| This study is conducted to investigate the AMF status and heavy metal (HM) accumulation characteristics of tree species grown in a lead-zinc mine area, Feng County, Shaanxi Provinence, China. The potential candidate trees that have adapted to local environment were selected for application in phytoremediation according to multiple criteria. The AMF composition and diversity in the root and rhizosphere of the candidate tree was analyzed using restriction fragment length polymorphism (RFLP) screening, and then the dominant and tolerant AMF group types were isolated. A greenhouse experiment was conducted to determine the roles of AMF in plant growth, Pb accumulation and traslocation, photosynthetic system and antioxidant enzyme system. The purpose of our study is to fully reveal the mechanisms of AMF improving the efficiency of phytoremediation and then provides a scientific basis for practical application of AMF in HM polluted soils. The main results are as follows:1. HM accumulation characteristics and AMF status of tree species grown in a lead-zinc mine areaFifteen different tree species were detected and all them could establish good symbiosis with AMF in Pb-Zn polluted area. The mycorrhizal frequency (MC), mycorrhizal colonization intensity (M%) and spore density (SD) reduced concomitantly with increased Pb and Zn levels, however, positive correlations were found between arbuscule density (A%) and soil total/DTPA-extractable Pb concentrations. The average concentrations of Pb, Zn, Cu and Cd in plant samples were 168.21 mg/kg,96.61 mg/kg,41.06 mg/kg and 0.79 mg/kg, respectively. Populus purdbmii Rehd. accumulated the highest concentrations of Zn (432.08 mg/kg) and Cu (140.85 mg/kg) in its leaves. Considerable amount of Pb (712.37 mg/kg) and Cd (3.86 mg/kg) were concentrated in the roots of Robinia pseudoacacia Linn. and Populus simonii Carr., respectively.Redundancy Analysis (RDA) showed that the efficiency of phytoremediation was enhanced by AM symbioses, and soil pH, Pb, Zn, Cd levels were the main factors influencing the HM accumulation characteristics of plants. After analyzing HM concentrations in all plant samples, we can conclude that the tree species absorb different HMs selectively to some extent:translocating more essential metals (Zn and Cu) into the aerial parts, while retaining more toxic heavy metals (Pb and Cd) in the roots to protect the above-ground parts from damage, which might be the primary mechanism for trees surviving in HM polluted soil. According to translocation factor (TF), bioconcentration factor (BCF), growth rate and biomass production, five tree species (Ailanthus altissima (Mill.) Swingle, Cotinus coggygria Scop., P. simonii, P. purdomii and R. pseudoacacia) were considered to be the most suitable candidates for phytoextraction and/or phytostabilisation purposes.2. AMF community in roots and rhizosphere soil of R. pseudoacacia grown in Pb-Zn mine areaTo better understand the adaptation of candidates plant and the related mycorrhizae to extreme environmental conditions, AMF colonization, spore density and community structure were analyzed in roots and rhizosphere soil of R. pseudoacacia. AMF could establish a good symbiotic relationship with roots of R. pseudoacacia, and the MC ranged form 21.7% to 39.3%. The MC declined with increased HM concentration. Samples of roots, soils and spores were analyzed by restriction fragment length polymorphism (RFLP) screening with AMF-specific primers (NS31 and AMI), and sequencing of rRNA small subunit (SSU). The phylogenetic analysis revealed 28 AMF group types (58 AMF sequences), including six AMF families:Glomeraceae, Claroideoglomeraceae, Diversisporaceae, Acaulosporaceae, Pacisporaceae, and Gigasporaceae. Of all AMF group types, six (21%) were detected based on spore samples alone, four (14%) based on root samples alone, and five (18%) based on samples from root, soil and spore.After alalyzing all detected AMF sequences, we found that Glo9 (Rhizophagus intraradices), Glo17(Funneliformis mosseae) and Acau3(Acaulospora sp.) were the three most abundant AMF group types in the current study. The results from RDA suggested that soil Pb and Zn concentrations, pH, organic matter content, and phosphorus levelshad significant correlations with the AMF species compositions in root and soil samples. In root samples, all AMF species were greatly inhibited by soil Pb and Zn concentrations, organic matter content, and phosphorus level, excepting for Glo6、Glo7、Glo8、Glo9、Glo17 and Divel. By contrast, in soil samples, only Glo4、Glo17 and Acau3 had high tolerance to soil Pb and Zn, while other AMF speies were considerably inhabited by soil Pb and Zn concentrations. Overall, the uncontaminated sites had higher species diversity than sites with heavy metal contamination. The species number and Margalef index of AMF community in unpolluted study sites were 1.43 and 1.49 higher than these in HM polluted study sites, respectively.3. The mechanisms of AMF on improving the Pb tolerance of R. pseudoacaciaA controlled greenhouse experiment was conducted to evaluate the effects of two AMF species (E mosseae and R. intraradices) on the growth, Pb accumulation, photosynthesis and antioxidant enzyme activity of a legume tree (R. pseudoacacid) at Pb addition levels of 0,500, 1,000 and 2,000 mg/kg in soil. AMF symbiosis decreased Pb concentrations in plant leaves and promoted biomass accumulation as well as the contents of photosynthetic pigments. The biomass, net photosynthetic rate, chlorophyll a (Ch1 a) and Ch1 b content of E mosseae plants increased 26.17%～37.11%,7.94%-49.86%,5.84%-41.27% and 8.31%～18.06% compared with non-mycorrhizal plants, respectively. On the contrary, The biomass, net photosynthetic rate, chlorophyll a (Ch1 a) and Ch1 b content of E mosseae plants increased 27.59%-56.02%, 6.50%～74.59%,9.85%-34.95% and 12.15%～15.11% compared with non-mycorrhizal plants, respectively.Furthermore, AM inoculation significantly enhanced the enzymatic activities of superoxide dismutase (SOD), ascorbate peroxidases (APX) and glutathione peroxidase (GPX) but decreased the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents of R. pseudoacacia. The H2O2 and MDA contents of E mosseae plants decreased 5.67%～29.53% and 27.92%-34.59% respectively, while the H2O2 and MDA contents of R. intraradices plants declined 520.47%-30.95% and 33.04%-36.75% respectively compared with non-mycorrhizal plants. Moreover, mycorrhizal dependency (MD) of plants increased with increasing Pb stress levels, and MD presented significantly positive corralations with soil Pb concentrations (/? mosseae:F=0.643, P=0.001; R. intraradices:F=0.815, P=0.000). However, AM inoculation showed no effects on enzymatic activities of peroxidase (POD), catalase (CAT) and glutathione reductase (GR) of R. pseudoacacia, which might result from different resistence of antioxidant enzymes for Pb pollution.Our results further clarify that AMF improve the tolerance of host to HM contamination probably through enhancing efficiencies of photosynthetic system and free radical scavenge system of host. Therefore, the application of the two Glomus species associated with R. pseudoacacia could be a promising strategy to enhance phytoremediation efficiency in Pb contaminated environments.4. The effects of AMF on phytoremediation and plant competition in Pb polluted soilA controlled greenhouse experiment was conducted to investigate the contributions of an AMF species, R. intraradices, to tree-herb competition (legume-legume or legume-grass) and phytoremediation in Pb polluted soil. When plants were grown individually, MDs of the legumes (0.13-0.27) were higher than that of grass (-0.03-0.03). Furthermore, the MDs of legumes increased with increase of Pb concentration (R. pseudoacacia:F=0.832, P=0.001; T.pratense:F=0.696, P=0.012;M. sativa:F=0.749, P=0.005), while the MD of Lolium perenne was negatively correated with Pb concentration, but was not siganificant (F=-0.261, P=0.412). The mycorrhizal legumes had much higher biomass compared with that of non-mycorrhizal plants (R. pseudoacacia,8.04%-28.21%; T. pratense,20.42%-32.48%; M. sativa,15.37%-27.82%), whereasAMF inoculation had no effects on biomass of grass.In co-culture planting pattern, MC of plant was enhanced by legume presence but inhibited by grass presence in co-culture system. N, P, S and Mg concentrations of mycorrhizal legumes were larger than these of non-mycorrhizal legumes, however, no differences in Na, K and Ca concentrations could be detected between non-mycorrhizal and mycorrhizal plants. Legume presence decreased soil pH (5.00%-8.64%) and thereby increased soil DTPA-Pb concentration, resulting in larger amount of Pb accumulated in both non-mycorrhizal and mycorrhizal plants (8.45%～40.75%). The interaction between legume tree and legume herbs supported the stress-gradient hypothesis (SGH):the relative interaction intensity between legume tree-herbs shifted from negative or neutral to positive with increasing Pb levels. However, the relationship between RII and Pb stress level did not exhibit a simple linear correlation:a hump-shaped curve could be found in legume tree-herbs planting patterns, while a valley-shaped curve could be observed in legume tree-grass planting pattern coincident with increasing Pb stress level, which probably since various tolerances, competitive ability and MD among plants.Our results highlight the important roles of AMF and legume herbs in assisting to improve phytoremediation efficiency in Pb polluted soil. The planting pattern with legume herbs that have high MD and the ability to improve Pb accumulation, together with legume tree with deep root systems and long life, can be instructive for optimizing in terms of maximizing both phytoremediation efficiency and biomass production. |
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