Assessment of Ni accumulation capability by fungi for a possible approach to remove metals from soils and waters.

Abandoned industrial sites and mines may constitute possible hazards for surrounding environment due to the presence of toxic compounds that may contaminate soils and waters. The possibility to remove metal contaminants, specifically nickel (Ni), by means of fungi was presented exploiting a set of fungal strains isolated from a Ligurian dismissed mine. The achieved results demonstrate the high Ni(II) tolerance, up to 500 mg Ni l-1, and removal capability of a Trichoderma harzianum strain. This latter hyperaccumulates up to 11,000 mg Ni kg-1, suggesting its possible use in a bioremediation protocol able to provide a sustainable reclamation of broad contaminated areas.

Overexpression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification.

MAIN CONCLUSION: The heterologous expression of AtPCS1 in tobacco plants exposed to arsenic plus cadmium enhances phytochelatin levels, root As/Cd accumulation and pollutants detoxification, but does not prevent root cyto-histological damages. High phytochelatin (PC) levels may be involved in accumulation and detoxification of both cadmium (Cd) and arsenic (As) in numerous plants. Although polluted environments are frequently characterized by As and Cd coexistence, how increased PC levels affect the adaptation of the entire plant and the response of its cells/tissues to a combined contamination by As and Cd needs investigation. Consequently, we analyzed tobacco seedlings overexpressing Arabidopsis phytochelatin synthase1 gene (AtPCS1) exposed to As and/or Cd, to evaluate the levels of PCs and As/Cd, the cyto-histological modifications of the roots and the Cd/As leaf extrusion ability. When exposed to As and/or Cd the plants overexpressing AtPCS1 showed higher PC levels, As plus Cd root accumulation, and detoxification ability than the non-overexpressing plants, but a blocked Cd-extrusion from the leaf trichomes. In all genotypes, As, and Cd in particular, damaged lateral root apices, enhancing cell-vacuolization, causing thinning and stretching of endodermis initial cells. Alterations also occurred in the primary structure region of the lateral roots, i.e., cell wall lignification in the external cortex, cell hypertrophy in the inner cortex, crushing of endodermis and stele, and nuclear hypertrophy. Altogether, As and/or Cd caused damage to the lateral roots (and not to the primary one), with such damage not counteracted by AtPCS1 overexpression. The latter, however, positively affected accumulation and detoxification to both pollutants, highlighting that Cd/As accumulation and detoxification due to PCS1 activity do not reduce the cyto-histological damage.

Pollination ecology in the narrow endemic winter-flowering Primula allionii (Primulaceae).

Reduction of pollen flow can affect plant abundance and population viability and cause selection on plant mating system and floral traits. Little is known on the effect of this phenomenon in species naturally restricted to small and isolated habitats, that may have developed strategies to cope with long-term isolation and small population size. We investigated the pollination ecology of the endemic distylous winter-flowering P. allionii to verify the possible limitation of female fitness due to reduced pollinator visits. We recorded a higher production of pollen grains in long-styled morph, and a higher seed set in short-styled morph. The high intra-morph variability of sexual organ position may explain the hybridization phenomena allowing and easier intra-morph pollination. The fruit set is constant, although its winter-flowering period might decrease pollen transfer. Nevertheless, the lower competition for pollinators with neighbouring plants and the long-lasting anthesis may offset its reproductive success. Even if our results show no evidence of imminent threats, changes in plant-pollinator interactions might increase inbreeding, resulting in an increased extinction risk.

Effects of Bacterial and Fungal Inocula on Biomass, Ecophysiology, and Uptake of Metals of Alyssoides utriculata (L.) Medik.

The inoculation of plants with plant-growth-promoting microorganisms (PGPM) (i.e., bacterial and fungal strains) is an emerging approach that helps plants cope with abiotic and biotic stresses. However, knowledge regarding their synergic effects on plants growing in metal-rich soils is limited. Consequently, the aim of this study was to investigate the biomass, ecophysiology, and metal accumulation of the facultative Ni-hyperaccumulator Alyssoides utriculata (L.) Medik. inoculated with single or mixed plant-growth-promoting (PGP) bacterial strain Pseudomonas fluorescens Migula 1895 (SERP1) and PGP fungal strain Penicillium ochrochloron Biourge (SERP03 S) on native serpentine soil (n = 20 for each treatment). Photosynthetic efficiency (Fv/Fm) and performance indicators (PI) had the same trends with no significant differences among groups, with Fv/Fms > 1 and PI up to 12. However, the aboveground biomass increased 4-5-fold for single and mixed inoculated plants. The aboveground/belowground dry biomass ratio was higher for plants inoculated with fungi (30), mixed (21), and bacteria (17). The ICP-MS highlighted that single and mixed inocula were able to double the aboveground biomass' P content. Mn metal accumulation significantly increased with both single and mixed PGP inocula, and Zn accumulation increased only with single PGP inocula, whereas Cu accumulation increased twofold only with mixed PGP inocula, but with a low content. Only Ni metal accumulation approached the hyperaccumulation level (Ni > 1000 mg/kg DW) with all treatments. This study demonstrated the ability of selected single and combined PGP strains to significantly increase plant biomass and plant tolerance of metals present in the substrate, resulting in a higher capacity for Ni accumulation in shoots.

Tomato (Solanum lycopersicum L.) accumulation and allergenicity in response to nickel stress.

Vegetables represent a major source of Ni exposure. Environmental contamination and cultural practices can increase Ni amount in tomato posing significant risk for human health. This work assesses the tomato (Solanum lycopersicum L.) response to Ni on the agronomic yield of fruits and the related production of allergens. Two cultivars were grown in pots amended with Ni 0, 30, 60, 120, and 300 mg kg-1, respectively. XRF and ICP-MS analyses highlighted the direct increase of fruit Ni content compared to soil Ni, maintaining a stable biomass. Leaf water content increased at Ni 300 mg kg-1. Total protein content and individual allergenic components were investigated using biochemical (RP-HPLC and N-terminal amino acid sequencing) and immunological (inhibition tests of IgE binding by SPHIAa assay on the FABER testing system) methodologies. Ni affected the fruit tissue concentration of pathogenesis-related proteins and relevant allergens (LTP, profilin, Bet v 1-like protein and TLP). This study elucidates for the first time that tomato reacts to exogenous Ni, uptaking the metal while changing its allergenic profiles, with potential double increasing of exposure risks for consumers. This evidence highlighted the importance of adequate choice of low-Ni tomato cultivars and practices to reduce Ni uptake by potentially contaminated matrices.

Root and Shoot Response to Nickel in Hyperaccumulator and Non-Hyperaccumulator Species.

The soil-root interface is the micro-ecosystem where roots uptake metals. However, less than 10% of hyperaccumulators' rhizosphere has been examined. The present study evaluated the root and shoot response to nickel in hyperaccumulator and non-hyperaccumulator species, through the analysis of root surface and biomass and the ecophysiological response of the related aboveground biomass. Ni-hyperaccumulators Alyssoides utriculata (L.) Medik. and Noccaea caerulescens (J. Presl and C. Presl) F.K. Mey. and non-hyperaccumulators Alyssum montanum L. and Thlaspi arvense L. were grown in pot on Ni-spiked soil (0-1000 mg Ni kg-1, total). Development of root surfaces was analysed with ImageJ; fresh and dry root biomass was determined. Photosynthetic efficiency was performed by analysing the fluorescence of chlorophyll a to estimate the plants' physiological conditions at the end of the treatment. Hyperaccumulators did not show a Ni-dependent decrease in root surfaces and biomass (except Ni 1000 mg kg-1 for N. caerulescens). The non-hyperaccumulator A. montanum suffers metal stress which threatens plant development, while the excluder T. arvense exhibits a positive ecophysiological response to Ni. The analysis of the root system, as a component of the rhizosphere, help to clarify the response to soil nickel and plant development under metal stress for bioremediation purposes.

A Mini-Review on the Co-growth and Interactions Among Microorganisms (Fungi and Bacteria) From Rhizosphere of Metal-Hyperaccumulators.

The co-growth and synergistic interactions among fungi and bacteria from the rhizosphere of plants able to hyper accumulate potentially toxic metals (PTMs) are largely unexplored. Fungi and bacteria contribute in an essential way to soil biogeochemical cycles mediating the nutrition, growth development, and health of associated plants at the rhizosphere level. Microbial consortia improve the formation of soil aggregates and soil fertility, producing organic acids and siderophores that increase solubility, mobilization, and consequently the accumulation of nutrients and metals from the rhizosphere. These microorganism consortia can both mitigate the soil conditions promoting plant colonization and increase the performance of hyperaccumulator plants. Indeed, microfungi and bacteria from metalliferous soils or contaminated matrices are commonly metal-tolerant and can play a key role for plants in the phytoextraction or phytostabilization of metals. However, few works deepen the effects of the inoculation of microfungal and bacterial consortia in the rhizosphere of metallophytes and their synergistic activity. This mini-review aimed to collect and report the data regarding the role of microbial consortia and their potentialities known to date. Moreover, our new data had shown an active fungal-bacteria consortium in the rhizosphere of the hyperaccumulator plant Alyssoides utriculata.

Frenemies: Interactions between Rhizospheric Bacteria and Fungi from Metalliferous Soils.

Is it possible to improve the efficiency of bioremediation technologies? The use of mixed cultures of bacteria and fungi inoculated at the rhizosphere level could promote the growth of the associated hyperaccumulating plant species and increase the absorption of metals in polluted soils, broadening new horizons on bioremediation purposes. This work investigates interactions between Ni-tolerant plant growth-promoting bacteria and fungi (BF) isolated from the rhizosphere of a hyperaccumulating plant. The aim is to select microbial consortia with synergistic activity to be used in integrated bioremediation protocols. Pseudomonas fluorescens (Pf), Streptomyces vinaceus (Sv) Penicilliumochrochloron (Po), and Trichoderma harzianum group (Th) were tested in mixes (Po-Sv, Po-Pf, Th-Pf, and Th-Sv). These strains were submitted to tests (agar overlay, agar plug, and distance growth co-growth tests), tailored for this aim, on Czapek yeast agar (CYA) and tryptic soy agar (TSA) media and incubated at 26 ± 1 °C for 10 days. BF growth, shape of colonies, area covered on plate, and inhibition capacity were evaluated. Most BF strains still exhibit their typical characters and the colonies separately persisted without inhibition (as Po-Sv) or with reciprocal confinement (as Th-Sv and Th-Pf). Even if apparently inhibited, the Po-Pf mix really merged, thus obtaining morphological traits representing a synergic co-growth, where both strains reached together the maturation phase and developed a sort of mixed biofilm. Indeed, bacterial colonies surround the mature fungal structures adhering to them without any growth inhibition. First data from in vivo experimentation with Po and Pf inocula in pot with metalliferous soils and hyperaccumulator plants showed their beneficial effect on plant growth. However, there is a lack of information regarding the effective co-growth between bacteria and fungi. Indeed, several studies, which directly apply the co-inoculum, do not consider suitable microorganisms consortia. Synergic rhizosphere BFs open new scenarios for plant growth promotion and soil bioremediation.

Zinc tolerance and accumulation in the ferns Polypodium cambricum L. and Pteris vittata L.

Zn uptake and compartmentalisation were studied in two ferns, the European Polypodium cambricum L., a possible Zn tolerant, and the sub-tropical Pteris vittata L., an As accumulator also able to accumulate Zn. Ferns growing in hydroponic systems were exposed to Zn concentrations ranging from non-toxic to lethal doses (0, 50, 125, 250, 500 mg kg(-1) as ZnSO4). After treatments, the following analyses were made: photosynthetic efficiency (Handy PEA), anatomical symptoms (optical and scanning electron microscopy), determination of Zn in fronds, rhizome and roots (atomic emission spectrometry, ICP-AES). Both species showed high bioconcentration and bioaccumulation factors, but low translocation factor, indicating Zn sequestration in the root/rhizome system. P. cambricum was more resistant to Zn, while P. vittata suffered from unrestricted uptake leading to macro- and microscopical damages and plant death. Data suggest that P. cambricum could be suitable for phytostabilisation of Zn-contaminated soils in temperate areas.

Rhizosphere response to nickel in a facultative hyperaccumulator.

This study faces the characterization of the culturable microbiota of the facultative Ni-hyperaccumulator Alyssoides utriculata to obtain a collection of bacterial and fungal strains for potential applications in Ni phytoextraction. Rhizosphere soil samples and adjacent bare soil associated with A. utriculata from serpentine and non-serpentine sites were collected together with plant roots and shoots. Rhizobacteria and fungi were isolated and characterized genotypically and phenotypically. Plants and soils were analyzed for total element concentration using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Serpentine and non-serpentine sites differ in terms of elements concentration in soil, plant roots and shoots. Ni and Co are significantly higher on serpentine site, while Ca is more abundant in non-serpentine site. Bacteria and fungi were significantly more abundant in rhizosphere than in bare soil and were dominated by genera Arthrobacter, Bacillus and Streptomyces, Penicillium and Mucor. The genus Pseudomonas was only found in rhizospheric serpentine soils (<2% of total serpentine isolates) and with Streptomyces sp. showed highest Ni-tolerance up to 15 mM. The same occurred for Trichoderma strain, belonging to the harzianum group (<2% of the total microfungal count) and Penicillium ochrochloron (<10% of the total microfungal count, tolerance up to Ni 20 mM). Among serpentine bacterial isolates, 8 strains belonging to 5 genera showed at least one PGPR activity (1-Aminocyclopropane-1-Carboxylic Acid (ACC) deaminase activity, production of indole-3-acetic acid (IAA), siderophores and phosphate solubilizing capacity), especially genera Pantoea, Pseudomonas and Streptomyces. Those microorganisms might thus be promising candidates for employment in bioaugmentation trials.

The impact of Ni on the physiology of a Mediterranean Ni-hyperaccumulating plant.

High nickel (Ni) levels exert toxic effects on plant growth and plant water content, thus affecting photosynthesis. In a pot experiment, we investigated the effect of the Ni concentration on the physiological characteristics of the Ni hyperaccumulator Alyssoides utriculata when grown on a vermiculite substrate in the presence of different external Ni concentrations (0-500 mg Ni L(-1)). The results showed that the Ni concentration was higher in leaves than in roots, as evidenced by a translocation factor = 3 and a bioconcentration factor = 10. At the highest concentration tested (500 mg Ni L(-1)), A. utriculata accumulated 1100 mg Ni per kilogram in its leaves, without an effects on its biomass. Plant water content increased significantly with Ni accumulation. Ni treatment did not, or only slightly, affected chlorophyll fluorescence parameters. The photosynthetic efficiency (FV/FM) of A. utriculata was stable between Ni treatments (always ≥ 0.8) and the photosynthetic performance of the plant under Ni stress remained high (performance index = 1.5). These findings support that A. utriculata has several mechanisms to avoid severe damage to its photosynthetic apparatus, confirming the tolerance of this species to Ni under hyperaccumulation.

Nickel phytoremediation potential of the Mediterranean Alyssoides utriculata (L.) Medik.

This study investigated the accumulation and distribution of nickel in the leaves and roots of the Mediterranean shrub Alyssoides utriculata to assess its potential use in phytoremediation of Ni contaminated soils. Total (AAS and ICP-MS) Ni, Ca and Mg contents were analyzed in the plants and related to their bioavailability (in EDTA) in serpentine and non-serpentine soils. To find the relationships between the soil available Ni and the Ni content of this species, we also evaluated possible interactions with Ca and Mg. The bioaccumulation factor (BF) and the translocation factor (TF) were determined to assess the tolerance strategies developed by A. utriculata and to evaluate its potential for phytoextraction or phytostabilization. The leaf Ni is higher than 1000 µg g(-1) which categorizes the species as a Ni-hyperaccumulator and a great candidate for Ni-phytoextraction purposes. In addition to the accumulation of Ni, the leaf Mg is also correlated with soil bioavailable concentrations. The Ca uptake and translocation were significantly lower in serpentine plants (higher Ni), as such, the leaf Ca is probably greatly influenced either by the soil's Ni or the soil Ca/Mg ratio. The BFs and TFs are strongly higher than 1 and generally did not significantly differed between plants from serpentine (higher Ni) and non-serpentine soils (lower Ni). The present study highlights for the first time that A. utriculata could be suitable for cleaning Ni-contaminated areas and provides a contribution to the very small volume of data available on the potential use of native Mediterranean plant species from contaminated sites in phytoremediation technologies.

Microfungi in highly copper-contaminated soils from an abandoned Fe-Cu sulphide mine: growth responses, tolerance and bioaccumulation.

Copper is one of the most dangerous soil contaminants. Soils affected by high copper concentrations show low biodiversity and, above all, inadequate environmental quality. Microorganisms such as fungi can play a key role in metal-polluted ecosystems via colonization and decontamination. The study is devoted to characterize the microfungal community in highly Cu-contaminated bare soil from derelict Fe-Cu sulphide mines and to isolate microfungal strains able to tolerate and accumulate Cu. 11 Different taxa to be isolated has been isolated during two sampling campaigns (in Autumn and in Spring). Among these, Clonostachys rosea, Trichoderma harzianum, and Aspergillus alliaceus were tested at increasing Cu(II) concentrations and showed a Cu(II)-tolerance capability ranging from 100 to 400 mg L(-1). Moreover, the strains of T. harzianum and C. rosea presented a high Cu(II)-bioaccumulation capability, 19628 and 22,222 mg kg(-1), respectively. These microfungi may be fruitfully exploited in mycoremediation protocols.

Effects of cadmium and arsenic on Pteris vittata under hydroponic conditions.

Pteris vittata is known to hyperaccumulate arsenic, and a large number of studies on this fern species can be found in the literature aimed at evaluating its behavior when coexposed to other toxic elements. In the present study, P. vittata was treated with different concentrations of As and/or Cd in a hydroponic system, that is, under complete bioavailability of the elements, with the objective of investigating the effects of these two elements and their interactions. The response of the plant was evaluated by measuring As, Cd, P, and Ca concentrations in different parts of the plant. Moreover, the symptoms of phytotoxicity were assessed in terms of biomass reduction and loss of photosynthetic efficiency related to necrosis of pinnae. The concentrations of As and Cd measured in the fronds and the root system were significantly dependent on the treatment, whereas P and Ca concentrations were not affected. Interaction effects between As and Cd were observed, with maximum toxicity symptoms after treatment with both elements. This could affect the potential use of this fern for phytoremediation. Although As treatment produced a significant effect on leaves (e.g., chlorosis and necrosis), Cd treatment produced a stronger negative impact on plant health, reducing significantly the biomass and photosynthetic efficiency.