| Phytoremediation which utilize hyperaccumulating plants to extract heavy metals has recently been proposed as a promising way to remove toxicants from contaminated soils. Mycorrhiza can promote absorption of plant nitrogen, phosphorus, mineral nutrients and water, thereby enhancing plant resistance to environmental stress. Therefore, the application of plant-endophyte symbiotic system is proposed to be a potential technique to improve efficiency of phytoremediation.Alnus nepalensis, belonging to Betulaceae, is fast growing, and colonizes denuded habitats freshly exposed soils, rocky and eroded slopes, and which is a common native species in the subtropical to temperate belts of the Himalaya. And, some reports indicated that Alnus species have the ability to accumulate heavy metals. In addition, the Alnus species may form symbiotic associations with dark septate endophytes (DSE), arbuscular mycorrhizal fungi (AMF), ectomycorrhizal fungi (EMF), and Frankia (actinomyces), and have the capability of fixing nitrogen by nodules. DSE are broadly classified as conidial or sterile septate fungal endophytes and comprise a heterogeneous assemblage of fungi, which form melanised structures such as inter-and intracellular hyphae and microsclerotia in the plant roots commonly occurred in some highly stress environments.In the present study, the accumulation capacity for Zn, Pb and Cd by Alnus nepalensis growing in a lead and zinc mining spoil heap in Munai, Lancang County, Yunnan province, southwest China was evaluated. In addition, we surveyed the colonization of DSE in plant roots, and isolated the DSE strains from the roots, also identified based on morphological and molecular analysis. The DSE strains were selected to test their tolerances to Cd2+in vitro, and the high Cd2+resistant DSE's EC50(median effect concentration) were determined. The potential application of Alnus nepalensis-DSE for phytoremediation of Zn, Pb and Cd contaminated soil was also discussed. Results of the thesis were as follows: 1. The colonization status of AMF and DSE in Alnus nepalensis naturely growing in Munai lead-zinc mine area were examined by alkaline lysising and acid fuchsin staining.The concentrations of Zn, Pb and Cd in rhizosphere soils were also examined. The results indicated that the contents of zinc, lead and cadmium, far exceeded the third class national standard in the plot1, plot2and plot3, thus the three sampled sites should be catalogued to heavily polluted areas; in plot4and plot5, zinc, lead is not exceeded, while cadmium exceeds the national soil polluted standard, thus be considered to be lightly polluted areas. DSE and AMF colonization were found in all investigated Alnus nepalensis root samples, and microsclerotia and typical AM structures were observed. Moderate colonisation levels of DSE from26.11%to35.51%in five plots were observed, and the highest level occurred in the moderately contaminated soil (P3), and decreased with higher (P1and P2) and lower (P4and P5) available metals in the soils which showed a normal distribution. AMF colonization (20.22%-29.20%) belongs to the moderate colonization too, but no any trend was found with the increase of metal concentrations in the soil.2. In total,153strains of DSE were isolated from48plant root samples collected in the five plots. The isolated ratio was3.5:1,4.4:1,3:1,2.5:1and2.75:1in P1, P2, P3, P4and P5, respectively. And phylogenic analysis based on their sequences of ITS rDNA showed that most of the strains belong to ascomyetes, including Cladosprium, Exophiala, Cladophialophora, Phialophora, Leptodontidium, Paraphoma, and some taxonomic status is not clear. Overall, Phialophora is the dominant genera in the roots of Alnus nepalensis, and31strains come from this genus.3. Resistance of the153DSE strains to cadmium in vitro were initially screened, and found that100strains from PI, P2and P3were more tolerance than the53strains from P4and P5. Among the tested DSE strains,24(19from P1, P2, P3;5from P4, P5) DSE strains grew well in the medium containing600mg/L Cd. In addition, the EC50(median effect concentration) values for the resistant strains of DSE to cadmium were determined. The result showed that the EC50of11strains are generally in150mg/L or so, but the strain of PB43reached234.42mg/L. Overall, Strains isolated from the heavily polluted areas are commonly higher than lightly polluted in cadmium resistance or biomass.4. The accumulation capacity for Zn, Pb and Cd by Alnus nepalensis growing in a lead and zinc mining spoil heap in Munai was evaluated, and the potential application of Alnus nepalensis for phytoremediation was also discussed. It was found that metal concentrations of the shoots ranged from92to263mg kg-1for Zn,24to219mg kg-1for Pb,10to59mg kg-1for Cd, respectively; and in the root heavy metal varied from116to574mg kg-1for Zn,28to3550mg kg-1for Pb, and22to95mg kg-1for Cd, respectively. Bioaccumulation factors and biotransfer factors ranged from0.19to21.92and0.34to0.79for Zn,0.01to7.02and0.04to1.44for Pb, and0.12to34.36and0.04to2.06for Cd, respectively. There was significant correlation between the root and soil metal concentration (Zn R=0.94, Pb R=0.99, Cd R=0.98; P<.01). Metal concentration in the shoot showed a good correlation with the soil Zn (R=0.97; P<0.01) and Pb (R=0.85;P<0.01), but Cd.The above results suggested that DSE and AMF colonized in all Alnus nepalensis root samples growing in lead and zinc mining spoil heap, and they could be the important components of the root system. Their biological and ecological functions deserved further studies. One hundred and fifty-three fungal strains were isoalated from the roots of A. nepalensis, among which Phialophora isolates were dominant. Both resistant and sensitive isolates were found in root samples from lightly-contaminated and heavily-contaminated plots, but fungal isolates from heavily-contaminated plots were generally more resistant. Moreover, A. nepalensis had a relatively high tolerance to Zn, Pb and Cd, and a stronger ability for absorption and accumulation of these metals in its roots. All in all, the use of A. nepalensis as a vegetation cover for the phytostabilization of land contaminated by heavy metals in Yunnan province does seem to have considerable potential. |
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