Assessment of heavy metal stress in the adaptation strategies of Tulipa luanica growing on serpentine soil through some biomarkers in comparison to Tulipa kosovarica.

The aim of this study was to gain a better understanding of how Tulipa luanica adapts to growth in soil with higher concentrations of heavy metals and to assess potential toxic effects using various biomarkers, in comparison to Tulipa kosovarica, a typical serpentine species. For this purpose, we analyzed the concentrations of Al, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb, and Zn in the soil, as well as their accumulation in plants and their associated stress effects. The results indicate that, despite the presence of some metals in very high concentrations in the soil (Al, Fe, Mn, and Ni), they are translocated in minimal amounts within plant organs, particularly in T. luanica. Nearly all metals exhibited significantly higher concentrations in T. kosovarica when compared to T. luanica. Based on the analysis of biomarkers, it is apparent that T. luanica shows greater sensitivity to these conditions. This is evident through the decreased activity of δ-aminolevulinic acid dehydratase and levels of δ-aminolevulinic acid, malondialdehyde, and glutathione observed in T. luanica. It appears that T. luanica effectively restricts the absorption of metals in serpentine soils; however, it experiences oxidative stress induced by these metals, setting it apart from the more resilient T. kosovarica.

Broomrape Species Parasitizing Odontarrhena lesbiaca (Brassicaceae) Individuals Act as Nickel Hyperaccumulators.

The elemental defense hypothesis supports that metal hyperaccumulation in plant tissues serves as a mechanism underpinning plant resistance to herbivores and pathogens. In this study, we investigate the interaction between Odontarrhena lesbiaca and broomrape parasitic species, in the light of the defense hypothesis of metal hyperaccumulation. Plant and soil samples collected from three serpentine sites in Lesbos, Greece were analyzed for Ni concentrations. Phelipanche nowackiana and Phelipanche nana were found to infect O. lesbiaca. In both species, Ni concentration decreased gradually from tubercles to shoots and flowers. Specimens of both species with shoot nickel concentrations above 1000 mg.kg-1 were found, showing that they act as nickel hyperaccumulators. Low values of parasite to O. lesbiaca leaf or soil nickel quotients were observed. Orobanche pubescens growing on a serpentine habitat but not in association with O. lesbiaca had very low Ni concentrations in its tissues analogous to excluder plants growing on serpentine soils. Infected O. lesbiaca individuals showed lower leaf nickel concentrations relative to the non-infected ones. Elevated leaf nickel concentration of O. lesbiaca individuals did not prevent parasitic plants to attack them and to hyperaccumulate metals to their tissues, contrary to predictions of the elemental defense hypothesis.

The effect of endophytic fungi on growth and nickel accumulation in Noccaea hyperaccumulators.

The role of endophytic fungi isolated from different populations of European Ni hyperaccumulators was investigated in regard to the microorganisms' ability to enhance the hyperaccumulation of Ni in Noccaea caerulescens. Effects of particular species of endophytic fungi on adaptation of N. caerulescens to excess Ni were tested by co-cultivation with single strains of the fungi. Seven of these had a positive effect on plant biomass production, whereas two of the tested species inhibited plant growth; biomass production of inoculated plants was significantly different compared to non-inoculated control. Inoculation with six fungal strains: Embellisia thlaspis, Pyrenochaeta cava, Phomopsis columnaris, Plectosphaerella cucumerina, Cladosporium cladosporioides and Alternaria sp. stimulated the plant to uptake and accumulate more Ni in both roots and shoots, compared to non-inoculated control. P. columnaris was isolated from all plant species sampled. Strains isolated from Noccaea caerulescens and Noccaea goesingensis increased Ni root and shoot accumulation of their native hosts (compared to non-inoculated control). Inoculation of different populations of Noccaea with P. columnaris of foreign origin did not cause its host to accumulate more Ni, with the exception of the Ni-unadapted ecotype of N. goesingensis. Inoculation with P. columnaris from N. caerulescens significantly improved Ni uptake, but the effect of the fungus was not as prominent as in the case of N. caerulescens. By comparing the transcriptomes of N. caerulescens and N. goesingensis from Flatz inoculated with P. columnaris, we showed that enhanced uptake and accumulation of Ni in the plants is accompanied by an upregulation of several genes mainly involved in plant stress protection and metal uptake and compartmentation.

Chromium, manganese, nickel, and cobalt mobility and bioavailability from mafic-to-ultramafic mine spoil weathering in western Massachusetts, USA.

Fragmented ultramafic bodies in New England were important mineral resources until the early twentieth century, yet few studies have addressed their potential to release trace metals to terrestrial and aquatic environments. Here, we evaluate the release of four trace metals (Cr, Co, Mn, and Ni) from a historic serpentine-talc "soapstone" quarry in Blandford, MA, USA. Soil pits, sediment and rock samples, and stream water samples were collected from upslope undisturbed areas, within the mine spoils and mine face, and downslope of the mine. In order to provide a bottom-up approach for understanding metal release, careful petrographic analysis, electron-dispersive spectroscopy, and wavelength-dispersive spectroscopy were employed to provide first-order insight into the mineralogy of the deposit and a determination of potential metal-bearing phases. Trace metals were primarily observed in ultramafic sheet silicates, primary Fe-oxides, and interstitial weathering-related sulfates. Bulk rock concentrations were Cr (1550 mg kg-1), Co (230 mg kg-1), Mn (1100 mg kg-1), and Ni (1960 mg kg1); Cr, Co, and Ni were elevated relative to the surrounding country rock. However, soils and sediments total concentrations were comparable to background soil concentrations: Cr (119 mg kg-1), Co (73 mg kg-1), Mn (894 mg kg-1), and Ni (65 mg kg-1). Moreover, < 0.5% of the total concentrations were bioavailable (0.1 M ammonium acetate extraction), implying that metals are present as insoluble forms. However, ~ 20% of the total Cr, Mn, Ni, and Co concentrations were strong acid extractable, suggesting mobilization over the coming decades. Stream water concentrations of Mn and Cr were < 50 µg L-1, below concentrations outlined by USEPA drinking water standards, and WHO water guidelines for Ni. These results suggest that transport of Cr, Mn, Ni, and Co from the serpentine-talc as dissolved compounds or sediments is limited by retention within silicate and oxides.

Phylogeny and historical biogeography of Lithospermeae (Boraginaceae): Disentangling the possible causes of Miocene diversifications.

Studies about the drivers of angiosperm clade diversifications have revealed how the environment continuously alters the species chances to adapt or to go extinct. This process depends on complex interactions between abiotic and biotic factors, conditioned to the geological and tectonic settings, the genetic variability of species and the rate at which speciation occurs. In this study, we aim to elucidate the timing of diversification of the Lithospermeae, the second largest tribe within Boraginaceae, and to identify the possible morphological and ecological characters associated with shifts in diversification rates of the most species-rich clades. Lithospermeae includes ca. 470 species and 26 genera, among which are some of the largest genera of the family such as Onosma (150 spp.), Echium (60 spp.), and Lithospermum (80 spp.). An exhaustive study of the whole clade is not available to date and its evolutionary history and diversification rates are incompletely known. In the present study, we provide the most comprehensive phylogeny of the group so far, sampling 242 species and all 26 genera. We found that crown-groups and diversification rates of Lithospermeae largely date back to the Mid-Miocene, with high diversification rates in the largest genera, though only significantly high in Onosma. Our analysis fails to associate any of the functional or morphological traits considered with significant shifts in diversification rates. The timing of the diversification of the species-rich clades corresponds with Miocene tectonic events and global climate changes increasing aridity across Eurasia and western North America. These results suggest a causal link between known ecological features of Lithospermeae (i.e., pre-adaptation to arid, open habitats, and mineral soils) and their diversification. Future studies should expand the sampling of individual subclades and detailed functional analyses to identify the contribution of adaptations to arid conditions and pollinator shifts.

Assessment of trace element accumulation potential of Noccaea kovatsii from ultramafics of Bosnia and Herzegovina and Serbia.

In this work, we present the results of the investigation of trace elements (Fe, Mg, Ni, Zn, Cu, Cr, Co, Cd, Pb) accumulation potential of Noccaea kovatsii (Heuff.) F. K. Mey., from the Balkan Peninsula. The study included eight populations from ultramafic soils, six from Bosnia and Herzegovina, and two from Serbia. Principal component analysis (PCA) was used to reveal relationships of elements in soil, and Pearson's correlation coefficients for analysing associations of available quantities of elements in soil and those in roots and shoots of N. kovatsii. Uptake and translocation efficiency was assessed by using bioconcentration (BCF) and translocation factors (TF). All the analysed populations of N. kovatsii emerged as strong Ni accumulators, with the highest shoot concentrations of 12,505 mg kg-1. Even thought contents of Zn in plant tissues of N. kovatsii were under the hyperaccumulation level (602 mg kg-1 and 1120 mg kg-1 respectively), BCF was up to 667, indicating that certain surveyed populations have strong accumulative potential for this element.

Assessing nickel tolerance of bacteria isolated from serpentine soils.

Serpentine soils present unique characteristics such as a low Ca/Mg ratio, low concentration of nutrients, and a high concentration of heavy metals, especially nickel. Soil bacterial isolates from an ultramafic complex located in the tropical savanna known as the Brazilian Cerrado were studied. Nickel-tolerant bacteria were obtained, and their ability to remove nickel from a culture medium was assessed. Bacterial isolates presented higher tolerance to nickel salts than previously reported for bacteria obtained from serpentine environments in other regions of the world. In addition, the quantification of nickel in cell pellets indicated that at least four isolates may adsorb soluble forms of nickel. It is expected that information gathered in this study will support future efforts to exploit serpentine soil bacteria for biotechnological processes involving nickel decontamination from environmental samples.

Major QTL controls adaptation to serpentine soils in Mimulus guttatus.

Spatially varying selection is a critical driver of adaptive differentiation. Yet, there are few examples where the fitness effects of naturally segregating variants that contribute to local adaptation have been measured in the field. Plant adaptation to harsh soil habitats provides an ideal study system for investigating the genetic basis of local adaptation. The work presented here identifies a major locus underlying adaptation to serpentine soils in Mimulus guttatus and estimates the strength of selection on this locus in native field sites. Reciprocal transplant and common-garden studies show that serpentine and nonserpentine populations of M. guttatus differ in their ability to survive on serpentine soils. We directly mapped these field survival differences by performing a bulk segregant analysis with F2 survivors from a field transplant study and identify a single QTL where individuals that are homozygous for the nonserpentine allele do not survive on serpentine soils. Genotyping the survivors from an independent mapping population reveals that this same QTL controls serpentine tolerance in a second, geographically distant population. Finally, we show that this QTL controls tolerance to soil properties, as opposed to some other aspect of the field sites that may differ, by performing a laboratory-based common-garden experiment in native serpentine soils that replicates the survival differences observed in the field. These results indicate that despite the myriad chemical and physical challenges plants face in serpentine habitats, adaptation to these soils in M. guttatus has a simple genetic basis.

Harnessing Rhizobia to Improve Heavy-Metal Phytoremediation by Legumes.

Rhizobia are bacteria that can form symbiotic associations with plants of the Fabaceae family, during which they reduce atmospheric di-nitrogen to ammonia. The symbiosis between rhizobia and leguminous plants is a fundamental contributor to nitrogen cycling in natural and agricultural ecosystems. Rhizobial microsymbionts are a major reason why legumes can colonize marginal lands and nitrogen-deficient soils. Several leguminous species have been found in metal-contaminated areas, and they often harbor metal-tolerant rhizobia. In recent years, there have been numerous efforts and discoveries related to the genetic determinants of metal resistance by rhizobia, and on the effectiveness of such rhizobia to increase the metal tolerance of host plants. Here, we review the main findings on the metal resistance of rhizobia: the physiological role, evolution, and genetic determinants, and the potential to use native and genetically-manipulated rhizobia as inoculants for legumes in phytoremediation practices.

Micro-edaphic factors affect intra-specific variations in trace element profiles of Noccaea praecox on ultramafic soils.

The aim of this study was to compare trace element profiles of Noccaea praecox (Wulfen) F. K. Mey. growing on ultramafic soils in different habitat types and to observe differences in uptake and translocation of trace elements. Physico-chemical characteristics of the soil and concentrations of P2O5, K2O, Ca, Fe, Mn, Zn, Cu, Ni, Cr, Pb, Cd, and Co in plant samples were presented. Biological concentration, accumulation, and translocation factors were calculated to estimate accumulation potential of different N. praecox accessions. All of the studied accessions were Ni hyperaccumulators (with shoot concentrations up to 14,593 mg kg-1), but with notable differences in accumulation and translocation rates. Significant differences in accumulation and translocation patterns of trace elements were observed among accessions from habitats characterized as serpentine steppes on dry, shallow soils in contrast to the accessions from habitats with higher soil moisture, and higher content of organic matter.

Serpentine soils affect heavy metal tolerance but not genetic diversity in a common Mediterranean ant.

Natural habitats with serpentine soils are rich in heavy metal ions, which may significantly affect ecological communities. Exposure to metal pollutants results, for instance, in a reduction of population genetic diversity and a diffused higher tolerance towards heavy metals. In this study, we investigated whether chronic exposure to metals in serpentine soils affect accumulation patterns, tolerance towards metal pollutants, and genetic diversity in ants. In particular, we studied colonies of the common Mediterranean ant, Crematogaster scutellaris, along a contamination gradient consisting of two differently contaminated forests and a reference soil with no geogenic contamination. We first evaluated the metal content in both soil and ants' body. Then, we tested for tolerance towards metal pollutants by evaluating the mortality of ants fed with nickel (Ni) solutions of increasing concentrations. Finally, differences in genetic diversity among ants from different areas were assessed using eight microsatellite loci. Interestingly, a higher tolerance to nickel solutions was found in ants sampled in sites with intermediate levels of heavy metals. This may occur, because ants inhabiting strongly contaminated areas tend to accumulate higher amounts of contaminants. Additional ingestion of toxicants beyond the saturation threshold would lead to death. There was no difference in the genetic diversity among ant colonies sampled in different sites. This was probably the result of queen mediated gene flow during nuptial flights across uncontaminated and contaminated areas of limited geographical extent.

Ectomycorrhizal fungal diversity associated with endemic Tristaniopsis spp. (Myrtaceae) in ultramafic and volcano-sedimentary soils in New Caledonia.

New Caledonian serpentine (ultramafic) soils contain high levels of toxic heavy metals, in particular nickel, (up to 20 g kg-1) and are deficient in essential elements like carbon, nitrogen and phosphorus while having a high magnesium/calcium ratio. Although previous studies showed that ectomycorrhizal symbioses could play an important role in the adaptation of the endemic plants to ultramafic soils (FEMS Microbiol Ecol 72:238-49, 2010), none of them have compared the diversity of microbial communities from ultramafic vs non-ultramafic soils in New Caledonia. We explored the impact of edaphic characteristics on the diversity of ectomycorrhizal (ECM) fungi associated with different endemic species of Tristaniopsis (Myrtaceae) growing under contrasting soil conditions in the natural ecosystems of New Caledonia. ECM root tips were thus sampled from two different ultramafic sites (Koniambo massif and Desmazures forest) vs two volcano-sedimentary ones (Arama and Mont Ninndo). The molecular characterization of the ECM fungi through partial sequencing of the ITS rRNA gene revealed the presence of different dominant fungal genera including, both soil types combined, Cortinarius (36.1%), Pisolithus (18.5%), Russula (13.4%), Heliotales (8.2%) and Boletellus (7.2%). A high diversity of ECM taxa associated with Tristaniopsis species was found in both ultramafic and volcano-sedimentary soils but no significant differences in ECM genera distribution were observed between both soil types. No link could be established between the phylogenetic clustering of ECM taxa and their soil type origin, thus suggesting a possible functional-rather than taxonomical-adaptation of ECM fungal communities to ultramafic soils.

Organic amendments for improving biomass production and metal yield of Ni-hyperaccumulating plants.

Ni phytomining is a promising technology for Ni recovery from low-grade ores such as ultramafic soils. Metal-hyperaccumulators are good candidates for phytomining due to their extraordinary capacity for Ni accumulation. However, many of these plants produce a low biomass, which makes the use of agronomic techniques for improving their growth necessary. In this study, the Ni hyperaccumulators Alyssum serpyllifolium ssp. lusitanicum, A. serpyllifolium ssp. malacitanum, Alyssum bertolonii and Noccaea goesingense were evaluated for their Ni phytoextraction efficiency from a Ni-rich serpentine soil. Effects of soil inorganic fertilisation (100:100:125kgNPKha(-1)) and soil organic amendment addition (2.5, 5 or 10% compost) on plant growth and Ni accumulation were determined. All soil treatments greatly improved plant growth, but the highest biomass production was generally found after addition of 2.5 or 5% compost (w/w). The most pronounced beneficial effects were observed for N. goesingense. Total Ni phytoextracted from soils was significantly improved using both soil treatments (inorganic and organic), despite the decrease observed in soil Ni availability and shoot Ni concentrations in compost-amended soils. The most promising results were found using intermediate amount of compost, indicating that these types of organic wastes can be incorporated into phytomining systems.

Albanian violets of the section Melanium, their morphological variability, genetic similarity and their adaptations to serpentine or chalk soils.

Violets of the section Melanium from Albanian serpentine and chalk soils were examined for their taxonomic affiliations, their ability to accumulate heavy metals and their colonization by arbuscular mycorrhizal fungi (AMF). The sequence analysis of the ITS1-5.8S rDNA-ITS2 region showed that all the sampled six Albanian violets grouped between Viola lutea and Viola arvensis, but not with Viola tricolor. The fine resolution of the ITS sequences was not sufficient for a further delimitation of the Albanian violets within the V. lutea-V. arvensis clade. Therefore, the Albanian violets were classified by a set of morphological characters. Viola albanica, Viola dukadjinica and Viola raunsiensis from serpentine soils as well as Viola aetolica from a chalk meadow were unambiguously identified, whereas the samples of Viola macedonica showed high morphological variability. All the violets, in both roots and shoots contained less than or similar levels of heavy metals as their harboring soils, indicating that they were heavy metal excluders. All the violets were strongly colonized by AMF with the remarkable exception of V. albanica. This violet lived as a scree creeper in shallow serpentine soil where the concentration of heavy metals was high but those of P, K and N were scarce.

Culturable endophytic bacteria enhance Ni translocation in the hyperaccumulator Noccaea caerulescens.

In this work, both culture-dependent and independent approaches were used to identify and isolate endophytic bacteria from roots of the Ni hyperaccumulator Noccaea caerulescens. A total of 17 isolates were cultured from root samples, selected for tolerance to 6mM Ni and grouped by restriction analysis of 16S rDNA. Bacterial species cultivated from roots belonged to seven genera, Microbacterium, Arthrobacter, Agreia, Bacillus, Sthenotrophomonas, Kocuria and Variovorax. The culture-independent approach confirmed the presence of Microbacterium and Arthrobacter while only other five clones corresponding to different amplified ribosomal DNA restriction patterns were detected. Five selected highly Ni-resistant bacteria showing also plant growth promoting activities, were inoculated into seeds of N. caerulescens, and in vivo microscopic analysis showed rapid root colonisation. Inoculated plants showed increased shoot biomass, root length and root-to-shoot Ni translocation. Root colonisation was also evident, but not effective, in the non-hyperaccumulating Thlaspi perfoliatum. Seed inoculation with selected Ni-resistant endophytic bacteria may represent a powerful tool in phytotechnologies, although transferring it to biomass species still requires further studies and screening.

Rinorea niccolifera (Violaceae), a new, nickel-hyperaccumulating species from Luzon Island, Philippines.

A new, nickel-hyperaccumulating species of Rinorea (Violaceae), Rinorea niccolifera Fernando, from Luzon Island, Philippines, is described and illustrated. This species is most similar to the widespread Rinorea bengalensis by its fasciculate inflorescences and smooth subglobose fruits with 3 seeds, but it differs by its glabrous ovary with shorter style (5 mm long), the summit of the staminal tube sinuate to entire and the outer surface smooth, generally smaller leaves (3-8 cm long × 2-3 cm wide), and smaller fruits (0.6-0.8 cm diameter). Rinorea niccolifera accumulates to >18,000 µg g(-1) of nickel in its leaf tissues and is thus regarded as a Ni hyperaccumulator.

Serpentine bacteria influence metal translocation and bioconcentration of Brassica juncea and Ricinus communis grown in multi-metal polluted soils.

The aim of this study was to assess the effects of inoculation of rhizosphere or endophytic bacteria (Psychrobacter sp. SRS8 and Pseudomonas sp. A3R3, respectively) isolated from a serpentine environment on the plant growth and the translocation and accumulation of Ni, Zn, and Fe by Brassica juncea and Ricinus communis on a multi-metal polluted serpentine soil (SS). Field collected SS was diluted to 0, 25, 50, and 75% with pristine soil in order to obtain a range of heavy metal concentrations and used in microcosm experiments. Regardless of inoculation with bacteria, the biomass of both plant species decreased with increase of the proportion of SS. Inoculation of plants with bacteria significantly increased the plant biomass and the heavy metal accumulation compared with non-inoculated control in the presence of different proportion of SS, which was attributed to the production of plant growth promoting and/or metal mobilizing metabolites by bacteria. However, SRS8 showed a maximum increase in the biomass of the test plants grown even in the treatment of 75% SS. In turn, A3R3 showed maximum effects on the accumulation of heavy metals in both plants. Regardless of inoculation of bacteria and proportion of SS, both plant species exhibited low values of bioconcentration factor (<1) for Ni and Fe. The inoculation of both bacterial strains significantly increased the translocation factor (TF) of Ni while decreasing the TF of Zn in both plant species. Besides this contrasting effect, the TFs of all metals were <1, indicating that all studied bacteria-plant combinations are suitable for phytostabilization. This study demonstrates that the bacterial isolates A3R3 and SRS8 improved the growth of B. juncea and R. communis in SS soils and have a great potential to be used as inoculants in phytostabilization scenarios of multi-metal contaminated soils.

Nickel-accumulating plants from the ancient serpentine soils of Cuba.

Extraordinary uptake of nickel (Ni), reaching concentrations of 0.1-5.0%, c. 1000 times greater than those usually found in flowering plants, has been observed previously in c, 190 species that grow on Ni-rich serpentine soils derived from ultramafic rocks in various parts of the world. These so-called hyperaccumulators of Ni include c. 50 species from the rich ultramafic flora of New Caledonia and c. 80 species from the Brassicaceae of Mediterranean Europe and Turkey. A study of a limited part (the families Buxaceae and Euphorbiaceae) of the very large ultramafic flora of Cuba has now identified this as the home of at least 80 hyperaccumulators, the largest number jet found in any one country. The more frequent incidence here of this unusual form of plant behaviour is linked to the very long period (r. 10-30 million years) during which some of the Cuban ultramafic substrata are believed to have been continuously available for colonization; the distribution of Ni hyperaccumulators between older and younger ultrarnafic soils in Cuba mirrors the overall incidence of endemic species in these areas.