CN103266073B - Sedum alfredii endophyte and application thereof - Google Patents
Sedum alfredii endophyte and application thereof Download PDFInfo
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Abstract
The invention discloses a sedum alfredii endophyte and an application thereof. The endophyte is named as Acinetobacter calcoaceticus Sasm3 with a preservation number of CGMCC NO.7334. The invention also discloses an application of the endophyte in plant-microorganism combined remediation on heavy metal contaminated soil. The application comprises the steps of: inoculating sedum alfredii with the endophyte and planting the sedum alfredii inoculated with the endophyte in the heavy metal contaminated soil to be remedied; and from the second growing season of the sedum alfredii inoculated with the endophyte, re-inoculating the sedum alfredii with the endophyte in each growing season, and harvesting the sedum alfredii. The endophyte can be used for enhancing the resistance of the sedum alfredii to high-concentration heavy metal so as to promote the healthy growth of the sedum alfredii in the environment of high-concentration heavy metal, thereby increasing the biomass of the sedum alfredii and enhancing the remediation efficiency of the sedum alfredii to the heavy metal contaminated soil.
Description
Technical field
The invention belongs to geobiont recovery technique field, particularly relate to a kind of Sedum alfredii Hance endophyte and application thereof.
Background technology
Along with the quick propelling of the high speed development of industry, the modernization of agricultural production and urbanization, heavy metal pollution of soil situation is day by day serious.
Heavy metal cannot be degraded by microorganisms, and most of heavy metal has the feature such as poor mobility, holdup time length, disguise, irreversibility in soil, and soil is once contaminated by heavy metals just extremely difficult reduction.Heavy metal in soil can affect the multiple metabolic activity such as plant growth, cell division on the one hand, suppresses the growth of crop thus reduces crop yield.On the other hand, the heavy metal in soil can enter food chain by Removed In Soil-crop System, brings great risk to human health.In the face of the Mass disturbance that tens heavy metals occurred in the past few years cause, reduce Mobility of Heavy Metals In Soil Environment content, reduce its pollution to agricultural product, ensure the ecosystem and human health extremely urgent.
In face of limited land resource, the reparation of heavy-metal contaminated soil becomes a vital task of agricultural sustainable development.Because heavy metal directly can not can carry out pollution abatement thing by the degraded in plant rhizosphere or plant as organic pollutant, the difficulty of the phytoremediation of heavy metal in soil is much bigger.
Phytoremediation (phyto-remediation) is the based technique for in-situ remediation utilizing pollutant in green plants transfer, accommodation or conversion environment, with conventional art as compared with the physics and chemistry methods such as soil moved in improve the original method, soil fixing, Soil leaching, have efficient, environmental friendliness, cost are low, do not cause the advantages such as secondary pollution.Sedum alfredii Hance finds at first on location, ancient Pb/Zn ore deposit, Quzhou City of Zhejiang Province, it is the primary Zn/Cd hyperaccumulative plant of only one China, effectively can extract Zn, Cd, Pb element in soil, be not only the good model material that research plant absorption, transhipment and ultraproduct tire out Zn/Cd mechanism, also be the good material promoting the heavy metal-polluted dyeing technique of phytoremediation, in its plant, heavy metal concentration is several times even tens times in soil.Although Sedum alfredii Hance is compared most heavy metal hyperaccumulative plant and is wanted some advantage in growth rate with biomass, but the efficiency that its repairing heavy metal in soil pollutes still be subject to growing slow, biomass is little, adaptability is weak and heavy metal-polluted soil to the restriction of property, therefore, how to promote that Sedum alfredii Hance grows, degeneration-resistant, improve biomass and to the absorption of heavy metal-polluted soil, transhipment, accumulation heavy metal ability, this practical application tool for application Sedum alfredii Hance restoration of soil polluted by heavy metal is of great significance.
Summary of the invention
The invention provides a kind of endophyte, the growth of Sedum alfredii Hance can be promoted, improve the absorbability of Sedum alfredii Hance heavy metal.
A kind of endophyte, Classification And Nomenclature is acinetobacter calcoaceticus (Acinetobactercalcoaceticus), complete called after acinetobacter calcoaceticus (Acinetobacter calcoaceticus) Sasm3, this bacterial strain has been deposited in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 03 19th, 2013, deposit number is CGMCC NO.7334.
Bacterial strain of the present invention is faint yellow circular colonies on LB culture medium, little, tasteless, surface wettability, transparent, the smooth of the edge is neat, Gram-negative, shaft-like, resistance to Zn concentration 25mM in LB fluid nutrient medium, resistance to Cd concentration 2.5mM, growth hormone (IAA) can be produced, produce acc deaminase, secrete addicted to iron element, anti-ampicillin 100 μ g/mL and tetracycline 15 μ g/mL, does not detect eccrine fiber element enzyme and pectase.
This bacterial strain is separated and obtains from Sedum alfredii Hance plant, surely can grow in the plant of Sedum alfredii Hance, promotes the growth of Sedum alfredii Hance and the absorbability of heavy metal.
Present invention also offers the application of described endophyte in plant-microorganism renovation of heavy metal polluted soil with combined.
Described heavy-metal contaminated soil is containing cadmium, zinc etc.
Described application specifically comprises:
(1) by the endophyte described in Sedum alfredii Hance inoculation, Sedum alfredii Hance is planted in heavy-metal contaminated soil to be repaired after inoculation;
(2) from postvaccinal second Growing season of Sedum alfredii Hance, each Growing season again to the endophyte described in Sedum alfredii Hance inoculation, and cradles Sedum alfredii Hance.
Sedum alfredii Hance can enduring high-concentration heavy metal pollution, absorb the heavy metals such as Zn, the Cd in soil and be accumulated in overground part tissue, it is the elite germplasm that repairing heavy metal in soil pollutes, but because its growth is slow, biomass is less, therefore remediation efficiency is not high, limit its application in practice.
The present invention to tire out in the root circle of environmental Sedum alfredii Hance and body concentration and separation to carrying out the good special endogenetic bacteria done mutually with Sedum alfredii Hance from growth in the ultraproduct of Proterozoic, then screening Promoting plant growth and strengthening can extract the bacterial strain of heavy metal efficiency, finishing screen chooses strain endophyte Acinetobacte rcalcoaceticus (Acinetobacter calcoaceticus) Sasm3, this bacterial strain is applied in plant soil restoration heavy metal pollution, set up plant-microorganism combined remediation technology system, Sedum alfredii Hance can be made better to utilize nutrition in soil, increase the biomass of Sedum alfredii Hance, improve the remediation efficiency of Sedum alfredii Hance heavy metal, give full play to the reparative potential of Sedum alfredii Hance.
In step (1), the inoculation method of described endophyte is: get Sedum alfredii Hance branch, after cuttage root-taking, immerses in the nutrient solution containing described endophyte, cultivates 5 ~ 7 days.
For ensureing the success rate of inoculation, the concentration of endophyte is generally 10
3~ 10
6cFU/mL, is preferably 10
5cFU/mL.
After inoculation, endophyte grows the inside at Sedum alfredii Hance root surely, with root growth symbiosis, Sedum alfredii Hance can be planted in contaminated by heavy metals soil.
9 annual ~ December and 3 ~ June are the Growing season of Sedum alfredii Hance, and Sedum alfredii Hance growth when just plantation is comparatively slow, does not need again to inoculate endophyte in a Growing season of primary vaccination endophyte, and later each season of growth inoculation once.
Because Sedum alfredii Hance grows in soil, therefore, from postvaccinal second Growing season of Sedum alfredii Hance, when again carrying out the inoculation of endophyte, fertile or Nutrition Soil as matrix, can impose in described heavy-metal contaminated soil after the bacterium liquid containing endophyte is mixed with matrix using machine.
The concentration of described endophyte is 10
8cFU/g matrix.
Wherein, the concentration of endophyte is 10
6~ 10
9cFU/g matrix, is preferably 10
8cFU/g matrix.
Due to bacterium survival and growth need certain humidity, after described bacterium liquid mixes with matrix, the humidity of matrix is 60% ~ 70%.
Application process can be executed for ditching deep placement or rainy day spill, and preferably, application process is ditching deep placement.Ditching deep placement is more conducive to endophyte and fully contacts with the root system of Sedum alfredii Hance, can also provide stable living environment for endophyte simultaneously.
Sedum alfredii Hance first time cradles from postvaccinal second Growing season, cradle when biomass is maximum, results overground part, when cradling, stem 4 ~ 8cm or reservation Sedum alfredii Hance 5 ~ 10 blades of retaining Sedum alfredii Hance overground part are advisable, and later each Growing season results once, i.e. 1 year 2 season of results, plant continuously for many years, realize gathering in continuously, high-efficiency and continuous reparation.
Compared with prior art, beneficial effect of the present invention is:
Endogenetic bacteria of the present invention can be grown surely in Sedum alfredii Hance body, the patience of Sedum alfredii Hance to high concentration heavy metal can not only be significantly improved, promote that it grows up healthy and sound in higher heavy metal concentration environment, the biomass of Sedum alfredii Hance can also be significantly improved and strengthen Sedum alfredii Hance to Zn, the absorbability of the heavy metals such as Cd, solve Sedum alfredii Hance poor growth in heavy-metal contaminated soil, the problem that biomass is little, enhance the environmental suitability of Sedum alfredii Hance, there is good social benefit, economic benefit and environmental benefit, significant for promoting the application of Sedum alfredii Hance in soil remediation.
The present invention is by inoculation endophyte, Sedum alfredii Hance seedlings plugging survival rate is improved greatly, survives artifact amount and significantly improve, stronger to high density pollution soil patience, absorb more heavy metal under same biomass, considerably improve the remediation efficiency of Sedum alfredii Hance heavy metal contaminated soil.In addition, after inoculation endophyte, the Sedum alfredii Hance grown in the wild is cradled causes the plant death rate significantly to reduce, and plant regrows more vigorous.
It is simple that the present invention inoculates process, maintenance cost is cheap, be conducive to long-term, stable heavy metal contaminated soil to repair, avoiding problems in chemurgy measure regulation and control the secondary pollution problem of adding chemical amelioration agent and causing, be conducive to improving the ecological environment.
Accompanying drawing explanation
Fig. 1 is Sedum alfredii Hance endophyte plate screening schematic diagram;
Fig. 2 is the phylogenetic tree analysis of Sedum alfredii Hance endophyte Sasm3;
Fig. 3 be in water planting environment endophyte Sasm3 on the impact of Sedum alfredii Hance root growth;
Fig. 4 is the impact that in earth culture environment, endophyte Sasm3 grows Sedum alfredii Hance;
Fig. 5 be in earth culture environment endophyte Sasm3 on the impact of Sedum alfredii Hance biomass;
Fig. 6 a is endophyte Sasm3 on the impact of Sedum alfredii Hance overground part heavy metal Zn and Cd concentration;
Fig. 6 b is endophyte Sasm3 on the impact of Sedum alfredii Hance root heavy metal Zn and Cd concentration;
Fig. 7 a is endophyte Sasm3 on the impact of Sedum alfredii Hance overground part heavy metal Zn and Cd accumulation;
Fig. 7 b is endophyte Sasm3 on the impact of Sedum alfredii Hance root heavy metal Zn and Cd accumulation.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further explained.
Embodiment 1 urgees the separation of the biological prosthetic endogenetic bacteria of Sedum alfredii Hance
Gather the Sedum alfredii Hance Wild plant grown fine from mining area, carry out the test of endophyte separation screening immediately.
Adopt conventional endophyte separation method.Whole strain Sedum alfredii Hance removes stale blade, then uses tap water 30min, then with distilling washing 3 times, each 3min; Suck plant surface moisture with aseptic blotting paper, then by sterile scissors, blade, stem and root are separated, by every some respectively with 70% alcohol leaching 3min, aseptic washing 3 times, 0.5%HgCl
2soak 1min, then use aseptic water washing more than 5 times, each 1min, and it is dull and stereotyped to confirm the germ-free condition on plant surface the sterilized water washed for the last time to be coated with LB.
Prepare LB solid medium (tryptone 10g, yeast extract 5g, NaCl10g, agar powder 1.5%, constant volume 1000mL, pH7.2,121 DEG C of sterilizings), be poured on by culture medium on 9cm culture dish, every block culture dish is about 20mL.In clean superclean bench, with aseptic operation cutter (cooling after flame calcination sterilizing) cutting plants sample thin slice, and thin slice is laid on the culture medium of culture dish, each culture medium about 9 (as shown in Figure 1), with sealed membrane seal culture dish be inverted in 30 DEG C of incubators cultivate one week.
Choose lower Sedum alfredii Hance section bacterial plaque around with oese, line isolates single bacterium colony three times repeatedly, is inoculated in LB test tube slant respectively, 4 DEG C of preservations (can preserve 2 months).
Embodiment 2 urgees the screening of the biological prosthetic endogenetic bacteria of Sedum alfredii Hance, purifying, preservation and qualification
Refrigerator takes out preserves bacterial classification, and be inoculated in LB fluid nutrient medium, 37 DEG C of incubated overnight, oese dips a small amount of bacterium liquid and again rules, and picking list bacterium colony is in LB fluid nutrient medium, and 30 DEG C grow to logarithmic phase.This bacterium liquid part is in order to preservation, and in 1.5mL sterile centrifugation tube, add 300 μ L sterile glycerols and 700 μ L logarithmic phase bacterium liquid ,-80 DEG C of preservations (can preserve 2 years), remaining bacterium liquid carries out the qualification of key physiological biochemical indicator.
1, pathogenic qualification
Above-mentioned logarithmic phase bacterium liquid is inoculated into fresh damaged blade face (Sedum alfredii Hance, tobacco, rape), compares with sterilized water, do not find blade Disease Characters; Extract 100 μ L bacterium liquid with asepsis injector to be expelled in the onion stem of fresh and healthy, compare with sterilized water, cultivate one week, do not find that onion has Disease Characters for 30 DEG C.
2, resistance to Zn/Cd feature
To containing Zn/Cd LB culture medium (respectively containing concentration gradient: Zn(1,2,4,5,8,10,12,15,20,25,30,35 or 40mM) or Cd(0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,1.0,1.2,1.5,2.0,2.5,3.0,3.5,4.0 or 5.0mM)) in inoculation 10 μ L logarithmic phase bacterium liquid, cross liquid for 37 DEG C to cultivate, observe endogenetic bacteria growing state under different Zn/Cd concentration.
3, acc deaminase ability is produced
When by the field planting of growth-promoting rhizosphere bacterium to seed coat or root system of plant time, can synthesize and producing IAA (auxin), the IAA of new synthesis will be absorbed by plant and form Endogenous auxin, thus the division of stimulating plant cell and prolongation.Meanwhile, IAA can excite the activity of ACC synzyme, S-adenosylmethionine is changed into 1-amino-cyclopropane-1-carboxylic acid.Acc deaminase is one of index measuring plant growth-promoting rhizobacteria Promoting plant growth ability.
With reference to Honma M and Shimomura T(Honma M & Shimomura T.Metabolismof 1-aminocyclopropane-1-carboxylic acid.Agric Biol Chem, 1978,42:1825-1831) set up to measure α-one butyric acid to detect the method for activity that endogenetic bacteria produces acc deaminase.
4, IAA ability is produced
Heteroauxin (IAA) be a kind of can coordinate plant growth grow auxin.
Adopt Salkowski colorimetric method (Sarwar & Kremer.Determination of bacteriallyderived auxins using a microplate method.Letters in Applied Microbiology, 2008,20:282-285) measure the ability that separator produces auxin class material IAA.
5, the mensuration addicted to iron element ability is secreted
Addicted to iron element be synthesized under low iron bar part by microorganism small molecule amount, can the chelate of special chelates ferric ions, the bacterium secreted addicted to iron element can be used for preventing and treating fungal disease.
Utilize CAS flat band method (Schwyn & Neilands.Universal chemical assay for thedetection and determination of siderophores.Analytical Biochemistry, 1987,160:47-56) bacterial detection secretion is addicted to the ability of iron element.
By detecting, screen the resistance to Zn/Cd ability of a strain, produce acc deaminase ability, produce IAA ability, secrete addicted to iron element ability endophyte bacterial strain all preferably, called after Sasm3, every physiological and biochemical index of this bacterial strain is see table 1.
Table 1 endophyte Sasm3 repairs relevant physiological and biochemical index to promotion
6, identification of strains and preservation
(1) morphological feature
This bacterial strain Sasm3 is faint yellow circular colonies on LB culture medium, little, tasteless, surface wettability, transparent, the smooth of the edge is neat, Gram-negative, shaft-like, resistance to Zn concentration 25mM in LB fluid nutrient medium, resistance to Cd concentration 2.5mM, growth hormone (IAA) can be produced, produce acc deaminase, secrete addicted to iron element, anti-ampicillin 100 μ g/mL and tetracycline 15 μ g/mL, does not detect cellulase and pectase.
(2) molecular biology identification
By the bacterium liquid 7000r/min collected by centrifugation thalline of Sasm3 bacterial strain, utilize Shanghai raw work biological bacterium DNA extraction kit to extract DNA, and utilize primer 2 7F and 1429R to increase the 16S rDNA of this bacterial strain, wherein, the base sequence of primer is:
27F：5′-AGAGTTTGATCCTGGCTCAG-3′；
1492R：5′-GGTTACCTTGTTACGACTT-3′。
Entrust Hua Da gene to check order by after the pcr amplification product purifying of acquisition, obtain through order-checking the 16S rDNA fragment that length is 1458bp, the number of registration in GenBank is KF002588, and nucleotide sequence is as shown in SEQ ID NO.1.
By Blast comparison, and utilize MEGA5.0 software to carry out Phylogenetic Analysis, result shows, the homology more than 99% (as Fig. 2) of the 16S rDNA nucleotide sequence of bacterial strain Sasm3 and acinetobacter calcoaceticus (Acinetobacter calcoaceticus).Therefore, identify that this Sasm3 bacterial strain is acinetobacter calcoaceticus (Acinetobacter calcoaceticus), called after acinetobacter calcoaceticus (Acinetobacter calcoaceticus) Sasm3, and this bacterial strain is delivered to the China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC NO.7334, and preservation date is on 03 19th, 2013.
Embodiment 3 endophyte is on Sedum alfredii Hance growth and the impact of heavy metal soil remediation
Nutrient solution consists of: K
2sO
4, 0.7mmol/L; KCl, 0.1mmol/L; Ca (NO
3)
24H
2o, 2mmol/L; MgSO
47H
2o, 0.5mmol/L; KH
2pO
4, 0.1mmol/L; H
3bO
3, 10 μm of ol/L; MnSO
4h
2o, 0.5 μm of ol/L; ZnSO
47H
2o, 1 μm of ol/L; (NH
4)
4moO
244H
2o, 0.01 μm of ol/L; FeSO
47H
2o is dissolved in the 20 μm of ol L boiled
-1na
2in EDTA solution, 20 μm of ol/L.Nutrition pH5.8 at night is adjusted) with NaOH or HCl
(1) water planting environment
Water culture is adopted to carry out Fast-propagation to Sedum alfredii Hance.The branch of clip Sedum alfredii Hance new life, every treaty 10cm is long, and only leave 5 ~ 8 blades of shoot apex, be inserted in 0.1 times of mill water culture nutrient solution, temperature is 23 ~ 25 DEG C, and illumination condition is 200 ~ 300 μ E/m
2s, after 3 weeks, shoot growth goes out a little root, then uses 1 times of above-mentioned Solution culture method, and ventilates in nutrient solution.Within every 3 ~ 4 days, change one time of nutrition liquid.In the 4th circumferential nutrient solution, add the thalline (collected by centrifugation logarithmic phase thalline, sterilized water washs 3 times) of endophyte Sasm3, the thalline in every milliliter of nutrient solution is 10
5cFU, changes nutrient solution afterwards and no longer accesses Sasm3 thalline.
Grow the growing state observing Sedum alfredii Hance after 1 month, as Fig. 3 shows, compared with not inoculating the contrast of endophyte Sasm3, the Sedum alfredii Hance growth of inoculation endophyte Sasm3 is more vigorous, and root system is more flourishing.
(2) earth culture environment
Adopt earth culture method plantation Sedum alfredii Hance.After water planting Sedum alfredii Hance inoculates one week, by its root washes clean, be transplanted to mine soil potted plant in the middle of, the physicochemical property of mine soil is in table 2, and keep the humidity of soil about 55%, growth time is 2 months.
As seen from Figure 4, after inoculation endophyte Sasm3, Sedum alfredii Hance the upperground part biomass obviously increases.After Sedum alfredii Hance is gathered in the crops, the biomass of overground part and underground part (i.e. root) is weighed and analyzed, find, compared with not connecing the contrast of endophyte Sasm3, after inoculation endophyte Sasm3, the overground part of Sedum alfredii Hance and the biomass of underground part all have remarkable increase in various degree, wherein, the increase by 18.35% of the upperground part biomass, underground part biomass increases 35.03%(as shown in Figure 5), illustrate that endophyte Sasm3 significantly can promote the growth of Sedum alfredii Hance overground part and underground part.
The basic physical and chemical of mine used soil tested by table 2
Measure the heavy metal quantitative analysis of Sedum alfredii Hance root and overground part and accumulation and analyze further, the detection of Zn and Cd adopts conventional method.As Fig. 6 a, Fig. 6 b, Fig. 7 a and Fig. 7 b, find, after inoculation endophyte Sasm3, heavy metal concentration and the accumulation of Sedum alfredii Hance overground part and underground part all significantly increase, wherein, overground part and root Zn content concn increase by 18.7% and 9.31% respectively, and content concn increase by 47.48%, the root Cd concentration increase of overground part Cd is not obvious; The accumulation of control group Sedum alfredii Hance overground part Zn is 401.52mg, and the accumulation of root Zn is 8.314mg, after inoculation endophyte Sasm3, the accumulation of Sedum alfredii Hance overground part Zn can reach 561.07mg, improve 39.73%, the accumulation of root Zn also reaches 12.22mg, improves 47.02%.Meanwhile, after inoculation endophyte Sasm3, the accumulation of Sedum alfredii Hance overground part Cd reaches 26.75mg, improves 74.17%, root accumulation raising 29.86%.
With do not inoculate compared with endophyte process, plant-microorganism recovery technique system significantly improves Sedum alfredii Hance biomass (during the soil process of mine, the level of signifiance of overground part and root biomass is respectively 0.027 and 0.034) and overground part heavy metal Cd concentration (during the soil process of mine, Zn and the Cd concentration level of signifiance is respectively 0.002 and 0.0001).
In addition, we are also found by test, under laboratory condition, do not inoculate endophyte plant survival rate and are about 71.0% ~ 90.1%, and after inoculating endophyte, make Sedum alfredii Hance seedlings plugging survival rate 92.8% ~ 99.5%.Because mine soil soil property is barren, if increase by about 20% Nutrition Soil microbial inoculum in mine soil, (pasture and water of rotting, concentration is CFU10
7~ 10
8the Sasm3 endophyte of/g) after, biomass is more increased to 94.7% ~ 136.2%.
Embodiment 4 urgees the application of the biological prosthetic endogenetic bacteria of Sedum alfredii Hance
Sedum alfredii Hance seedling strain Fast-propagation (method with reference to embodiment 3): get Sedum alfredii Hance top branch and take root in nutrient solution about 45 days.LB fluid nutrient medium shakes Sasm3 bacterium liquid and spends the night, and sterilized water washes away culture medium, by 10
5cFU/mL is inoculated into takes root in seedling basket, ventilate cavity 1 week.Now Sedum alfredii Hance endophyte is inner by surely growing at root, with root growth symbiosis.
Cottage propagation: Sedum alfredii Hance inoculation endophyte is after 1 week, and endophyte is inner by surely growing at its root, plants in heavy-metal composite pollution mine soil by the strain of Sedum alfredii Hance seedling, seedling density is every square metre of plantation 70 ~ 100 strains.During firm plantation, growth is comparatively slow, and 9 annual ~ December and 3 ~ June are its Growing season.Again need not inoculate endophyte in a first plantation Growing season, the Sasm3 bacterium liquid washing away culture medium once, during inoculation, mixes as matrix with fertilizer or Nutrition Soil, keeps cell concentration to be 10 by later each season of growth inoculation
8cFU/g matrix, humidity 60% ~ 70%, ditching deep placement (also can spill the rainy day and execute), ditching deep placement consumption is 0.12 ~ 0.15kg/m
2, after two season of growth, more new plant can be grown.
Cradle results: first time cradles from postvaccinal second Growing season, cradle when biomass is maximum, results overground part, during harvesting, overground part retains stem about 4 ~ 8cm, later each Growing season results once, i.e. in 1 year 2 season of results, are planted for many years continuously, realize harvesting continuously, high-efficiency and continuous reparation.
Statistics finds, after inoculation endophyte, the survival rate of Sedum alfredii Hance is close to 100%, and usually not inoculating endophyte plant survival rate is 71.0 ~ 90.1%, and by harvesting causing the seedling strain death rate significantly reduce about 50% after the Sedum alfredii Hance grown inoculation in the wild, seedling strain regrows more vigorous.The Sedum alfredii Hance inoculation endophyte grown in the wild is after 3 months, and same biomass overground part absorbs heavy metal Zn, Cd amount improves 18.7% and 47.48% respectively.
Claims (9)
1. an endophyte, is characterized in that, called after acinetobacter calcoaceticus (Acinetobactercalcoaceticus) Sasm3, and deposit number is CGMCC NO.7334.
2. endophyte as claimed in claim 1 repairs the application in zinc and/or cadmium pollution soil at Sedum alfredii Hance-microbial association.
3. apply as claimed in claim 2, it is characterized in that, comprising:
(1) by the endophyte described in Sedum alfredii Hance inoculation, Sedum alfredii Hance is planted in heavy-metal contaminated soil to be repaired after inoculation;
(2) from postvaccinal second Growing season of Sedum alfredii Hance, each Growing season again to the endophyte described in Sedum alfredii Hance inoculation, and cradles Sedum alfredii Hance.
4. apply as claimed in claim 3, it is characterized in that, in step (1), the inoculation method of described endophyte is: get Sedum alfredii Hance branch, after cuttage root-taking, immerses in the nutrient solution containing described endophyte, cultivates 5 ~ 7 days.
5. apply as claimed in claim 3, it is characterized in that, in step (2), the inoculation method of described endophyte is: mixed with matrix by the bacterium liquid containing endophyte, impose in described heavy-metal contaminated soil.
6. apply as claimed in claim 5, it is characterized in that, the concentration of described endophyte is 10
6~ 10
9cFU/g matrix.
7. apply as claimed in claim 5, it is characterized in that, after described bacterium liquid mixes with matrix, the humidity of matrix is 60% ~ 70%.
8. apply as claimed in claim 5, it is characterized in that, application process is spill ditching deep placement or rainy day to execute.
9. apply as claimed in claim 3, it is characterized in that, described in when cradling, retain the stem 4 ~ 8cm of Sedum alfredii Hance overground part or retain Sedum alfredii Hance 5 ~ 10 blades.
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