Phytoremediation of Metal Contaminated Soil Using Willow: Exploiting Plant-Associated Bacteria to Improve Biomass Production and Metal Uptake

Short rotation coppice (SRC) of willow and poplar is proposed for economic valorization and concurrently as remediation strategy for metal contaminated land in northeast-Belgium. However, metal phytoextraction appears insufficient to effectuate rapid reduction of soil metal contents. To increase both biomass production and metal accumulation of SRC, two strategies are proposed: (i) in situ selection of the best performing clones and (ii) bioaugmentation of these clones with beneficial plant-associated bacteria. Based on field data, two experimental willow clones, a Salix viminalis and a Salix alba x alba clone, were selected. Compared to the best performing commercial clones, considerable increases in stem metal extraction were achieved (up to 74% for Cd and 91% for Zn). From the selected clones, plant-associated bacteria were isolated and identified. All strains were subsequently screened for their plant growth-promoting and metal uptake enhancing traits. Five strains were selected for a greenhouse inoculation experiment with the selected clones planted in Cd-Zn-Pb contaminated soil. Extraction potential tended to increase after inoculation of S. viminalis plants with a Rahnella sp. strain due to a significantly increased twig biomass. However, although bacterial strains showing beneficial traits in vitro were used for inoculation, increments in extraction potential were not always observed.     

Variation in the uptake of Arsenic, Cadmium, Lead, and Zinc by different species of willows Salix spp. grown in contaminated soils

The experiment assessed the variability of in seven clones of willow plants of high biomass production (Salix smithiana S-218, Salix smithiana S-150, Salix viminalis S-519, Salix alba S-464, Salix ’Pyramidalis’ S-141, Salix dasyclados S-406, Salix rubens S-391). They were planted in a pots for three vegetation periods in three soils differing in the total content of risk elements. Comparing the calculated relative decrease of total metal contents in soils, the phytoextraction potential of willows was obtained for cadmium (Cd) and zinc (Zn), moderately contaminated Cambisol and uncontaminated Chernozem, where aboveground biomass removed about 30% Cd and 5% Zn of the total element content, respectively. The clones showed variability in removing Cd and Zn, depending on soil type and contamination level: S. smithiana (S-150) and S. rubens (S-391) demonstrated the highest phytoextraction effect for Cd and Zn. For lead (Pb) and arsenic (As), the ability to accumulate the aboveground biomass of willows was found to be negligible in both soils. The results confirmed that willow plants show promising results for several elements, mainly for mobile ones like cadmium and zinc in moderate levels of contamination. The differences in accumulation among the clones seemed to be affected more by the properties of clones, not by the soil element concentrations or soil properties. However, confirmation and verification of the results in field conditions as well as more detailed investigation of the mechanisms of cadmium uptake in rhizosphere of willow plants will be determined by further research.     

Development of a hydroponic screening technique to assess heavy metal resistance in willow (Salix)

A nutrient thin film hydroponic system has been developed which allows rapid screening of willow (Salix) clones for their resistance to heavy metals, and hence their use in phytoremediation. Two clones known to be different in their resistance to heavy metals (Salix burjatica (Germany) and S. triandra x viminalis (Q83)), could be distinguished on the basis of leaf biomass, root biomass and stem height after 6 weeks. There were also differences in the uptake of heavy metals between the two clones.     

Cuticular wax composition of Salix varieties in relation to biomass productivity

The leaf cuticular waxes of six Salix clones (one Salix miyabeana, one Salix dasyclados, one Salix eriocephala, two Salix purpurea, and one interspecific hybrid of Salix eriocephala x interior) with different biomass productivities were characterized by gas chromatography-mass spectrometry. Total wax content ranged from 6.3 to 16.8 microg cm(-2), and two distinct patterns of wax were measured. The wax from leaves of S. dasyclados 'SV1' differed from all other clones and was dominated by fatty acids (42%), high concentrations of n-alkanes (25%) and n-alcohols (28%), with low n-aldehyde content (4%). All other clones produced cuticular wax dominated by n-alcohols (32-51%), particularly 1-hexacosanol, with fatty acids (14-37%) and n-aldehydes (19-26%) present in lower abundances. Clones of Salix grown under identical environmental conditions produce noticeably different amounts of cuticular wax. In contrast to previous studies of Salix, total wax content was independent of biomass productivity, measured as basal area, suggesting that wax production is not directly linked with woody biomass production by shrub willows under these site conditions.     

Cadmium and copper uptake and translocation in five willow (Salix L.) species

The efficacy for phytoremediation of five willow species was tested by experimental copper and cadmium uptake in a greenhouse hydroponic system. Five treatments included two concentrations (5 and 25 microM for each metal) and a control. Metal concentrations in solution as well as solution uptake were monitored. Metal resistance was assessed through effects on the dry weight of roots and shoots. The willow species tested were generally resistant of increased Cu and Cd content. Metal accumulation was found in all plant organs of all species. Growth and transpiration were not decreased by 5 microM of copper and 25 microM of cadmium in the solution for most species. 25 microM copper caused injury and reduced the dry weight for all species after 21 d. Salix nigra was highly resistant of both Cu and Cd and accumulated more metals than other species. Future field study should be conducted to confirm the findings and feasibility of the phytoremediation technology using those species.     

Combined enhancements of temperature and UVB influence growth and phenolics in clones of the sexually dimorphic Salix myrsinifolia

Although several climatic factors are expected to change simultaneously in the future, the effect of such combined changes on plants have seldom been tested under field conditions. We report on a field experiment with dark-leaved willow, Salix myrsinifolia, subjected to enhancements in ultraviolet-A (UVA), UVB radiation and temperature, setup in Joensuu, Eastern Finland. S. myrsinifolia is a dioecious species, known as an important food plant for many herbivores. Cuttings of eight clones, four of each sex, of dark-leaved willow were planted in the field in spring 2009. In both 2009 and 2010, the total biomass increased significantly with temperature, and in 2010 there was an additive effect of UVB radiation. Both height and diameter increased with temperature in 2009, while the effect on height growth ceased in 2010. Males had greater diameter growth than females in 2010. Most phenolic compounds in the leaves decreased under enhanced temperature in both growing seasons. In 2010, four of six salicylates increased in response to enhanced temperature. Some quercetin derivatives increased under enhanced UVB radiation. Females had higher concentrations of chlorogenic acids than males, and while enhanced temperature reduced chlorogenic acid in females only, luteolins were reduced only in males. In summary, the combined enhancements gave no effects in addition to those that appeared under the single-factor treatments, except for the additive effect of UVB on temperature-increased biomass. The few gender-related differences found in response to climate change do not allow any marked expectations of future climate-induced changes in sex ratios.     

Selection ofAzolla forms resistant to the water lily aphid,Rhopalosiphum nymphaeae Life history of Rhopalosiphum nymphaeae

Genotypic and environmentally determined differences inSalix viminalis L. suitability forDasineura marginemtorquens (Bremi) (Diptera: Cecidomyiidae) growth and survival were investigated in laboratory and field experiments. Earlier studies have documented high neonate larval mortality on certainS. viminalis genotypes. Here we present results from laboratory experiments which show no difference in larval establishment behaviour on resistant and susceptible willow genotypes. These data indicate that larvae are not able to detect the plant characteristic responsible for resistance. In a field study insect performance was measured on willow shoots of dissmilar size. Adult body size was positively correlated to willow shoot length. In turn, body size was positively correlated to potential fecundity. These results are discussed in relation to observed high densities ofD. marginemtorquens in vigorously growing willows cultivated for biomass production.     

Efficiency of selected heavy metals accumulation by Salix viminalis roots

The efficiency of the absorption from the medium and accumulation by plant of ions of the heavy metals depends on many factors including plant age and its genotype. The heavy metals accumulation by 1-, 2- and 3-year-old plants was studied in the aspect of reclamation and revitalization effectiveness of demoted areas. Results of this study answer the question concerning the accumulation of seven heavy metals (Cd, Co, Cr, Cu, Ni, Pb and Zn) by willow cuttings (Salix viminalis) in environments with different concentrations of these elements. Cuttings used were originally grown on a substrate not contaminated with the metals and rich in nutrients. In order to eliminate the effect of soil physicochemical factors, the experiment was carried out using a hydroponic system. Results indicated significant effects of the investigated metal concentrations on their accumulation by willow. The extent of metal accumulation as an indicator of the remediation capacity of willow depended on the age of the cuttings used at planting.     

Genetic variation in resistance and tolerance to insect herbivory in Salix cordata

1. This study investigated whether sand-dune willow Salix cordata , exhibits genetic variation in resistance and tolerance to herbivory.
2. A field experiment using cuttings from nine willow clones demonstrated genetic variation in resistance to the specialist herbivore Altica subplicata , as measured by beetle densities. Willow clones differed significantly in both total biomass and leaf trichome densities, and herbivore densities were marginally correlated with both of these parameters.
3. Tolerance to herbivory was measured in a greenhouse experiment by comparing growth response of plants experiencing 50% artificial defoliation and plants experiencing no defoliation. Clones showed significant differences in tolerance to herbivory for some growth measures (changes in height and number of leaves), but not for other growth measures (stem diameter growth and final biomass).
4. Despite the significant genetic variation in both resistance and tolerance, no trade-off was found between resistance and tolerance to herbivory.     

Physiological and Proteomic Responses of Different Willow Clones (Salix fragilis X alba) Exposed to Dredged Sediment Contaminated by Heavy Metals

High biomass producing species are considered as tools for remediation of contaminated soils. Willows (Salix spp.) are prominent study subjects in this regard. In this study, different willow clones (Salix fragilis x alba) were planted on heavy-metal polluted dredging sludge. A first objective was assessment of the biomass production for these clones. Using a Gupta statistic, four clones were identified as high biomass producers (HBP). For comparison, a group of four clones with lowest biomass production were selected (LBP). A second objective was to compare metal uptake as well as the physiological and proteomic responses of these two groups. All these complementary data's allow us to have a better picture of the health of the clones that would be used in phytoremediation programs. Cd, Zn, and Ni total uptake was higher in the HBPs but Pb total uptake was higher in LBPs. Our proteomic and physiological results showed that the LBPs were able to maintain cellular activity as much as the HBPs although the oxidative stress response was more pronounced in the LBPs. This could be due to the high Pb content found in this group although a combined effect of the other metals cannot be excluded.     

不同能源柳无性系对NaCl胁迫的生理响应

为了探索不同能源柳无性系的耐盐潜力,在盆栽条件下,对1年生能源柳无性系2、4、C、E扦插苗设置NaCl盐分梯度,使其土壤含盐量保持在0、0.2%、0.4%、0.6%、0.8%,并以乡土旱柳为对照,胁迫45d后,对5个无性系的气体交换参数、保护酶活性和有机渗透调节物质进行测定。研究发现:(1)在低浓度盐胁迫下(土壤含盐量0 0.4%),导致各能源柳光合速率降低的是气孔因素;在高浓度盐胁迫下(土壤含盐量大于0.4%),导致各能源柳无性系光合速率下降的主要是非气孔因素。(2)随着土壤盐浓度的增加,各能源柳无性系的过氧化氢酶(CAT)活性逐渐减小,过氧化物酶(POD)活性先增加后减小,而超氧化物歧化酶(SOD)活性呈现增加的趋势,并且4个能源柳无性系在盐胁迫条件下保护酶活性强于旱柳。(3)在土壤含盐量小于0.6%时,4个能源柳无性系的可溶性蛋白含量始终大于旱柳;在整个胁迫过程中,能源柳C的脯氨酸含量始终大于旱柳。(4)与旱柳相比,4个能源柳无性系在土壤含盐量0 0.4%的土壤中都能良好生长,其中能源柳C的耐盐潜力更大,可在土壤含盐量为0.6%的土壤中正常生长。这说明,4个能源柳无性系均可在天津轻、中度盐渍化地区栽培。     

Phytoextraction of heavy metals by two Salicaceae clones in symbiosis with arbuscular mycorrhizal fungi during the second year of a field trial

We evaluated the potential of Salix viminalis (5027) and Populus × generosa for the phytoextraction of heavy metals (HM) inoculated or not with an arbuscular mycorrhizal (AM) fungus Glomus intraradices during a second year of growth in a randomized complete block field trial on a slightly contaminated site. Both plant clones produced high aboveground biomass yields, however P. × generosa produced significantly more biomass than S. viminalis. The two plant clones accumulated high concentrations of Cd and Zn in their shoots, while Cu and Pb were stored in their roots. In general, S. viminalis accumulated higher concentrations of HM. The inoculation of G. intraradices in the previous year did not influence plant clones’ biomass yields during the second growing season. However, Cu and Cd translocation to shoots was limited, and Cu was preferentially concentrated in roots of inoculated plants, compared to non-inoculated plants, which were also colonized by native AM fungi taxa. Efficiency of S. viminalis and P. × generosa for Cd and Zn rehabilitation in slightly contaminated soil has been demonstrated, but mycorrhizal inoculation did not significantly increase HM extraction.     

Melampsora rust species on biomass willows in central and north-eastern Germany

Melampsora willow rusts are the most important fungal pathogens in short rotation coppices of biomass willows. In the past, breeding programmes for rust resistant biomass willows concentrated on the distinction of races within the forma specialis Melampsora larici-epitea f. sp. larici-epitea typica that colonized Salix viminalis and related clones. In a new breeding program that is based on a wider range of willow species it is necessary to identify further Melampsora species and formae specialis that are pathogens of willow species other than S. viminalis. Therefore, three stock collections with Salix daphnoides, Salix purpurea, and other shrub willow species (including S. viminalis) species were sampled in north-eastern Germany. A fourth stock collection in central Germany contributed rusts of tree willows (Salix fragilis and Salix alba) and the large shrub Salix caprea. Out of 156 rust samples, 149 were successfully sequenced for ITS rDNA. A phylogenetic analysis combining Neighbour-Joining, Maximum-Likelihood and Bayesian analysis revealed six species: Melampsora ribesii-purpureae, Melampsora allii-salicis-albae, Melampsora sp. aff. allii-fragilis, Melampsora larici-pentandrae, Melampsora larici-caprearum, and Melampsora larici-epitea. The first four species were found exclusively on the expected hosts. Melampsora larici-caprearum had a wider host range comprising S. caprea and S. viminalis hybrids. Melampsora larici-epitea can be further differentiated into two formae speciales. The forma specialis larici-epitea typica (59 samples) colonized Salix viminalis clones, Salix purpurea, Salix×dasyclados, and Salix×aquatica. In contrast to this relatively broad host range, f. sp. larici-daphnoides (65 samples) was found exclusively on Salix daphnoides. With the distinction and identification of the rust species/formae speciales it is now possible to test for race-specific resistances in a more targeted manner within the determined pairings of rust and willow species.     

Does nitrogen fertilization have an impact on the trade-off between willow growth and defensive secondary metabolism?

The effect of nitrogen fertilization on the phytomass production, shoot length and leaf secondary phenolics in nine Salix myrsinifolia clones was investigated. Cuttings taken from 1-year-old and 2-year-old shoot parts of field cultivated clones were grown at three concentrations of nitrogen (7, 150 and 300 ppm) in a greenhouse for one growing season. The willow clones differed significantly in phytomass yield and secondary phenolics content. Nitrogen fertilization affected significantly the growth and secondary metabolism of willow clones. In most clones, the addition of nitrogen from a sub-optimum concentration (7 ppm) to an optimum concentration (150 ppm) appeared to reduce the amounts of salicortin, chlorogenic acid and unknown salicylate and increased shoot phytomass, but a supraoptimum nitrogen concentration (300 ppm) resulted in highly variable growth and secondary phenolic responses. A significantly negative correlation between leaf phytomass and amount of total phenolics at sub-optimum and optimum N-treatments indicates trade-off between growth and secondary metabolism in willow clones at these treatments. However, the leaf phytomass:total amount of phenolics ratio varied significantly among clones, and in all clones it was not significantly lower at sub-optimum N-treatment than at optimum N-treatment.     

Stress tolerance of five willow clones after irrigation with different amounts of landfill leachate

Short-rotation willow (Salix sp.) coppice, a commercial crop grown for energy purposes, is used for phytoremediation of landfill leachate in Sweden. However, the leachate's chemical composition can cause plant damage mainly due to high NaCl and NH4 concentrations. A pot experiment was conducted in order to quantify the growth responses of five different willow clones ("78-183", "Jorr", "Loden", "Olof", "Tora") to irrigation with different leachate mixtures (corresponding to 240, 180, and 120 mg Cl l(-1)) and to assess the applicability of leaf length and leaf fluctuating asymmetry as stress indicators. A series of plant traits (shoot, root and leaf dry weight, leaf area, leaf length and leaf fluctuating asymmetry) were measured. The irrigation with leachate resulted in reduced relative growth rates but there were no clear differences between the different concentrations. The clones "Jorr" and "Loden" performed best in terms of differences in relative growth rate between control and leachate treatments. Leaf length appeared to be a useful stress diagnostic tool for use in situ showing a high correlation to growth, whereas fluctuating asymmetry showed no such correlation. Higher N, lower P and higher Na concentrations in plant stems treated with leachate than control plants were observed.     

Lead uptake and translocation by willows in pot and field experiments

Plant growth and lead (Pb) uptake by seven willow varieties were investigated in pot and field experiments to assess the suitability of willows for phytoremediation of Pb at heavily contaminated sites such as skeet ranges. Differences in uptake and translocation of Pb in Salix were observed between pot and field experiments. In the pot experiment, willows grown in Pb-contaminated field soil for 6 months showed tolerance to very high soil Pb concentration (21,360 mg kg(-1)), and with the addition of EDTA were able to take up and translocate more than 1000 mg kg(-1) Pb into above-ground tissues. In the field experiment, all willow varieties showed tolerance to heterogeneously high soil Pb concentrations. Plants were also able to take up and translocate Pb into above-ground tissues. However, after 4.5 months, the lead concentration in the above-ground tissues of willows grown in soil amended with EDTA was less than 200 mg kg(-1). The results from the pot experiment suggest that Salix varieties have the potential to take up and translocate significant amounts of Pb into above-ground tissues using EDTA. However, to verify the phytoextraction abilities of Salix in the field, additional research is needed.     

Assessment of willow (Salix sp.) as a woody heavy metal accumulator: field survey and in vivo X-ray analyses

Trees that accumulate metals are important plants for restoring contaminated soil because of their high biomass. In our previous study, we discovered that Salix miyabeana has the capability to take up high levels of Cd, and identified the several accumulation sites of the endogenous metals in the leaf parts of plants. To analyze the detailed localization of Cd in apoplastic and symplastic compartments in S. miyabeana, synchrotron radiation-based micro X-ray fluorescence (SR-μ-XRF) analysis and micro X-ray absorption near edge structure (μ-XANES) measurements were performed on beam line 37XU of the SPring-8 (Hyogo, Japan). The two-dimensional metal distribution of segments of young stems was obtained by μ-XRF with approximately 2 μm(2) X-ray beams and showed the predominant localization of Cd in the apoplastic region. μ-XANES analyses suggest that the apoplastic detoxification of Cd in willow depends on Cd-oxygen, but not on Cd-sulfur, interaction. S. miyabeana growing near an old mining site in Japan was then examined to evaluate the metal accumulating ability of this plant in the field. The metal concentration in the leaves of the plants was compared to that in the soil and enrichment factors (EFs) were calculated for Cu, Zn, Cd and Pb. Results showed efficient removal of Cd and Zn from the contaminated sites by the willow plants. In order to discuss the combined and long-term effect of multiple heavy metals on S. miyabeana grown in soil, variable-pressure scanning electron microscopy fitted with energy dispersive X-ray analysis (VPSEM-EDX) and SR-μ-XRF was used to characterize the serration of leaves. The combination of 2D elemental images revealed metal accumulation in the tip cells in serrations without any exudation of heavy metals from the hydathodes.     

The use of non-dormant rods as planting material: A new approach to establishing willow for environmental applications

In this trial we tested a new planting technique for willows used in environmental applications such as green structures in urban settings. Three commercial willow clones were planted in spring 2008 at different periods as non-dormant woody rods. Comparing the main growth parameters at the end of the season, we found that planting date may affect plant establishment depending on the clone. Based on our results, the use of non-dormant willow material collected and planted in spring is possible in our environment with several different clones. Best results were obtained with Salix viminalis (Sv 5027), which can be planted with a delay of about one month without showing any significant decrease in biomass production or survival rate.     

Host selection and performance of the giant willow aphid, Tuberolachnus salignus Gmelin – implications for pest management

1 The recent increase in planting of selected willow clones as energy crops for biomass production has resulted in a need to understand the relationship between commonly grown, clonally propagated genotypes and their pests. 2 For the first time, we present a study of the interactions of six willow clones and a previously unconsidered pest, the giant willow aphid Tuberolachnus salignus. 3 Tuberolachnus salignus alatae displayed no preference between the clones, but there was genetic variation in resistance between the clones; Q83 was the most resistant and led to the lowest reproductive performance in the aphid 4 Maternal effects buffered changes in aphid performance. On four tested willow clones fecundity of first generation aphids on the new host clone was intermediate to that of the second generation and that of the clone used to maintain the aphids in culture. 5 In the field, patterns of aphid infestation were highly variable between years, with the duration of attack being up to four times longer in 1999. In both years there was a significant effect of willow clone on the intensity of infestation. However, whereas Orm had the lowest intensity of infestation in the first year, Dasyclados supported a lower population level than other monitored clones in the second year.     

Screening willow clones for Radiocesium uptake at varying potassium supply in solution culture

Transfer of radiocesium from soils to the wood of willows is generally low. Therefore, willow short rotation coppice for energy production is a possible alternative land use in areas contaminated by radiocesium. A large number of willow clones are available differing in, for example, biomass production or nutrient uptake. In order to select a clone with a high biomass production and a low radiocesium uptake, 12 clones were screened in nutrient solutions, spiked with ¹³⁴Cs. Radiocesium concentrations in the plants varied less than twofold between the clones. Shoot radiocesium concentrations were significantly related to shoot potassium concentrations (R² = 0.55).

In a second experiment, four of these clones were grown in solution culture at varying K concentrations (0.08 to 2 mM). The radiocesium uptake was more affected by K supply than by the type of clone. The shoot radiocesium concentrations were reduced between 3.5‐ and 5.2‐fold by increasing the K supply from 0.08 to 0.4 mM, A further increase to 2 vaM did not affect radiocesium uptake. We conclude that intervarietal differences between willow clones were of less significance in determining radiocesium concentrations in the shoots than the impact of external K concentrations.