 quantity can reach 2.5 * 10 respectively in the solid-state microbial inoculum
9With 1.2 * 10
10CFU/g.
Use embodiment 2 microbial inoculums strengthening the Indian mustard cadmium pollution soil repair, its concrete steps are:
Potted plant soil picks up from Chang'an, Shaanxi Province county agricultural land soil, and supplying the total Cd content of examination soil is 0.25mg/kg, and soil is established 4 and added Cd
2+Level is respectively 0,20,40, and 80mg/kg is the CdCl of respective amount
2Wiring solution-forming makes to supply examination soil and heavy metal to mix repeatedly, leaves standstill balance 30d.Experiment is established the blank group respectively, is added the urea group and with the microbial inoculum group.After emerging, every basin keeps 3 strains., with the liquid bacterial agent water of above-mentioned preparation dilution proportion according to 1:200; The every basin of method that adopts root to irritate waters the bacterium liquid of 20ml dilution; Whenever water once at a distance from 3 weeks, the group that does not add bacterium is watered the dead thalline of Isodose, and the concentration that adds urea group urea is 0.12g/L.Plant strain growth was gathered in the crops after 50 days, measured the dry weight of overground part and root respectively, HNO
3-HClO
4Digestion, Cd concentration in each sample of aas determination. calculate overground part and the suction Cd amount of root and the soil sanitation rate of each processing of different treatment.
Visible like Fig. 4-7 result, with microbial inoculum with add the growth that urea all can promote Indian mustard, increased 13.4-42.7% with the dry weight of microbial inoculum group Indian mustard than control group, increased 4.2-29.9% and add the urea group than control group; With microbial inoculum with add urea and all can promote the Indian mustard over-ground part Cd
2+Enrichment, with microbial inoculum group Indian mustard cumulative Cd
2+Amount has increased 16.5-33.6% than control group, has only increased 3.7-13.6% and add the urea group than control group; With microbial inoculum group Indian mustard to Cd in the soil
2+Clearance increased 41.7-77.3% than control group, only increased 8.06-30.2% and add the urea group than control group.Therefore, this microbial inoculum can significantly improve the living weight of Indian mustard and to Cd
2+Enrichment, quicken Cd in the soil
2+Removal.
Use solid fungicide provided by the invention to be used to strengthen the alfalfa cadmium pollution soil repair, its concrete steps are:
Potted plant soil is identical with embodiment 3, and soil is established 3 and added Cd
2+Level is respectively 0,20,50mg/kg, the experiment establish respectively the blank group with the microbial inoculum group.Solid fungicide and soil are added soil and abundant mixing according to the consumption of 10g/kg soil, alfalfa treated, after emerging, every basin keeps 10 strains, and plant strain growth was gathered in the crops after 45 days, measured the overground part dry weight respectively, HNO
3-HClO
4Digestion, Cd concentration in each sample of aas determination. the suction Cd amount of the overground part of calculating different treatment and the soil sanitation rate of each processing.
Visible like Fig. 8-10 result, can promote the growth of alfalfa with microbial inoculum, increased 14.6-24.4% with the dry weight of microbial inoculum group alfalfa than control group; Microbial inoculum can promote the Indian mustard over-ground part to Cd
2+Enrichment, with microbial inoculum group alfalfa cumulative Cd
2+Amount has increased 26.4-51.9% than control group; With microbial inoculum group alfalfa to Cd in the soil
2+Purification rate increased 54.3-74.2% than control group.
Use solid fungicide provided by the invention to be used to strengthen the rape cadmium pollution soil repair, its concrete steps are:
Potted plant soil is identical with embodiment 3, and soil is established 3 and added Cd
2+Level is respectively 0,20,50mg/kg, experiment establish respectively the blank group with the microbial inoculum group.Rape is first at no Cd
2+Grow seedlings in the soil; Select the identical Brassica campestris L seedling of growing way and soak root with the liquid bacterial agent diluent of above-mentioned 1:50, control group soaks root with the dead bacterium liquid of same dose, after three weeks of growth, dilutes liquid irrigating root with the liquid bacterial agent of 1:200 again; Control group adds the dead microbial inoculum with dosage; All the other conditions are identical with embodiment 3, and plant strain growth was gathered in the crops after 45 days, Cd concentration in the dry weight of measuring overground part respectively and each sample.
Figure 11-13 result is visible, can obviously promote the growth of rape with microbial inoculum, has increased 15.1-36.4% with the dry weight of microbial inoculum rape than control group; Can promote the rape over-ground part to Cd with microbial inoculum
2+Enrichment, with microbial inoculum group rape cumulative Cd
2+Amount has increased 14.3-41.7% than control group; With microbial inoculum group rape to Cd in the soil
2+Purification rate increased 31.6-92.5% than control group.
Claims (8)
1. edaphic bacillus with spontaneous nitrogen fixing capacity, its classification called after edaphic bacillus (
Agrobacterium sp.) Ymu6-2, being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.5007.
2. the edaphic bacillus with spontaneous nitrogen fixing capacity according to claim 1 is characterized in that this bacterial strain has following characteristic: Ymu6-2 bacterium colony on solid medium A and is creamy white, circular protrusions, and neat in edge, periphery of bacterial colonies has transparent circle, G
-, aerobic or amphimicrobian, it is shaft-like that the ESEM demonstration is tubbiness, 28 ~ 32 ℃ of its righttest growth temperatures, the righttest pH value is 6.0 ~ 8.0; On solid medium A, can tolerate the Cd of 400-800mg/l
2+Grow, can tolerate the Cd of 224-448mg/l among the liquid medium within A
2+Growth, its nitrogen fixing capacity is at 0.56 ~ 0.73nmol10
7Cfu
-1H
-1, separating the inorganic phosphorus ability is 264.5mg/l, and the ability of producing indolylacetic acid is 15.25. ~ 30.6 μ g/ml, and the ability of producing siderophore is 0.806 ~ 0.402, and no acc deaminase is active;
Described solid medium A consists of: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2O 0.005-0.01g, Na
2MoO
42H
2O 0.0025-0.005 g, agar powder 15-20g, zero(ppm) water 1000ml, pH 7.0 ~ 7.2.
3. the microbiobacterial agent that contains the said edaphic bacillus of claim 1.
4. the preparation method of the said microbiobacterial agent of claim 3 may further comprise the steps:
1) strain fermentation
1. one-level triangular flask liquid culture
With frozen Ymu6-2 quick-thawing under 37 ℃ of conditions, insert respectively in the triangular flask that liquid culture A is housed according to the inoculum size of 0.5-1%, 28-32 ℃, shaking culture 15-20hrs;
Culture medium A consists of: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2O 0.005-0.01g, Na
2MoO
42H
2O 0.0025-0.005 g, zero(ppm) water 1000ml, p H 7.0 ~ 7.2;
2. second order fermentation jar fermentation
1) the one-level nutrient solution of the Ymu6-2 inoculum size according to 5-10% is inserted respectively in the fermentor tank that liquid nutrient medium E is housed, carry out fermentation culture, jar warm 28-32 ℃, cultivate pH7-8, aerobic culture 20-30 hrs;
Described substratum E consists of: sucrose 10-15g, starch 2-3g, dregs of beans 20-30g, KH
2PO
40.2-0.41 g, K
2HPO
40.3-0.52 g, CaCl
20.1-0.2 g, MgSO
47H
2O 0.1-0.2 g, Na
2MoO
42H
2O 0.0025-0.005 g, water 1000ml, p H 7.0 ~ 7.2;
2) straw powder, turfy soil, rice chaff being pulverized 60 mesh sieves, is the ratio uniform mixing of 50-70:20-30:10-20 according to mass percent, adds Sodium orthomolybdate and ferrous sulfate again, and wherein Sodium orthomolybdate is 0.2 ~ 0.5g/Kg, and ferrous sulfate is 0.05 ~ 0.08g/Kg;
3) the Ymu6-2 fermented liquid that obtains in the step 1) is directly squeezed into container for storing liquid; Promptly get liquid high-efficiency nitrogen-fixing microbial inoculum; With dosage and the step 2 of the liquid high-efficiency nitrogen-fixing microbial inoculum that obtains according to 50-100ml/kg) in the solid state substrate mixing of gained, 28-32 ℃ of fermentation got final product
in 5-7 days.
5. the application of the said edaphic bacillus of claim 1 in the heavy metal pollution of soil environmental improvement.
6. according to the said application of claim 5, it is characterized in that heavy metal is a cadmium ion.
7. according to the said application of claim 5; It is characterized in that method of use is: when plant is soaked seed, soak seed or plant root 2-5hrs with 50-100 liquid bacterial agent diluent doubly; Irritate root in nursery stage or vegetative period and adopt 100-500 liquid bacterial agent diluent 20-40ml/Kg soil dosage pouring diluent doubly, the whole growth phase is irritated root 1-3 time; Solid-state microbial inoculum is as the base manure and the use of topdressing, and using dosage is a 5-20g/Kg soil.
8. the application of the said edaphic bacillus of claim 1 in soil ecology is repaired.
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| CN103789224A (en) * | 2013-11-14 | 2014-05-14 | 西北大学 | Agrobacterium with free living nitrogen fixation, phosphate dissolution and potassium dissolution capacity and application of agrobacterium |
| CN108485998A (en) * | 2018-02-12 | 2018-09-04 | 暨南大学 | The agrobacterium T29 of one plant height effect activation mineral element and heavy metal cadmium |
| CN108496616A (en) * | 2018-02-12 | 2018-09-07 | 暨南大学 | A method of promote agrobacterium to be colonized in plant rhizosphere |
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| CN103789224B (en) * | 2013-11-14 | 2016-08-17 | 西北大学 | A kind of edaphic bacillus having from growing nitrogen-fixing, phosphorus decomposing and ability of dissolving potassium and application |
| CN103789224A (en) * | 2013-11-14 | 2014-05-14 | 西北大学 | Agrobacterium with free living nitrogen fixation, phosphate dissolution and potassium dissolution capacity and application of agrobacterium |
| CN108485998B (en) * | 2018-02-12 | 2021-08-24 | 暨南大学 | Agrobacterium T29 for efficiently activating mineral elements and heavy metal cadmium |
| CN108485998A (en) * | 2018-02-12 | 2018-09-04 | 暨南大学 | The agrobacterium T29 of one plant height effect activation mineral element and heavy metal cadmium |
| CN108496616A (en) * | 2018-02-12 | 2018-09-07 | 暨南大学 | A method of promote agrobacterium to be colonized in plant rhizosphere |
| CN108496616B (en) * | 2018-02-12 | 2020-03-17 | 暨南大学 | Method for promoting agrobacterium tumefaciens to fix plant rhizosphere |
| CN113905998A (en) * | 2019-02-05 | 2022-01-07 | 皮沃特生物股份有限公司 | Crop yield consistency enhancement by biological nitrogen fixation |
| CN112522138A (en) * | 2020-11-25 | 2021-03-19 | 山东大学 | Multifunctional agrobacterium strain and application thereof |
| CN112522138B (en) * | 2020-11-25 | 2022-06-17 | 山东大学 | Multifunctional agrobacterium strain and application thereof |
| CN112625960A (en) * | 2020-12-28 | 2021-04-09 | 山东大学 | Agrobacterium DP3, microbial inoculum thereof and application thereof in field of biological fertilizer preparation |
| CN113337445A (en) * | 2021-07-06 | 2021-09-03 | 福建省农业科学院农业生物资源研究所 | Gemonad RF4 and application thereof |
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