Strategies in a metallophyte species to cope with manganese excess.

The effect of exposure to high Mn concentration was studied in a metallophyte species, Erica andevalensis, using hydroponic cultures with a range of Mn concentrations (0.06, 100, 300, 500, and 700 mg L-1). At harvest, biomass production, element uptake, and biochemical indicators of metal stress (leaf pigments, organic acids, amino acids, phenols, and activities of catalase, peroxidase, superoxide dismutase) were determined in leaves and roots. Increasing Mn concentrations led to a decrease in biomass accumulation, and tip leaves chlorosis was the only toxicity symptom detected. In a similar way, photosynthetic pigments (chlorophylls a and b, and carotenoids) were affected by high Mn levels. Among organic acids, malate and oxalate contents in roots showed a significant increase at the highest Mn concentration, while in leaves, Mn led to an increasing trend in citrate and malate contents. An increase of Mn also induced an increase in superoxide dismutase activity in roots and catalase activity in leaves. As well, significant changes in free amino acids were induced by Mn concentrations higher than 300 mg L-1, especially in roots. No significant changes in phenolic compounds were observed in the leaves, but root phenolics were significantly increased by increasing Mn concentrations in treatments. When Fe supply was increased 10 and 20 times (7-14 mg Fe L-1 as Fe-EDDHA) in the nutrient solutions at the highest Mn concentration (700 mg Mn L-1), it led to significant increases in photosynthetic pigments and biomass accumulation. Manganese was mostly accumulated in the roots, and the species was essentially a Mn excluder. However, considering the high leaf Mn concentration recorded without toxicity symptoms, E. andevalensis might be rated as a Mn-tolerant species.

Phytostabilization potential of Erica australis L. and Nerium oleander L.: a comparative study in the Riotinto mining area (SW Spain).

Phytostabilization is a green, cost-effective technique for mine rehabilitation and ecological restoration. In this study, the phytostabilization capacity of Erica australis L. and Nerium oleander L. was assessed in the climatic and geochemical context of the Riotinto mining district, southwestern Spain, where both plant species colonize harsh substrates of mine wastes and contaminated river banks. In addition to tolerating extreme acidic conditions (up to pH 3.36 for E. australis), both species were found to grow on substrates very poor in bioavailable nutrients (e.g., N and P) and highly enriched with potentially phytotoxic elements (e.g., Cu, Cd, Pb, S). The selective root absorption of essential elements and the sequestration of potentially toxic elements in the root cortex are the main adaptations that allow the studied species to cope in very limiting edaphic environments. Being capable of a tight elemental homeostatic control and tolerating extreme acidic conditions, E. australis is the best candidate for use in phytostabilization programs, ideally to promote early stages of colonization, improve physical and chemical conditions of substrates and favor the establishing of less tolerant species, such as N. oleander.

EFFECTS OF COLD STRATIFICATION AND GA3 ON GERMINATION OF ARBUTUS UNEDO SEEDS OF THREE PROVENANCES.

BACKGROUND: Arbutus unedo is a valuable Mediterranean shrub as an ornamental plant as well as fruit tree. Fresh fruits of A. unedo are a good source of antioxidants, of vitamins C, E and carotenoids and also are characterized by the high content of mineral elements. MATERIALS AND METHODS: The effects of gibberellic acid (GA3) and cold stratification (CS) on seed germination performance were investigated in A. unedo seeds collected from three provenances in the Northern part of Greece. Seeds of each provenance were soaked in solutions of GA3 (500, 1000 or 2000 ppm) for 24 h and subsequently were subjected to CS at 3 - 5°C for 0, 1, 2, and 3 months. RESULTS: Non-stratified seeds of the three A. unedo provenances which were not treated with GA3 solutions exhibited very low germination. However, seed germination was significantly improved after a one-month period of CS. Similarly, the non-stratified seeds of all three provenances became non-dormant after the treatment with 2000 ppm GA3 and they germinated at high percentages. However, in untreated seeds with GA3, after a one-month CS period the seeds of the Pieria provenance exhibited higher germination percentage than that of Rodopi provenance seeds. Furthermore, in non-stratified seeds, the Pieria provenance seeds treated with GA3 germinated at higher percentages and more rapidly than those of the other two provenances. CONCLUSION: The results indicated that untreated seeds exhibited very low germination at 20/25°C. However, in all three provenances seed germinability was significantly improved by a one-month period of CS or treatment of seeds with 2000 ppm GA3. Furthermore, there was a considerable variability among seed provenances in response to the treatments which were applied.

Tolerance to high Zn in the metallophyte Erica andevalensis Cabezudo & Rivera.

The tolerance to high Zn was studied in the metallophyte Erica andevalensis Cabezudo & Rivera grown in nutrient solutions at different Zn concentrations (5, 500, 1,000, 1,500 and 2,000 µM Zn). Plant growth and nutrient uptake were determined. Metabolic changes were assessed by the analysis of peroxidase activity, organic metabolites related to metal chelation (amino acids, organic acids (malate, citrate) or protection (polyamines). While plants tolerated up to 1,500 µM Zn, despite presenting of low growth rates, the concentration of 2,000 µM Zn was toxic producing high mortality rates. Roots accumulated high Zn concentration (11,971 mg/kg) at 1,500 µM external Zn) apparently avoiding metal transfer into shoots. After 30 days of treatment with high Zn (1,000 and 1,500 µM Zn), the leaves accumulated high levels of glutamine. Short-term treatment with 500 µM Zn, significantly increased the concentration of asparagine and glutamine in roots. Citrate concentration was also considerably increased when exposing roots to Zn excess. Metal immobilization in the root system, low interference with the uptake of nutrients and an increased production of putative organic ligands (amino acids, citrate) might have provided the Zn tolerance displayed by Erica andevalensis.

Evaluation of lead toxicity in Erica andevalensis as an alternative species for revegetation of contaminated soils.

Although revegetation using native flora is a low cost way to stabilize soil and restore the landscape contaminated with metals, little is known regarding the Pb-tolerance of many of these species. For this purpose, we evaluated the tolerance of Erica andevalensis to Pb by growing plants in nutrient solutions with increasing concentrations of Pb (up to 100 microM). Plant growth and different physiological parameters were determined to ascertain tolerance to metal stress. Additionally, an electron microscopy study coupled with EDX-analysis was performed to get clues on the Pb uptake and translocation from roots into stem and leaves. The LOEC (the lowest observed effect concentration) of Pb was 40 microM while the IC50 (inhibition concentration) was 80 microM Pb. Chemical analysis revealed a root > stem > leaf accumulation pattern. There was a severe reduction in fresh biomass and chlorophyll concentration at the highest Pb dose. The SEM-EDX study indicated that Pb was mostly located in root epidermal tissues. The blockage of Pb on the root probably avoided its toxic effects by limiting Pb transport to other tissues.

The origin of lead in the organic horizon of tundra soils: atmospheric deposition, plant translocation from the mineral soil or soil mineral mixing?

Knowledge of the anthropogenic contribution to lead (Pb) concentrations in surface soils in high latitude ecosystems is central to our understanding of the extent of atmospheric Pb contamination. In this study, we reconstructed fallout of Pb at a remote sub-arctic region by using two ombrotrophic peat cores and assessed the extent to which this airborne Pb is able to explain the isotopic composition ((206)Pb/(207)Pb ratio) in the O-horizon of tundra soils. In the peat cores, long-range atmospheric fallout appeared to be the main source of Pb as indicated by temporal trends that followed the known European pollution history, i.e. accelerated fallout at the onset of industrialization and peak fallout around the 1960s-70s. The Pb isotopic composition of the O-horizon of podzolic tundra soil ((206)Pb/(207)Pb=1.170 ± 0.002; mean ± SD) overlapped with that of the peat ((206)Pb/(207)Pb=1.16 ± 0.01) representing a proxy for atmospheric aerosols, but was clearly different from that of the parent soil material ((206)Pb/(207)Pb=1.22-1.30). This finding indicated that long-range fallout of atmospheric Pb is the main driver of Pb accumulation in podzolic tundra soil. In O-horizons of tundra soil weakly affected by cryoturbation (cryosols) however, the input of Pb from the underlying mineral soil increased as indicated by (206)Pb/(207)Pb ratios of up to 1.20, a value closer to that of local soil minerals. Nevertheless, atmospheric Pb appeared to be the dominant source in this soil compartment. We conclude that Pb concentrations in the O-horizon of studied tundra soils - despite being much lower than in boreal soils and representative for one of the least exposed sites to atmospheric Pb contaminants in Europe - are mainly controlled by atmospheric inputs from distant anthropogenic sources.

Arsenic, antimony, and other trace element contamination in a mine tailings affected area and uptake by tolerant plant species.

The study was conducted to characterize mineralogical and elemental composition of mine tailings in order to evaluate the environmental hazards, and identify the metal accumulation potential of native plant species from São Domingos mine, one of the long-term activity mines of the Iberian Pyrite Belt dating back to pre-Roman times. The mine tailings including soils and different plant species from São Domingos were analyzed for determination of tailings characteristics and chemical element contents in tailings and plants. The large amounts of mining wastes are causing significant adverse environment impacts due to acid mine drainage production and mobilization of potentially toxic metals and metalloids in residential areas, agricultural fields, downstreams, and rivers. The typical mineralogical composition is as follows: quartz, micas, K-feldspar, olivine-group minerals, magnetite, goethite, hematite, jarosite, and sulfides. The mine tailings were highly contaminated by As, Ag, Cr, Hg, Sn, Sb, Fe, and Zn; and among them, As and Sb, main contaminants, attained the highest concentrations except Fe. Arsenic has exhibited very good correlations with Au, Fe, Sb, Se, and W; and Sb with As, Au, Fe, Se, Sn, and W in tailings. Among the all plant species, the higher concentrations of all the metals were noted in Erica andevalensis, Erica australis, Echium plantagium, and Lavandula luisierra. Considering the tolerant behavior and abundant growth, the plant species Erica australis, Erica andevalensis, Lavandula luisierra, Daphne gnidium, Rumex induratus, Ulex eriocladus, Juncus, and Genista hirsutus are of major importance for the rehabilitation and recovery of degraded São Domingos mining area.

Effects of silicon on copper toxicity in Erica andevalensis Cabezudo and Rivera: a potential species to remediate contaminated soils.

The influence of silicon on responses to copper excess was studied in plants of Erica andevalensis. Plantlets were grown in nutrient solutions containing two Cu (1 and 500 µM) and three Si concentrations (0, 0.5 and 1 mM). Plant growth, water content, and mineral nutrient concentration were determined. Plants grown with 500 µM Cu showed differences in growth and shoot water content depending on Si supply. The addition of 1 mM Si in high-Cu nutrient solutions significantly improved plant growth and reduced water loss preventing plant death related to Cu-excess. Silicon supply reduced significantly leaf Cu concentration (up to 32%) and increased Cu concentration in roots. Phytoliths isolated from leaves were analysed by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy. Such phytoliths consisted in silica deposits associated with Cu and other elements (K, Ca, P). Improvement by Si of Cu tolerance in E. andevalensis was clearly related to the inhibition of Cu upward transport. The leaf phytoliths formed in Si-treated plants might have some contribution to tolerance by Cu immobilisation and inactivation.

Antioxidants in Erica andevalensis: a comparative study between wild plants and cadmium-exposed plants under controlled conditions.

Erica andevalensis is an endemic species from SW Iberian Peninsula, always growing in metal-enriched and acid soils. In the present study, a comparison was made between wild E. andevalensis plants collected from the field and cultivated ones exposed to different cadmium levels (0, 0.5, 5 and 50 µM). Wild plants contain higher levels of ascorbic acid (around 8000 nmol g(-1) FW) than lab-cultivated control plants (around 3000 nmol g(-1) FW). Glutathione levels follow an opposite trend being smaller in wild plants than lab-cultivated ones. Moreover, the total antioxidant capacity of wild plants is 90 times higher than in cultivated plants non-exposed to cadmium. Cadmium treatment of lab-cultivated plants did not affect the growth of E. andevalensis or the glutathione levels. However, the total antioxidative capacity increased in plants exposed to 50 µM of cadmium. Cadmium was added to the soil and it was transported into leaves reaching levels of 3.299 ± 0.781 µg Cd/g DW in plants exposed to 50 µM. These results underline a possible importance of antioxidants in the metal tolerance show by the high antioxidant capacity detected in both wild and lab-cultivated plants exposed to high cadmium levels.

Effect of plant growth regulators and genotype on the micropropagation of adult trees of Arbutus unedo L. (strawberry tree).

Arbutus unedo grows spontaneously around the Mediterranean basin. The species is tolerant to drought and has a strong regeneration capacity following fires making it interesting for Mediterranean forestation programs. Considering the sparse information about the potential of this fruit tree to be propagated in vitro, a project to clone selected trees based on their fruit production was initiated a few years ago. The role of several factors on A. unedo propagation was evaluated. The results showed that 8.9 µm kinetin gave the best results although not significantly different from those obtained with benzyladenine or zeatin. The inclusion of thidiazuron or 1-naphthaleneacetic acid promoted callus growth and had deleterious effects on the multiplication rate. The genotype of the donor plants is also a factor interfering with the multiplication. The results also indicated that the conditions used for multiplication influenced the behavior of shoots during the rooting phase.

Stress responses of Erica andevalensis Cabezudo & Rivera plants induced by polluted water from Tinto River (SW Spain).

The effects of Tinto River water on Erica andevalensis growth, biochemical indicators and elemental concentration and distribution were investigated under laboratory conditions. High levels of toxic elements such as B, Fe and S and acidic pH characterized the river water. Plant analysis revealed that the concentration of Al, B, S and Fe increased in all plant organs reaching in some cases values in the toxicity range. Plants transferred into river water stopped growing and stress was manifested by plant water loss, increase in peroxidase activity and decrease of chlorophyll a concentration. Significant decreases of free amino acid concentration were found in shoots and roots of plants grown in diluted river water. The results indicated that Tinto River water acidity and its excess in soluble elements produced altogether severe alterations in roots affecting plant water and nutrient uptake and leading to the massive entry of some metals (e.g. Fe, Al) with toxic effects. Scanning-electron microscopy (cryoSEM and ESEM) observations showed that E. andevalensis had not exclusion mechanisms of Cu, Fe and S therefore it was not able to reduce translocation to aerial parts.

Phenolics composition in Erica sp. differentially exposed to metal pollution in the Iberian Southwestern Pyritic Belt.

The phenolic composition of different populations of Erica andevalensis,E. australis and E. arborea was analyzed according to the different degree of exposition to metals in soils. E. andevalensis populations, highly exposed to metal pollution, had the lowest total phenol content and the lowest antioxidant activity measured as trolox equivalents. The HPLC analysis of leaf extracts revealed that phenolic composition in all analyzed populations of E. australis and E. arborea was very similar, although wild populations were differentially exposed to metal pollutions. However, E. andevalensis showed a phenolic profile characterized by the absence of many phenolic compounds quantified in the other species, although two compounds derived from cinnamic and coumaric acids were exclusively observed in this species.

Evaluation of anilinopyrimidine and other fungicides for control of grey mould (Botrytis cinerea) in container-grown Calluna vulgaris.

BACKGROUND: Grey mould (Botrytis cinerea Pers. ex Fr.) is a damaging disease affecting container-grown Calluna vulgaris (L.) Hull. Trials were conducted to test anilinopyrimidine fungicides (pyrimethanil, cyprodinil and mepanipyrim), azoxystrobin and tolylfluanid for crop safety on cultivars, and to evaluate spray programmes based on alternating these fungicides with different modes of action for disease control. Timing and application at different spray intervals were also investigated. RESULTS: Cyprodinil was slightly phytotoxic on only one of the seven cvs tested. Alternating nine-spray programmes of azoxystrobin with an anilinopyrimidine or tolylfluanid, or an anilinopyrimidine with tolylfluanid, with the first spray applied immediately after potting, were equally effective in reducing disease incidence and severity compared with untreated plants. Spray programmes of five or eight sprays, using an anilinopyrimidine and at least one other fungicide with a different mode of action, applied alternately or in blocks of two sprays of the same fungicide and immediately after potting, were equally effective in reducing disease. Omission of early fungicide sprays until 9 weeks after potting and subsequent alternate application of pyrimethanil and tolylfluanid with a different mode of action at 6 week intervals gave reduced disease control. CONCLUSION: There is potential to use spray programmes based on alternating anilinopyrimidines with fungicides with a different mode of action to provide effective control of B. cinerea on C. vulgaris.

Comparison of arsenic resistance in Mediterranean woody shrubs used in restoration activities.

Myrtus communis, Arbutus unedo and Retama sphaerocarpa are Mediterranean shrubs widely used in revegetation of semiarid degraded soils. The aim of this work is to study the resistance of these plants to arsenic under controlled conditions, in order to evaluate their potential use in revegetation and/or phytoremediation of As-polluted soils. R. sphaerocarpa showed higher resistance to As than M. communis or A. unedo according to its higher EC50, P status and P/As molar ratio in both, roots and shoots, and the lower increases in lipid peroxidation and decrease of chlorophyll levels in response to arsenic, while the highest arsenate sensitivity was obtained for A. unedo. Arsenic was mainly retained in roots, and, although M. communis accumulated higher arsenic amounts than the other two species, R. sphaerocarpa showed the highest root to shoot transfer. Most of the studied parameters (chlorophylls, MDA and total thiols) showed significant correlation with arsenic concentration in roots and leaves of plants, so they can be useful indexes in the diagnosis of arsenic toxicity in these species. According to our results, both M. communis and R. sphaerocarpa could be used in the revegetation of moderately arsenic contaminated sites.

Effect of a long-term fire retardant (Fire Trol 934) on the germination of nine Mediterranean-type shrub species.

Fire Trol 934 is a long-term fire retardant commonly used in fire prevention and extinction. Our objective was to determine the effect of this chemical on seed germination of nine plant species from Mediterranean-type shrublands, where these chemicals are potentially used. Seeds were exposed to five different Fire Trol concentrations, (0 (control) to 10%, on a log scale) and monitored in a germination chamber for nine weeks. Seeds from four Cistus species were subjected to an additional heat treatment that simulated thermal scarification caused by fire. Retardant exposure caused a significant decrease in total germination in all species, and exposure to the highest Fire Trol concentration (10%) resulted in complete inhibition of germination. However, the sensitivity to Fire Trol varied across species and this differential species sensitivity may potentially lead to different impacts in the soil seed banks depending on whether sites are burned or unburned. Exposure to Fire Trol 934 may affect recruitment of shrubland species particularly during dry autumns, due to limited leaching of these chemicals from the soil surface. Consequently, its use should be avoided in sites where particularly sensitive plant species are present.

Ozone effects on the ultrastructure of peatland plants: Sphagnum mosses, Vaccinium oxycoccus, Andromeda polifolia and Eriophorum vaginatum.

BACKGROUND AND AIMS: Ozone effects on peatland vegetation are poorly understood. Since stress responses are often first visible in cell ultrastructure, electron microscopy was used to assess the sensitivity of common peatland plants to elevated ozone concentrations. METHODS: Three moss species (Sphagnum angustifolium, S. magellanicum and S. papillosum), a graminoid (Eriophorum vaginatum) and two dwarf shrubs (Vaccinium oxycoccus and Andromeda polifolia), all growing within an intact canopy on peat monoliths, were exposed to a concentration of 0, 50, 100 or 150 ppb ozone in two separate growth chamber experiments simulating either summer or autumn conditions in central Finland. After a 4- or 5-week-long exposure, samples were photographed in a transmission electron microscope and analysed quantitatively using image processing software. KEY RESULTS: In the chlorophyllose cells of the Sphagnum moss leaves from the capitulum, ozone exposure led to a decrease in chloroplast area and in granum stack thickness and various changes in plastoglobuli and cell wall thickness, depending on the species and the experiment. In E. vaginatum, ozone exposure significantly reduced chloroplast cross-sectional areas and the amount of starch, whereas there were no clear changes in the plastoglobuli. In the dwarf shrubs, ozone induced thickening of the cell wall and an increase in the size of plastoglobuli under summer conditions. In contrast, under autumn conditions the cell wall thickness remained unchanged but ozone exposure led to a transient increase in the chloroplast and starch areas, and in the number and size of plastoglobuli. CONCLUSIONS: Ozone responses in the Sphagnum mosses were comparable to typical ozone stress symptoms of higher plants, and indicated sensitivity especially in S. angustifolium. The responses in the dwarf shrubs suggest stimulation of photosynthesis by low ozone concentrations and ozone sensitivity only under cool autumn conditions.