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Two plant pathogenic fungi, Botrytis cinerea and Alternaria alternata , isolated from crop plants, were exposed to Cu in ionic (Cu 2+ ), microparticulate (MP, CuO) or nanoparticulate (NP, Cu or CuO) form, in solid and liquid culturing media in order to test fungal response and toxic effects of the mentioned compounds for the potential use as fungicides. B. cinerea has shown pronounced growth and lower levels of lipid peroxidation compared to A. alternata . Its higher resistance/tolerance is attributed mainly to biotransformation of CuO and Cu NPs and CuO MPs into a blue compound at the fungal/culturing media interface, recognized by Cu K-edge EXAFS analysis as Cu-oxalate complex. The pronounced activity of catechol-type siderophores and organic acid secretion in B. cinerea induce leaching and mobilization of Cu ions from the particles and their further complexation with extracellularly secreted oxalic acid. The ability of pathogenic fungus to biotransform CuO MPs and NPs hampers their use as fungicides. However the results show that B. cinerea has a potential to be used in degradation of Cu(O) nanoparticles in environment, copper extraction and purification techniques. [ABSTRACT FROM AUTHOR]

Abstract: The effects of biofumigation and soil heating on arbuscular mycorrhizal fungi (AMF) colonisation, strawberry growth and strawberry yield in pot experiments compared with untreated soil and chemical fumigation with dazomet were tested. Three different Brassicaceae species (Brassica juncea, Eruca sativa, Sinapis alba) were used as biofumigant plant green manure and soil heating was applied to simulate soil solarisation. Half of the plants were inoculated with indigenous arbuscular mycorrhizal fungi inoculum. With one exception (E. sativa) among the uninoculated plants, the treatments significantly decreased the mycorrhizal colonisation parameters compared with the untreated control. Dazomet displayed the greatest inhibitory effects on AMF establishment. In addition, the intensity and number of bands corresponding to Glomus spp. obtained with temporal temperature-gradient gel electrophoresis were lower for strawberry plants from biofumigant treatments than from the control. For the inoculated plants, there were almost no significant differences among the mycorrhizal colonisation parameters. The mass of leaves for the uninoculated and inoculated plants was higher for almost all non-chemical soil fumigant treatments compared with the control, except for heating of the uninoculated treatments. The number of strawberry fruits for the uninoculated biofumigant treatments was the highest, being higher than the values observed for the heating treatments, the chemical disinfection treatments and the control. There were no significant differences among the inoculated treatments. Biofumigation with Brassicaceae species resulted in higher soil organic matter and mineral nutrients and had a relatively small effect on AMF colonisation (F% = 59.0, 80.3, 47.3 for Bj, Es and Sa, respectively) compared with uninoculated controls (F% = 84.3). Despite the reduced AMF colonisation, biofumigation resulted in a higher fruit number and mass of leaves. Therefore, it represents a non-chemical soil fumigation method that should be applied in sustainable strawberry production. [Copyright &y& Elsevier]

Cd, Zn and Pb accumulation, spatial distribution within seeds and germinating seedlings, and seeds fitness of metal hyperaccumulating Thlaspi praecox were investigated in order to gain more knowledge on plant reproductive success at metal polluted sites. The seeds contained up to 1351μgg−1 (dry weight) of Cd, 121μgg−1 of Zn and 17μgg−1 of Pb. Seed fitness was negatively influenced by seed Cd hyperaccumulation. Nevertheless, the viability of seeds was decreased by maximally 20%, indicating very efficient tolerance of the plant embryos to Cd. Localisation by micro-PIXE revealed preferential storage of most elements in the embryonic axis. Cd and Zn were preferentially localised in the epidermis of cotyledons. The restriction of seed Pb and Zn uptake and hyperaccumulation of Cd, accompanied by partitioning of Cd in the epidermal tissues of cotyledons, may enable the survival of T. Praecox embryos and seedlings in Cd polluted environments. [Copyright &y& Elsevier]

Abstract: Arbuscular mycorrhizal fungi (AMF) have repeatedly been demonstrated to alleviate heavy metal stress of plants. The current manuscript summarizes results obtained to date on the colonization of plants by AMF in heavy metal soils, the depositions of heavy metals in plant and fungal structures and the potential to use AMF-plant combinations in phytoremediation, with emphasis on pennycresses (Thlaspi ssp.). The focus of this manuscript is to describe and discuss studies on the expression of genes in plants and fungi under heavy metal stress. The summary is followed by data on differential gene expression in extraradical mycelia (ERM) of in vitro cultured Glomus intraradices Sy167 supplemented with different heavy metals (Cd, Cu or Zn). The expression of several genes encoding proteins potentially involved in heavy metal tolerance varied in their response to different heavy metals. Such proteins included a Zn transporter, a metallothionein, a 90kD heat shock protein and a glutathione S-transferase (all assignments of protein function are putative). Studies on the expression of the selected genes were also performed with roots of Medicago truncatula grown in either a natural, Zn-rich heavy metal “Breinigerberg” soil or in a non-polluted soil supplemented with 100μM ZnSO4. The transcript levels of the genes analyzed were enhanced up to eight fold in roots grown in the heavy metal-containing soils. The data obtained demonstrate the heavy metal-dependent expression of different AMF genes in the intra- and extraradical mycelium. The distinct induction of genes coding for proteins possibly involved in the alleviation of damage caused by reactive oxygen species (a 90kD heat shock protein and a glutathione S-transferase) might indicate that heavy metal-derived oxidative stress is the primary concern of the fungal partner in the symbiosis. [Copyright &y& Elsevier]

Abstract: Plant communities on plots with different metal pollution levels were compared in a field study in order to select the most suitable plant species for the direction of secondary succession toward the targeted grassland vegetation. The vegetational succession showed a gradual increase in plant cover and the number of plant species on the less polluted locations. Two predominant grass species Calamagrostis varia and Sesleria caerulea were selected for phytostabilisation, but a severely reduced seed germination capacity obstructed their use in practice. The mycorrhizal succession showed a gradual replacement of non-mycorrhizal with mycorrhizal plant species. Similar levels of arbuscular mycorrhizal colonisation of a particular plant species may be developed within each growing season regardless of the levels of pollution, with the exception of vesicle/intraradical spore formation. The results suggest that lower overall mycorrhizal colonisation levels and increased vesicle/spore formation may be a part of a mycorrhizal strategy at the most polluted locations. [Copyright &y& Elsevier]

Abstract: Plants of the Zn, Cd and Pb hyperaccumulator Thlaspi praecox Wulfen (Brassicaceae) inoculated or not with indigenous arbuscular mycorrhizal (AM) fungal mixture were grown in a highly Cd, Zn and Pb contaminated substrate in order to evaluate the functionality of symbiosis and assess the possible impact of AM colonisation on heavy metal uptake and tolerance. The results suggest AM development in the metal hyperaccumulating T. praecox is favoured at elevated nutrient demands, e.g. during the reproductive period. AM colonisation parameters positively correlated with total soil Cd and Pb. Colonised plants showed significantly improved nutrient and a decreased Cd and Zn uptake as revealed by TRXRF, thus confirming the functionality of the symbiosis. Reduced heavy metal uptake, especially at higher soil metal contents, indicates a changed metal tolerance strategy in colonised T. praecox plants. This is to our knowledge the first report on AM colonisation of the Zn, Cd and Pb hyperaccumulator T. praecox in a greenhouse experiment. [Copyright &y& Elsevier]

Significant hyperaccumulation of Zn, Cd and Pb in field samples of Thlaspi praecox Wulf. collected from a heavy metal polluted area in Slovenia was found, with maximal shoot concentrations of 14 590mgkg-1 Zn, 5960mgkg-1 Cd and 3500mgkg-1 Pb. Shoot/root ratios of 9.6 for Zn and 5.6 for Cd show that the metals were preferentially transported to the shoots. Shoot bioaccumulation factors exceeded total soil Cd levels 75-fold and total soil Zn levels 20-fold, further supporting the hyperaccumulation of Cd and Zn. Eighty percent of Pb was retained in roots, thus indicating exclusion as a tolerance strategy for Pb. Low level colonisation with arbuscular mycorrhizal fungi (AMF) of a Paris type was observed at the polluted site, whereas at the non-polluted site Arum type colonisation was more common. To our knowledge this is the first report of Cd hyperaccumulation and AMF colonisation in metal hyperaccumulating T. praecox. [Copyright &y& Elsevier]

Crops' wild relatives host a wide range of microorganisms, including some beneficial species that are not found or are under-represented in the domesticated crops. Our goal was to study the underexplored composition of root-associated fungal communities in endangered wild grapevines. We found high taxonomic diversity representing multiple trophic guilds that include beneficial symbiotrophs and endophytes. Soil factors explain a relatively small part of their overall variability. In contrast, the majority of the associated fungal taxa shows a close fit to the neutral model for prediction of their distributions. Only beneficial arbuscular mycorrhizal fungi and the pathogenic Ilyonectria depart from the neutral distribution model and form intimate interactions with the plant host. In addition, pathogenic fungi rarely occurred in samples that included ectomycorrhizal fungi, which suggested potentially applicable inter-microorganism interactions. High abundance and diversity of fungal endophytes on the wild grapevine roots highlight the need for their careful consideration in future studies. • Wild grapevine roots host high diversity of fungi from multiple trophic guilds. • Root-associated fungi fit mainly into neutral model for distribution prediction. • Arbuscular mycorrhizal fungi and Ilyonectria most intimately relate to the host. • Pathogenic fungi rarely occurred in samples that included ectomycorrhizal fung. [ABSTRACT FROM AUTHOR]

Seed-associated fungal communities affect multiple parameters of seed quality at all stages of production, from seed development to post-harvest storage and germination. We therefore investigated the diversity and dynamics of fungal communities in the seeds of common buckwheat ( Fagopyrum esculentum ) and Tartary buckwheat ( F. tataricum ) from harvest to 1 y of storage. Fungal populations in seeds were relatively stable, comprised mainly of field fungi. Incidence of fungi was most likely determined by fungal interspecies direct interactions, as well as by their synthesis of volatile organic compounds. Most prominent antagonistic interactions were seen for two plant pathogens , Alternaria alternata on Botrytis cinerea. Detrimental effects of the fungi on seed germination and seedling development were related to fungal extracellular enzyme activity, and in particular to amylase, cellulase and, polyphenol oxidase. Polyphenol and tannin concentrations in buckwheat seedlings were related to fungal growth rate and intensity of fungal cellulase activity, respectively, which suggests that physical penetration of the fungi through the host tissues is probably the stimulus for the activation of plant defence reactions in these seedlings. [ABSTRACT FROM AUTHOR]

Abstract: The different buckwheat species are gluten-free pseudocereals, and they are amongst the most important alternative foodstuffs as they have an excellent nutrient profile. The grain of tartary buckwheat (Fagopyrum tataricum Gaertn.) is known to be rich in antioxidants (especially rutin) and mineral elements. In addition to the spatial distributions of Mg, P, S, K, Ca, Mn, Fe, Cu and Zn at the tissue level that have been determined using micro-proton-induced X-ray emission, we have also assessed the distributions of C, O, Mg, P, Mn, Fe, Cu and Zn at the cellular and sub-cellular levels using synchrotron radiation-based low-energy X-ray fluorescence. The highest concentrations of all of the studied mineral elements were in the cotyledons, except for Ca, which dominated in the pericarp. The relative mineral-element distributions calculated on dry weight basis confirm the observed mineral distribution profiles. In the cotyledons, P clearly partitions to the mesophyll and is mainly ascribed to phytate. In the mesophyll, the P spatial distribution strongly correlates with Mg and, in decreasing order, with Cu>Fe>Mn>Zn. These spatial distributions of the mineral elements, their concentrations and their co-localisation are discussed in relation to other pseudocereal and cereal grain. [Copyright &y& Elsevier]

Abstract: Maize root residues that remain in the field after harvesting are a source of water-soluble compounds, which can inhibit growth of succeeding maize crops through autotoxicity. The aim of this study was to test whether the formation of symbiosis between maize and arbuscular mycorrhizal fungi (AMF) alleviates negative effects of autotoxicity. Maize root extracts containing water-soluble compounds were used to simulate the release of autotoxic compounds from decomposing root residues in soil under experimental conditions and were applied at increasing concentrations to maize seedlings. The 0.15% (w/v) root extract treatment decreased root biomass and increased guaiacol peroxidase (G-POD) activity and concentrations of the major water-soluble compound benzoxazinone 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) in roots of maize seedlings. As such, 0.15% concentration was used in subsequent inoculation experiment. The effects of maize root extracts seen in non-inoculated seedlings were lost in AMF-inoculated maize, even though the AMF colonization was reduced in the root extract-treated seedlings. These data indicate that AMF can alleviate the negative effects induced in maize seedlings by water-soluble compounds from maize roots and show potential importance of AMF in extensive agricultural practice. [Copyright &y& Elsevier]

Abstract: A better understanding of the mechanisms that govern copper (Cu) uptake, distribution and tolerance in Brassica carinata plants in the presence of chelators is needed before significant progress in chelate-assisted Cu phytoextraction can be made. The aims of this study were therefore to characterise (S,S)-N,N′-ethylenediamine disuccinic acid (EDDS)-assisted Cu uptake, and to compare the spatial distribution patterns of Cu in the roots and leaves of B. carinata plants. The plants were treated with 30μM or 150μM CuSO4 or CuEDDS in hydroponic solution. Quantitative Cu distribution maps and concentration profiles across root and leaf cross-sections of the desorbed plants were obtained by micro-proton induced X-ray emission. In roots, the 30μM treatments with both CuSO4 and CuEDDS resulted in higher Cu concentrations in epidermal/cortical regions. At 150μM CuSO4, Cu was mainly accumulated in root vascular bundles, whereas with 150μM CuEDDS, Cu was detected in endodermis and the adjacent inner cortical cell layer. Under all treatments, except with a H+-ATP-ase inhibitor, the Cu in leaves was localised mainly in vascular tissues. The incubation of plants with 150μM CuEDDS enhanced metal translocation to shoots, in comparison to the corresponding CuSO4 treatment. Inhibition of H+-ATPase activity resulted in reduced Cu accumulation in 30μM CuEDDS-treated roots and 150μM CuEDDS-treated leaves, and induced changes in Cu distribution in the leaves. This indicates that active mechanisms are involved in retaining Cu in the leaf vascular tissues, which prevent its transport to photosynthetically active tissues. The physiological significance of EDDS-assisted Cu uptake is discussed. [Copyright &y& Elsevier]

Abstract: Sečovlje salterns are an important protected area of biotic diversity in the Mediterranean. They represent an extreme environment with high salinity and drought that severely influence the growth of organisms. In the present study, diversity of plant halophytes and their mycorrhizal status were screened at eight different locations, which were mostly dikes and salt ponds, and which were deliberately selected for their distinct properties (e.g. soil salinity ranging from 105 to 2627 μS cm−1, vegetation type and management practice of the salterns). Twelve different halophytic plant species were recorded, of which eleven are designated as vulnerable. With few exceptions, they were found at the abandoned (Fontanigge) and sustained (Lera) locations of the Sečovlje salterns, distributed according to their tolerance to the salinity and waterlogging. The highest diversity of halophytes was listed at Fontanigge, in the abandoned, periodically flooded and gradually overgrown salt ponds. All of the examined species were colonised with either arbuscular mycorrhizal fungi (AMF) and/or dark septate endophytes (DSEs). High levels of colonisation were however detected only for species belonging to the Asteraceae and Plantaginaceae families. Higher root colonisation frequencies were generally seen for plants growing in the abandoned parts, when compared to the managed parts, whereas there was little correlation of the colonisation parameters with physicochemical parameters of rhizospheric soil properties. Molecular analysis by temporal temperature gradient gel electrophoresis (TTGE) of roots of halophytic plant species with confirmed AMF colonisation (arbuscules present) revealed the occurrence of at least six different AMF species, related to Glomus geosporum, Glomus caledonium and Glomus intraradices, and to different Glomus sp. clades and the Diversispora clade. This is to the best of our knowledge the first report of AMF and DSE mycorrhizal status of most of the halophyte plant species examined and of the brother scale identification of AMF species based on molecular analyses of roots of diverse halophytes from high saline environments. [Copyright &y& Elsevier]

Abstract: Phytoremediation is an emerging technology that employs the use of higher plants for the clean-up of contaminated environments. Progress in the field is however handicapped by limited knowledge of the biological processes involved in plant metal uptake, translocation, tolerance and plant–microbe–soil interactions; therefore a better understanding of the basic biological mechanisms involved in plant/microbe/soil/contaminant interactions would allow further optimization of phytoremediation technologies. In view of the needs of global environmental protection, it is important that in phytoremediation and plant biology studies the analytical procedures for elemental determination in plant tissues and soil should be fast and cheap, with simple sample preparation, and of adequate accuracy and reproducibility. The aim of this study was therefore to present the main characteristics, sample preparation protocols and applications of X-ray fluorescence-based analytical techniques (energy dispersive X-ray fluorescence spectrometry—EDXRF, total reflection X-ray fluorescence spectrometry—TXRF and micro-proton induced X-ray emission—micro-PIXE). Element concentrations in plant leaves from metal polluted and non-polluted sites, as well as standard reference materials, were analyzed by the mentioned techniques, and additionally by instrumental neutron activation analysis (INAA) and atomic absorption spectrometry (AAS). The results were compared and critically evaluated in order to assess the performance and capability of X-ray fluorescence-based techniques in phytoremediation and plant biology studies. It is the EDXRF, which is recommended as suitable to be used in the analyses of a large number of samples, because it is multi-elemental, requires only simple preparation of sample material, and it is analytically comparable to the most frequently used instrumental chemical techniques. The TXRF is compatible to FAAS in sample preparation, but relative to AAS it is fast, sensitive and multi-elemental. The micro-PIXE technique requires rather expensive instrumentation, but offers multi-elemental analysis on the tissue and cellular level. [Copyright &y& Elsevier]

Elemental uptake and arbuscular mycorrhizal (AM) colonisation were studied during the life cycle of field collected Cd/Zn hyperaccumulating Thlaspi praecox (Brassicaceae). Plant biomass and tissue concentrations of Cd, Pb, Zn, Fe and Ni were found to vary during development, while no variation in P, K, Ca, Mn and Cu tissue concentrations were observed. The lowest Cd bioaccumulation in rosette leaves (BAFRL) observed during seeding was partially attributed to lower translocation from roots to rosette leaves and partially to high translocation to stalks, indicating a high Cd mobility to reproductive tissues, in line with our previous studies. The highest intensity of AM colonisation (M%) was observed in the flowering phase and was accompanied by increased root Cd, Zn, Pb and Fe contents. In addition, a positive correlation between AM colonisation and Fe contents in rosette leaves was found. The results indicate developmental dependence of AM formation, accompanied by selective changes in nutrient acquisition in T. praecox that are related to increased plant needs, and the protective role of AM colonisation on metal polluted sites during the reproductive period. [Copyright &y& Elsevier]

During tea preparation mineral elements are extracted from the dried leaves of tea (Camellia sinensis (L.) Kuntze) plants into the solution. Micro-particle induced X-ray emission was employed to investigate the spatial distribution of magnesium (Mg), calcium (Ca) and manganese (Mn) in the young and old leaves of tea plants grown in the absence and presence of aluminium (Al) in the substrate. Results revealed that in tea leaves the largest concentrations of Mg occurred in the epidermis, of Ca in oxalate crystals and of Mn in epidermis and oxalate crystals; there was a leaf-age effect on tissue-specific concentrations of Mg, Ca and Mn with all tissues of old leaves containing larger concentrations of Mg, Ca and Mn than young leaves; supplementation of substrate with Al reduced concentrations of Mg, Ca and Mn in the old leaves, and a link between the distribution of Mg, Ca and Mn in the tea leaves with the extraction efficiencies of these elements into the tea was possible. We conclude that old leaves of tea plants cultivated under conditions of low Al availability will have the largest concentrations of Mg, Ca and Mn and may represent most acceptable ingredient for the preparation of tea. Image 1 • Spatial distribution of Mg, Ca and Mn was investigated in the young and old leaves of tea. • The largest concentration of Mg was found in epidermis of both young and old leaves. • Ca was mainly associated with oxalate crystals. • Mn was found in epidermis and in oxalate crystals. • High extraction efficiency reported for Mg and Mn may be related with their allocation to epidermis. [ABSTRACT FROM AUTHOR]