Description, identification, and growth of Tuber borchii Vittad. mycorrhized Pinus sylvestris L. seedlings on different lime contents.

Tuber borchii forms ectomycorrhiza with oaks, hazel, and pines, including Pinus sylvestris. However, its ectomycorrhiza morphotype with P. sylvestris was not comprehensively described so far, and molecular analyses are missing despite a high danger of misidentification of T. borchii ectomycorrhiza with other closely related and less valuable truffle species. We described for the first time the morphology and anatomy of T. borchii-P. sylvestris ectomycorrhiza using differential interference contrast technique and semi-thin sections in combination with molecular confirmation of identity. Color of ectomycorrhiza is reddish to dark brown, and morphotypes are unevenly but densely covered by warts-bearing pin-like cystidia. All layers of the hyphal mantle are pseudoparenchymatous with outer mantle layer formed of epidermoid cells. T. borchii ectomycorrhiza was identified by a molecular comparison with fruitbodies used for inoculation and its respective ectomycorrhizae. T. borchii has a wide ecological amplitude. To get a better insight in mycorrhization requirements, we investigated growth of P. sylvestris and its ectomycorrhiza infection rate with T. borchii in substrate with different lime content. The mycorrhization of P. sylvestris with T. borchii in the mycorrhization substrate and cultivation in greenhouse conditions was successful, with colonization of P. sylvestris varying between 36.5 and 48.1%. There was no significant correlation of mycorrhization to applied lime contents, and consequently to pH in substrate, while the increased levels of lime improved growth of the P. sylvestris seedlings.

Habitat specialisation controls ectomycorrhizal fungi above the treeline in the European Alps.

Alpine habitats are one of the most vulnerable ecosystems to environmental change, however, little information is known about the drivers of plant-fungal interactions in these ecosystems and their resilience to climate change. We investigated the influence of the main drivers of ectomycorrhizal (EM) fungal communities along elevation and environmental gradients in the alpine zone of the European Alps and measured their degree of specialisation using network analysis. We sampled ectomycorrhizas of Dryas octopetala, Bistorta vivipara and Salix herbacea, and soil fungal communities at 28 locations across five countries, from the treeline to the nival zone. We found that: (1) EM fungal community composition, but not richness, changes along elevation, (2) there is no strong evidence of host specialisation, however, EM fungal networks in the alpine zone and within these, EM fungi associated with snowbed communities, are more specialised than in other alpine habitats, (3) plant host population structure does not influence EM fungal communities, and (4) most variability in EM fungal communities is explained by fine-scale changes in edaphic properties, like soil pH and total nitrogen. The higher specialisation and narrower ecological niches of these plant-fungal interactions in snowbed habitats make these habitats particularly vulnerable to environmental change in alpine ecosystems.

Ectomycorrhizal community composition of organic and mineral soil horizons in silver fir (Abies alba Mill.) stands.

Vertical ectomycorrhizal (ECM) community composition was assessed on silver fir (Abies alba Mill.) in beech-silver fir forests in Bosnia and Herzegovina. Organic and upper mineral horizons were described by pedological analyses. Silver fir root tips were divided into vital ECM, old and non-mycorrhizal for each horizon separately. Morpho-anatomical classification of vital ECM root tips with an assessment of abundance was followed by ITS-based molecular characterization and classification into exploration types. The percentage of vital ECM root tips was not affected by the soil horizon. Altogether, 40 ECM taxa were recorded. Several taxa have not previously been reported for silver fir: Hebeloma laterinum, Inocybe fuscidula, I. glabripes, Lactarius acris, L. albocarneus, L. blennius, L. fluens, Ramaria bataillei, Russula badia, R. lutea, R. mairei, Sistotrema sp., Tarzetta catinus, Tomentella atroarenicolor, T. badia, T. cinerascens, T. bryophylla, and T. ramosissima, indicating high potential for diversity of ECM fungi in silver fir stands. No significant differences in community composition and species richness and diversity were detected between mineral and organic horizons. Community composition was affected by CaCO3, organic carbon concentration, organic carbon stock, total nitrogen stock, C/N ratio and soil bulk density. No significant effects of soil parameters were detected for exploration types. The contact exploration type was dominant in both soil horizons. Significantly different relative abundances of dominant taxa Tomentella stuposa, Cenococcum geophilum and Piloderma sp. 1 were detected in the two horizons. Twelve taxa were limited to the organic horizon and eight to the mineral horizon.

Intra-annual dynamics of phloem formation and ultrastructural changes in sieve tubes in Fagus sylvatica.

Despite increased interest in the timing and dynamics of phloem formation, seasonal changes in the structure of phloem sieve elements remain largely unexplored. To understand better the dynamics of phloem formation and the functioning of sieve tubes in the youngest phloem in Fagus sylvatica L., we investigated repeatedly taken phloem samples during the growing season of 2017 by means of light microscopy, and transmission and scanning electron microscopy. Phloem formation started with the expansion of the overwintered early phloem sieve tubes adjacent to the cambium and concurrent cambial cell production. The highest phloem growth rate was observed in general 1 week after the onset of cambial cell production, whereas the transition from early to late phloem occurred at the end of May. Cambial cell production ceased at the end of July. The final width of the phloem increment was 184 ± 10 µm, with an early phloem proportion of 59%. Collapse of older phloem tissue is a progressive process, which continuously occurred during the sampling period. Collapse of early phloem sieve tubes started shortly after the cessation of cambial cell production. Prior to the onset of radial growth, late phloem from the previous year represented 80% of the total non-collapsed part; during the growth period, this percentage decreased to 20%. Differences were observed in both sieve tube ultrastructure and sieve plate geometry between the youngest and older phloem. However, sieve plates were never completely occluded by callose, suggesting that processes affecting the functionality of sieve tubes may differ in the case of regular collapse or injury. The youngest parts of the phloem increment from the previous year (i.e., previous late phloem) continue functioning for some time in the current growing season, but the two-step development of overwintered phloem cells also ensures a sufficient translocation pathway for photosynthates to the actively growing tissues.

Abscisic acid enhances lead translocation from the roots to the leaves and alleviates its toxicity in Populus × canescens.

To shed light on physiological mechanisms underlying abscisic-acid (ABA)-mediated lead (Pb) uptake, translocation and detoxification, we exposed Populus × canescens saplings to either 0 or 3 mM Pb2+ in combination with either 0 or 10 µM exogenous ABA. Pb was taken up by the roots and accumulated mainly in the cortex. A fraction of the Pb in the roots was translocated to the leaves, thereby resulting in decreased photosynthesis and biomass. Pb accumulation caused a burst of reactive oxygen species (ROS), with higher concentrations of total thiols, glutathione, and ascorbate in the roots and/or leaves. Exogenous ABA stimulated Pb uptake, decreased Pb deposition in the cortex, and enhanced Pb vascular loading in the roots. Exogenous ABA alleviated the Pb-induced reductions in photosynthesis and root biomass, and decreased Pb-triggered ROS overproduction in the roots and/or leaves. Correspondingly, exogenous ABA stimulated the mRNA levels of a few genes involved in Pb uptake, transport, and detoxification, including NRAMP1.4, ABCG40, FRD3.1, PCS1.1, and ABCC1.1. These results suggest that exogenous ABA enhances Pb uptake and translocation, and alleviates Pb toxicity in poplars through the ABA-induced movement of Pb from the root cortex to the vascular stele, and transcriptionally regulated key genes involved in Pb tolerance.

Different belowground responses to elevated ozone and soil water deficit in three European oak species (Quercus ilex, Q. pubescens and Q. robur).

Effects on roots due to ozone and/or soil water deficit often occur through diminished belowground allocation of carbon. Responses of root biomass, morphology, anatomy and ectomycorrhizal communities were investigated in seedlings of three oak species: Quercus ilex L., Q. pubescens Willd. and Q. robur L., exposed to combined effects of elevated ozone (ambient air and 1.4 × ambient air) and water deficit (100% and 10% irrigation relative to field capacity) for one growing season at a free-air ozone exposure facility. Effects on root biomass were observed as general reduction in coarse root biomass by -26.8% and in fine root biomass by -13.1% due to water deficit. Effect on coarse root biomass was the most prominent in Q. robur (-36.3%). Root morphological changes manifested as changes in proportions of fine root (<2 mm) diameter classes due to ozone and water deficit in Q. pubescens and due to water deficit in Q. robur. In addition, reduced fine root diameter (-8.49%) in Q. robur was observed under water deficit. Changes in root anatomy were observed as increased vessel density (+18.5%) due to ozone in all three species, as reduced vessel tangential diameter (-46.7%) in Q. ilex due to interaction of ozone and water, and as generally increased bark to secondary xylem ratio (+47.0%) due to interaction of ozone and water. Water deficit influenced occurrence of distinct growth ring boundaries in roots of Q. ilex and Q. robur. It shifted the ectomycorrhizal community towards dominance of stress-resistant species, with reduced relative abundance of Tomentella sp. 2 and increased relative abundances of Sphaerosporella brunnea and Thelephora sp. Our results provide evidence that expression of stress effects varies between root traits; therefore the combined analysis of root traits is necessary to obtain a complete picture of belowground responses.

1-Aminocyclopropane-1-Carboxylate Oxidase Induction in Tomato Flower Pedicel Phloem and Abscission Related Processes Are Differentially Sensitive to Ethylene.

Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0-12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.

Scleroderma areolatum ectomycorrhiza on Fagus sylvatica L.

Despite its broad host range and distribution and its potential applications in commercial plantation forests, comprehensive descriptions of Scleroderma ectomycorrhizae are available only for Scleroderma citrinum, Scleroderma bovista and Scleroderma sinnamariense. This study provides a morphological and anatomical description of tree nursery derived ectomycorrhizae of Scleroderma areolatum on Fagus sylvatica, grown for several years in a climatized room. Ectomycorrhizae of S. areolatum were silvery white with abundant rhizomorphs; all mantle layers were plectenchymatous, rhizomorphs of type E, with prominent emanating hyphae with thick cell wall. The distal ends of emanating hyphae of rhizomorphs were inflated and often merged with other emanating hyphae. All parts of the mycorrhiza were clampless. In hyphae of the outer mantle layer, rhizomorphs and emanating hyphae, oily droplets were observed that did not stain in sulfo-vanillin and disappeared in lactic acid after a few hours. Although the phylogenetic analysis positioned the newly described ectomycorrhiza together with Scleroderma verrucosum and Scleroderma cepa in a single clade with a taxon name SH005470.07FU, the ectomycorrhizae of these three species can be morphologically well separated based on rhizomorph type.

Biomonitoring with epiphytic lichens as a complementary method for the study of mercury contamination near a cement plant.

The study was focused on understanding the mercury contamination caused by a cement plant. Active and passive biomonitoring with epiphytic lichens was combined with other instrumental measurements of mercury emissions, mercury concentrations in raw materials, elemental mercury concentrations in air, quantities of dust deposits, temperatures, precipitation and other measurements from the cement plant's regular monitoring programme. Active biomonitoring with transplanted lichens Pseudevernia furfuracea (L.) Zopf was performed at seven of the most representative sites around the cement plant and one distant reference site for periods of 3, 6 and 12 months. In situ lichens of different species were collected at the beginning of the monitoring period at the same sites. Mercury speciation of the plant exhaust gas showed that the main form of emitted mercury is reactive gaseous mercury Hg²âº, which is specific for cement plants. Elemental mercury in air was measured in different meteorological conditions using a portable mercury detector. Concentrations in air were relatively low (on average below 10 ng m⁻³). In situ lichens showed Hg concentrations comparable to lichens taken from the background area for transplantation, indicating that the local pollution is not severe. Transplanted lichens showed an increase of mercury, especially at one site near the cement plant. A correlation between precipitation and Hg uptake was not found probably due to a rather uniform rainfall in individual periods. Dust deposits did not influence Hg uptake significantly. Lichens vitality was affected over longer biomonitoring periods, probably due to some elements in dust particles, their alkalinity and the influence of other emissions. Mercury uptake measured in vital transplanted lichens was in a good correlation with the working hours (i.e. emitted Hg quantity) of the kiln. The study showed that selected lichens could be used to detect low to moderate Hg emissions from a cement plant and that the biomonitoring procedure could be further standardized and used as part of an environmental monitoring programme.

Arsenic accumulation and thiol status in lichens exposed to As(V) in controlled conditions.

Thalli of epiphytic lichen Hypogymnia physodes (L.) Nyl. and terricolous Cladonia furcata (Huds.) Schrad., collected from an area with background arsenic concentrations, were exposed to 0, 0.1, 1 and 10 microg mL(-1) arsenate (As(V)) solutions for 24 h. After exposure they were kept in the metabolically active state for 0, 24 and 48 h in a growth chamber. In the freeze dried samples glutathione (GSH), glutathione disulphide (GSSG), cysteine (Cys) and cystine were analysed and induction of phytochelatin (PC) synthesis measured by reversed-phase high-performance liquid chromatography in combination with fluorescence detection or UV spectrometry. Total arsenic content in thalli was measured by instrumental neutron activation analysis (INAA). In H. physodes, which contained higher amounts of arsenic compared to C. furcata, total glutathione content significantly decreased in samples exposed to 10 microg mL(-1) As(V), whereas in C. furcata a significant increase was observed. In both species PC synthesis was induced in thalli exposed to 10 microg mL(-1).

Uptake and biotransformation of arsenate in the lichen Hypogymnia physodes (L.) Nyl.

The uptake and metabolism of arsenate, As(V), as a function of time and concentration were examined in the lichen Hypogymnia physodes (L.) Nyl. Lichen thalli were exposed to As(V) in the form of a solution. Exponential uptake of As(V) from 4 microg mL(-1) As(V) solution was accompanied by constant arsenite, As(III), excretion back into the solution. Arsenate taken up into the lichens from 0, 0.1, 1, 10 microg mL(-1) As(V) solutions was partially transformed into As(III), dimethylarsinic acid (DMA) and (mono)methylarsonic acid (MA). 48 h after exposure, the main arsenic compound in the lichens was DMA in 0.1, As(III) in 1 and As(V) in 10 microg mL(-1) treatment. The proportion of methylated arsenic compounds decreased with increasing arsenate concentration in the exposure solution. These results suggest that at least two types of As(V) detoxification exist in lichens; arsenite excretion and methylation.

Characteristics of suspended matter in the River Sava watershed, Slovenia.

A combination of C/N ratios, delta(13)C and delta(15)N values in suspended matter was used to examine the seasonal (late summer 2004 and spring 2005) relationship with hydrological characteristics of the River Sava watershed in Slovenia. The values of C/N ratios range from 1.2 to 19.1, delta(13)C values range from-29.2 to-23.0 per thousand and delta(15)N values from 0.5 to 16.7 per thousand and indicate that the samples are a mixture of two end members: modern soils and plant litter. A simple mixing model was used to indicate that soil organic carbon prevails over plant litter and contributes more than 50% of suspended material. The calculated annual particulate organic carbon flux is estimated as 5.2x10(10) g C/year, the annual particulate nitrogen flux 8.5x10(9) g N/year and the total suspended solid flux is estimated to be 1.3x10(12) g/year. Anthropogenic impact was detected only in a tributary stream of the River Sava, which is located in an agriculture-industrial area and is reflected in higher delta(15)N values in suspended matter and high nitrate concentrations in the late summer season.

Extraction of arsenic compounds from lichens.

Different extraction procedures were applied to improve the extraction efficiency of arsenic compounds from lichens. Two lichen species were chosen from an arsenic-contaminated environment: epiphytic Hypogymnia physodes (L.) Nyl. and terricolous Cladonia rei Schaer. Samples were extracted with water at temperatures of 20, 60 and 90 degrees C, using mixtures of methanol/water (9:1, 1:1 and 1:9), Tris buffer and acetone and the extracts speciated. Water and Tris buffer showed the best extraction efficiency of all extractants used; however, the extraction efficiency was still less than 23%. Since a major fraction of arsenic appeared to be associated with trapped soil particles, a sequential extraction procedure originally designed for soils (extraction steps: (1) 0.05 mol l(-1) (NH(4))(2)SO(4); (2) 0.05 mol l(-1) (NH)(4)H(2)PO(4); (3) 0.2 mol l(-1) NH(4)-oxalate buffer, pH 3.25; (4) mixture of 0.2 mol l(-1) NH(4)-oxalate buffer and 0.1 mol l(-1) ascorbic acid, pH 3.25; (5) 0.5 mol l(-1) KOH) was applied and found to remove 45% of the total arsenic from H. physodes and 83% from C. rei. The lipid-soluble fraction of arsenic was estimated by k(0)-INAA analysis of diethylether extracts and was found to be negligible. An HPLC-UV-HGAFS system was used to determine the arsenic compounds extracted. In both lichen species, arsenous acid, arsenic acid, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, trimethylarsine oxide and glycerol-ribose were detected. In addition, phosphate-ribose was found in H. physodes.