Polycyclic aromatic hydrocarbon (PAH) phytoaccumulation in urban areas by Platanus × acerifolia, Celtis australis, and Tilia grandifolia leaves and branches.

Polycyclic aromatic hydrocarbon (PAH) concentrations in the leaves and 1-year-old branches of three common tree species growing in a middle-sized city located in a moderate climate zone were estimated. For this purpose, PAH phytoaccumulation in Platanus × acerifolia, Celtis australis, and Tilia grandifolia species from highly urbanized, traffic congested, and highly PAH-contaminated streets was compared with trees from non-contaminated parks in the same urban core. The gathered data was used to define 17 PAH profiles, identify the main PAH pollution emission sources, and determine the organ and species specificity of PAHs accumulation. Due to the direct absorption of polluted air via stomata, the leaves accumulated up to 30% more PAHs compared to the 1-year-old branches. As expected, PAH concentrations were much higher in street trees, while heavy weight PAHs (with five and six rings) were accumulated in the highest concentrations. The highest foliar Σ17 PAH concentrations were detected in street-grown C. australis, followed by P. acerifolia and T. grandifolia (502.68, 488.45, and 339.47 ng g-1 dry weight (DW), respectively). The same pattern was noted for Σ17 PAHs in branches (414.89, 327.58, and 342.99 ng g-1 DW, respectively). Thus, T. grandifolia emerged as the least effective PAH sink as it accumulated up to ~ 40% less PAHs than P. acerifolia and C. australis leaves/branches. Among the 17 tracked PAHs, benzo[a]anthracene, benzo[a]pyrene, dibenzo[a,h]anthracene, and pyrene were found to have accumulated in the highest concentrations in all analyzed species irrespective of the site, and accounted for more than 50% of the total detected PAHs. Finally, a "black box" about species and organ specificity, as well as specific drivers that limit PAHs uptake capacity by trees, was opened, while this work provides insights into further PAH phytoremediation strategies.

In vitro selection of drought-tolerant white poplar clones based on antioxidant activities and osmoprotectant content.

Introduction: In light of upcoming climate change, there is an urgent requirement for tree improvement regarding adaptability to drought-caused stress and the development of quick and reliable screening methodologies for genotypes' drought tolerance. White poplar is, despite its high adaptability, considered to be an endangered tree species in Serbia, which gives it special importance in the preservation and improvement of biodiversity of riparian ecosystems. The main goal of this research was to evaluate the tolerance of five white poplar clones to the presence of polyethylene glycol (PEG 6000 molecular weight 6000) (different concentrations (e.g. 0 g/L, 1 g/L, 10 g/L, 20 g/L, and 50 g/L) in Aspen Culture Medium (ACM). Methods: The tolerance of the clones was evaluated by using morphological parameters (shoot fresh and dry weight, root fresh and dry weight), photosynthetic pigments (contents of chlorophyll a, chlorophyll b, carotenoids, and chlorophyll a+b), and biochemical parameters (total phenolic content, total flavonoid content, ferric reducing antioxidant power, antioxidant activities (DPPH activity and ABTS assay), free proline content and glycine betaine content. Results and Discussion: The values of morphological and photosynthetic pigments declined with an increase in the concentration of PEG 6000. At a concentration of 50 g/L, the content of shoot fresh mass decreased by 41%, the content of Chl a by 68%, Chl b by 65%, and Car by 76% compared to the control. Also, at the same medium, there was an increase in the content of total phenols, accumulation of proline, the content of glycine betaine as well as in antioxidant activity. Based on the obtained results, it can be assumed that more drought-tolerant clones are characterized by high values for biomass, high content of photosynthetic pigments, and high content of proline and glycine betaine in conditions similar to drought in vitro. Clone L-80 showed better results in most of the tested parameters, especially compared to the reference clone Villafranca.

Environmental drivers of ground-floor bryophytes diversity in temperate forests.

Bryophytes play important role in forest ecosystem functioning and their distribution and diversity are driven by numerous environmental factors. The aim of the present study was to bring new insights in deeper understanding of terrestrial bryophytes diversity in temperate forests, as well as to determine the environmental factors which have predominant influence on ground-floor bryophytes. The survey was conducted in Fruska Gora Mountain (Serbia) across seven forest sites where ground-bryophytes were sampled. Soil moisture, temperature, and pH were measured as soil characteristics, while herbaceous cover, litter cover, stream distance, number of trees and shrubs were used as characteristics of stand structure. Species richness, Shannon diversity index, and evenness index were used as diversity measures. Generalised linear model and canonical correspondence analysis (CCA) were used to test the influence of environmental variables on bryophyte diversity. Litter cover was the most important explanatory variable, followed by soil moisture, stream distance and tree number, respectively. Overall, the stand structure was found to have a greater impact on ground-floor bryophyte diversity compared to soil characteristics. Identification of the most significant ecological factors affecting the diversity and distribution of bryophytes in forest ecosystems is of great importance in forest ecology with the aim of defining adequate management methods to preserve the biodiversity of forests, with particular emphasis on endangered and rare bryophyte species.

The Genus Heterogynis Rambur, 1866 (Heterogynidae, Lepidoptera): Congruence of Molecular, Morphological and Morphometric Evidence Reveal New Species in Serbia.

The Heterogynidae are a small family of moths consisting of a single genus Heterogynis and sixteen described species distributed in the Mediterranean region. A species new to science, Heterogynis serbica sp. nov., is described from the locality of Srebrenac, Mt. Kopaonik, Republic of Serbia, Balkan Peninsula, by applying an integrative taxonomic approach using morpho-anatomical characteristics, wing morphometics and DNA barcoding. Male genitalia, scanning electron micrographs of adult male head anatomy, abdominal tergites/sternites, cocoons and habitats of the closely related species H. serbica sp. nov. and H. zikici are discussed and illustrated. Photographs of adult males and females, cocoons, plants in which the cocoons were found and habitats are shown. Importantly, marked differences in genital structure and other morphological characters were noted. These differences were confirmed with forewing morphometrics and COI-based DNA barcoding results. Additionally, DNA barcodes for H. serbica sp. nov. and H. zikici were compared against previously available data for the genus to evaluate the phylogenetic relationships. We conclude that deep, previously unknown and unexpected intrageneric morphological diversity exists in the genus Heterogynis.

Metal- and Organ-Specific Response to Heavy Metal-Induced Stress Mediated by Antioxidant Enzymes' Activities, Polyamines, and Plant Hormones Levels in Populus deltoides.

Besides anthropogenic factors, climate change causes altered precipitation patterns that indirectly affect the increase of heavy metals in soils due to hydrological effects and enhanced leaching (i.e., Cd and Ni), especially in the vicinity of mines and smelters. Phytoextraction is a well-known, powerful "green" technique for environmental clean-up that uses plants to extract, sequester, and/or detoxify heavy metals, and it makes significant contributions to the removal of persistent inorganic pollutants from soils. Poplar species, due to their growth features, high transpiration rate, large biomass, and feasible reproduction represent great candidates for phytoextraction technology. However, the consequences of concomitant oxidative stress upon plant metabolism and the mechanism of the poplar's tolerance to heavy metal-induced stress are still not completely understood. In this study, cuttings of poplar species (Populus deltoides W. Bartram ex Marshall) were separately exposed to two heavy metals (Cd2+ and Ni2+) that were triple the maximum allowed amount (MAA) (according to national legislation). The aim of the study was to estimate the effects of heavy metals on: (I) the accumulation of free and conjugated polyamines, (II) plant hormones (including abscisic acid-ABA and indole-3-acetic acid-IAA), and (III) the activities of different antioxidant enzymes at root and leaf levels. By using the selected ion monitoring (SIM) mode of gas chromatography with mass spectrometry (GC/MS) coupled with the isotopically labeled technique, amounts of ABA and IAA were quantified, while polyamine amounts were determined by using high-performance liquid chromatography (HPLC) with fluorometric detection after derivatization. The results showed that P. deltoides responded to elevated concentrations of heavy metals in soils by exhibiting metal- and organ-specific tolerance. Knowledge about tolerance mechanisms is of great importance for the development of phytoremediation technology and afforestation programs for polluted soils.

Ectomycorrhizal Fungi Modulate Pedunculate Oak's Heat Stress Responses through the Alternation of Polyamines, Phenolics, and Osmotica Content.

The physiological and biochemical responses of pedunculate oaks (Quercus robur L.) to heat stress (HS) and mycorrhization (individually as well in combination) were estimated. One-year-old Q. robur seedlings were grown under controlled conditions in a pot experiment, inoculated with a commercial inoculum of ectomycorrhizal (ECM) fungi, and subjected to 72 h of heat stress (40 °C/30 °C day/night temperature, relative humidity 80%, photoperiod 16/8 h) in a climate chamber, and they were compared with seedlings that were grown at room temperature (RT). An in-depth analysis of certain well-known stress-related metrics such as proline, total phenolics, FRAP, ABTS, non-protein thiols, and lipid peroxidation revealed that mycorrhized oak seedlings were more resistant to heat stress (HS) than non-mycorrhized oaks. Additionally, levels of specific polyamines, total phenolics, flavonoids, and condensed tannins as well as osmotica (proline and glycine betaine) content were measured and compared between four treatments: plants inoculated with ectomycorrhizal fungi exposed to heat stress (ECM-HS) and those grown only at RT (ECM-RT) versus non-mycorrhized controls exposed to heat stress (NM-HS) and those grown only at room temperature (NM-RT). In ectomycorrhiza inoculated oak seedlings, heat stress led to not only a rise in proline, total phenols, FRAP, ABTS, non-protein thiols, and lipid peroxidation but a notable decrease in glycine betaine and flavonoids. Amounts of three main polyamines (putrescine, spermine, and spermidine) were quantified by using high-performance liquid chromatography coupled with fluorescent detection (HPLC/FLD) after derivatization with dansyl-chloride. Heat stress significantly increased putrescine levels in non-mycorrhized oak seedlings but had no effect on spermidine or spermine levels, whereas heat stress significantly increased all inspected polyamine levels in oak seedlings inoculated with ectomycorrhizal inoculum. Spermidine (SPD) and spermine (SPM) contents were significantly higher in ECM-inoculated plants during heat stress (approximately 940 and 630 nmol g-1 DW, respectively), whereas these compounds were present in smaller amounts in non-mycorrhized oak seedlings (between 510 and 550 nmol g-1 DW for Spd and between 350 and 450 nmol g-1 DW for Spm). These findings supported the priming and biofertilizer roles of ectomycorrhizal fungi in the mitigation of heat stress in pedunculate oaks by modification of polyamines, phenolics, and osmotica content.

Species-Level Differences in Osmoprotectants and Antioxidants Contribute to Stress Tolerance of Quercus robur L., and Q. cerris L. Seedlings under Water Deficit and High Temperatures.

The general aim of this work was to compare the leaf-level responses of different protective components to water deficit and high temperatures in Quercus cerris L. and Quercus robur L. Several biochemical components of the osmotic adjustment and antioxidant system were investigated together with changes in hormones. Q. cerris and Q. robur seedlings responded to water deficit and high temperatures by: (1) activating a different pattern of osmoregulation and antioxidant mechanisms depending on the species and on the nature of the stress; (2) upregulating the synthesis of a newly-explored osmoprotectant, dimethylsulphoniopropionate (DMSP); (3) trading-off between metabolites; and (4) modulating hormone levels. Under water deficit, Q. cerris had a higher antioxidant capacity compared to Q. robur, which showed a lower investment in the antioxidant system. In both species, exposure to high temperatures induced a strong osmoregulation capacity that appeared largely conferred by DMSP in Q. cerris and by glycine betaine in Q. robur. Collectively, the more stress-responsive compounds in each species were those present at a significant basal level in non-stress conditions. Our results were discussed in terms of pre-adaptation and stress-induced metabolic patterns as related to species-specific stress tolerance features.

Developing Forest Therapy Programmes Based on the Health Benefits of Terpenes in Dominant Tree Species in Tara National Park (Serbia).

Numerous medical studies have shown the positive effects of forests on different aspects of human health. This study deals with the content of major terpenes in dominant coniferous species in Tara National Park, Serbia, in order to explore the potential for the development of a novel health tourism programme based on forest therapy. Main terpenes were analysed using a headspace-sampling technique coupled with gas-chromatography-mass spectrometry (Head-space-GC/MS). Needles of fir and spruce growing in the vicinity of hiking trails were investigated for possibilities to perform such therapy. Major detected terpenes were α-cadinol and spathulenol previously described as antiviral, antitumor, antimicrobial and immunomodulatory agents. The results of the study were favourable and worked well with the existing walking infrastructure in the observed area of the Tara Mountain, as they act as invaluable resources for designing the structured forest bathing walks. The study not only adds to the knowledge in the environmental and public health realm but also to tourism and sustainability studies.

Different tree-ring width sensitivities to satellite-based soil moisture from dry, moderate and wet pedunculate oak (Quercus robur L.) stands across a southeastern distribution margin.

Associations of pedunculate oak (Quercus robur L.) radial growth with satellite-based soil moisture (SM) during the intensive tree growth period over a 30-year time span (1980-2010) were analyzed. This study included tree-ring width (TRW) chronologies from 22 stands located in four southeastern (SE) European countries (Slovenia, Croatia, Serbia and Bulgaria), which were grouped into three wetness groups (WGs): dry (<650 mm), moderate (650-750 mm), and wet (>750 mm), following the annual sum of precipitation. High correlation strengths during the intensive growth period-late spring and early summer months (April to June) was noted, which was opposite to the trend in late summer months. Variations in detrended TRW (TRWi) sensitivity to SM were also observed among the WGs. Specifically, the TRWi chronologies from the dry and wet WGs provided a greater number of significant correlations (p < 0.01) than trees from the moderate WG did. In wetter stands, TRWi correlated more negatively in the wettest (spring) months, while the correlation was weaker in summer months; these trends were opposite to those of trees growing in drier conditions that had the strongest responses to SM. A generalized additive mixed model (GAMM) based on 38 variables indicated that the fit for SM and radial growth was as strong as the fits for other traditionally measured parameters (temperature, precipitation, and river water level) and calculated drought indices (standardized precipitation index and the Ellenberg index) and TRW. Additionally, radial growth chronologies from drier sites had stronger fits with surrounding environmental factors. In conclusion, our findings suggest that SM can potentially be used as a reliable remote sensing indicator of the soil wetness in oak forests, which affects tree productivity and radial growth patterns and provides a new opportunity in dendrochronology research on larger scales.

Citric acid as soil amendment in cadmium removal by Salix viminalis L., alterations on biometric attributes and photosynthesis.

During the past decade, the target in cleaning polluted sites is an application of chelating agents, such as citric acid (CA), which is proposed as a good candidate in the promotion of phytoremediation. Among heavy metals, cadmium (Cd) is one of the most common and dangerous elements, which strongly disturbs morphophysiological properties in plants. A pot experiment was assessed to evaluate the influence of CA in Cd phytoremediation in alkaline soil by Salix viminalis (clone SV068). The effects of CA on Cd bioavailability, mobility, and distribution in plants, various morphometric measurements, and physiological performances as photosynthesis, transpiration, water use efficiency, and pigment content were tested. The highest Cd accumulation was evident after 60 days of growing, in plants subjected to combined treatment of CA with a higher dose of Cd. Application of CA showed a beneficial effect in maintaining the photosynthetic rate as well as gas exchange capacity in willows grown in Cd-contaminated soil. Furthermore, CA slightly increased plant growth and biomass production, depending on applied Cd dose and harvest period. A chelating agent like CA showed strong influence in plant response to combat Cd toxicity.

Growth and physiological responses of three poplar clones grown on soils artificially contaminated with heavy metals, diesel fuel, and herbicides.

We tested the growth and physiological responses of three poplar clones [Populus deltoides Bartr. ex Marsh. 'Bora', 'PE 19/66'; Populus × euramericana (Dode) Guinier 'Pannonia'] grown for 3 years on soils artificially contaminated with heavy metals, diesel fuel, and herbicides at the Experimental Estate of the Institute of Lowland Forestry and Environment (ILFE), University of Novi Sad, Serbia. Within three field blocks, clonal whole-plots were divided into seven subplots containing a non-contaminated control and six artificially-contaminated soil treatments: (1) 10.6 kg Cd ha-1, (2) 247 kg Cu ha-1, (3) 183.3 kg Ni ha-1, (4) 6,667 L diesel fuel ha-1, (5) 236 g Oxyfluorfen ha-1, and (6) 1,320 g Pendimethalin ha-1. Significant clone × treatment interactions governed growth and physiology throughout the study (p < 0.05), and the influence of inorganics versus organics varied with tree age. Heavy metals had a more substantial influence on growth and physiology as the trees matured, while diesel and herbicide treatments were most pronounced during the first growing season (p < 0.0001). Clones 'Bora' and 'PE 19/66' exhibited greater biomass than 'Pannonia', with trees growing in the control soils exhibiting 13.8 and 19.6 times greater biomass than 'Pannonia', respectively.

Potential Elevation Shift of the European Beech Stands (Fagus sylvatica L.) in Serbia.

According to climate projection models, the global temperature is expected to rise by at least 1.5°C by the end of this century. According to some studies the expected rise in Serbia is even higher. Global warming may result in creating new areas for forest growth. Although creating new forests would be a positive outcome in some areas, global warming can cause negative impacts in other areas, and this can lead to forest loss and the shift of geographical ranges, or even extinction, of plant species. The European beech is the dominant forest tree species in Serbia, featuring high ecological importance and economic value. In mixed or pure stands, beech forests cover approximately 660,400 ha, accounting for 29.3% of the total Serbian forest area. In the present study, the effects of climate change on the distribution of the European beech stands in Serbia, with an emphasis on their elevation shifts, were examined using species distribution models (SDMs). Data for the present tree cover in Serbia, climate projections, and environmental data were used for model building. The models were first tested against present inventory data. In these tests, the models were found to provide accurate projections, as shown by their true skills statistics (TSS) values ranging from 0.652 to 0.736 and area under the curve (AUC) values ranging from 0.868 to 0.937. The potential distribution patterns predicted by the models indicate that the European beech elevational distribution in Serbia would decrease, exhibiting a significant upward shift in elevation during the first part of this century. Current beech stand locations could be changed, and other areas at higher elevations may be more suitable for beech growth. After 2071, European beech stands at elevations below 500 m would be even smaller. This change is caused by temperature rise and occurrence of climate extremes. However, on the highest elevations, further upward shift of the species is not expected.

Growth, physiology, and phytoextraction potential of poplar and willow established in soils amended with heavy-metal contaminated, dredged river sediments.

Phytotechnologies have been used worldwide to remediate and restore damaged ecosystems, especially those caused by industrial byproducts leaching into rivers and other waterways. The objective of this study was to test the growth, physiology, and phytoextraction potential of poplar and willow established in soils amended with heavy-metal contaminated, dredged river sediments from the Great Backa Canal near Vrbas City, Serbia. The sediments were applied to greenhouse-grown trees of Populus deltoides Bartr. ex Marsh. clone 'Bora' and Salix viminalis L. clone 'SV068'. Individual pots with trees previously grown for two months were amended with 0, 0.5 and 1.0 kg of sediment containing 400 mg Cr kg-1, 295 mg Cu kg-1, 465 mg Zn kg-1, 124 mg Ni kg-1, 1.87 mg Cd kg-1, and 61 mg Pb kg-1. Following amendment, trees were grown for two seasons (i.e., 2014, 2015), with coppicing after the first season. In addition to growth parameters, physiological traits related to the photosynthesis and nitrogen metabolism were assessed during both growing seasons. At the end of the study, trees were harvested for biomass analysis and accumulation of heavy metals in tree tissues and soils. Application of sediment decreased aboveground biomass by 37.3% in 2014, but increased height (16.4%) and leaf area (19.2%) in 2015. Sediment application negatively impacted the content of pigments and nitrate reductase activity, causing them to decrease over time. Generally, the effect of treatments on growth was more pronounced in poplars, while willows had more pronounced physiological activity. Accumulation patterns were similar to previously-published results. In particular, Zn and Cd were mostly accumulated in leaves of both poplar and willow, which indicated successful phytoextraction. In contrast, other metals (e.g., Cr, Ni, Pb, Cu) were mostly phytostabilized in the roots. Differences in metal allocation between poplar and willow were recorded only for Cu, while other metals followed similar distribution patterns in both genera. Results of this study indicated that the composition of heavy metals in the sediments determined the mechanisms of the applied phytoremediation technique.

Hydroponic screening of black locust families for heavy metal tolerance and accumulation.

Present work examines phytoextraction potential of four black locust families (half-sibs 54, 56, 115, and 135) grown hydroponically. Plants were treated with 6 ppm of cadmium (Cd), 100 ppm of nickel (Ni), and 40 ppm of lead (Pb) added in Hoagland nutrient solution, accompanying with simultaneously applied all three metals. Responses to metals exposure among families were different, ranging from severe to slight reduction of root and shoot biomass production of treated plants. Calculated tolerance indices are indicating tested families as highly tolerant (Ti > 60). Family 135 had the lowest tolerance index, pointing that it was highly susceptible to applied metals. Comparing photosynthetic activities of tested families it has been noticed that they were highly sensitive to stress induced by heavy metals. Net photosynthetic rate of nickel treated plants was the most affected by applied concentration. Cadmium and nickel concentrations in stems and leaves of black locust families exceeded 100 mg Cd kg(-1) and 1000 mg Ni kg(-1), in both single and multipollution context. On the contrary, accumulation of lead in above ground biomass was highly affected by multipollution treatment. Tf and BCF significantly varied between investigated treatments and families of black locust. Concerning obtained results of heavy metals accumulation and tolerance of black locust families can be concluded that tested families might be a promising tool for phytoextraction purposes, but it takes to be further confirmed in field trials.

Antioxidant characterization of oak extracts combining spectrophotometric assays and chemometrics.

Antioxidant characteristics of leaves, twigs, and acorns from two Serbian oak species Quercus robur L. and Quercus petraea L. from Vojvodina province (northern Serbia) were investigated. 80% ethanol (in water) extracts were used for antiradical power (ARP) determinations against DPPH(•), (•)NO, and O2 (•-) radicals, ferric reducing antioxidant power (FRAP), total phenol, tannin, flavonoid, and proanthocyanidin contents. Permanganate reducing antioxidant capacity (PRAC) was determined using water extracts. Beside, mentioned parameters, soluble proteins, lipid peroxidation (LP), pigments and proline contents were also determined. The data of different procedures were compared and analyzed by multivariate techniques (correlation matrix calculation and principal component analysis (PCA)). PCA found that investigated organs of two different oak tree species possess similar antioxidant characteristics. The superior antioxidant characteristics showed oak leaves over twigs and acorns and seem to be promising source of antioxidants with possible use in industry and pharmacy.

Variability in chemical composition and abundance of the rare tertiary relict Pinus heldreichii in Serbia.

The particular significance of the whitebark pine (Pinus heldreichii Christ.) stems from the fact that it is a tertiary relict and Balkanic subendemite covering a very narrow and intermittent area in Serbia. A representative pool of 48 adult trees originating from three populations, one recently discovered natural (Population I) and two planted populations (Populations II and III) was investigated in order to evaluate the intra- and interpopulation variability of the essential oil of the complete fund of P. heldreichii in Serbia. In the pine-needle-terpene profile, 104 compounds were detected, 84 of which could be identified. Among the essential-oil constituents, monoterpenes and sesquiterpenes dominated, comprising ca. 90% of the essential oil. The terpenic profile of Population I was characterized by a predominance of monoterpenes (e.g., limonene (1), α-pinene, and Δ(3) -carene (4)), while sesquiterpenes (e.g., germacrene D (2) and ß-caryophyllene (3)) obviously preponderated in the profile of Populations II and III. This study also demonstrated that the abundance of whitebark pines in Serbia had significantly changed over the last few decades. The number of individuals in the natural population had increased, while the number of individuals in the planted populations had decreased. Today, the whitebark pine fund in Serbia comprises less than 250 trees.

Cadmium phytoextraction potential of poplar clones (Populus spp.).

Biomass production, leaf number and area, photosynthetic and dark respiration rates, leaf concentration of photosynthetic pigments, nitrate reductase activity, as well as cadmium concentrations in leaves, stem, and roots were measured in poplar clones PE 4/68, B-229, 665, and 45/51. Plants were grown hydroponically under controlled conditions and treated with two different cadmium (Cd) concentrations (10(-5) and 10(-7) M) in the same background solution (Hoagland's solution). The presence of Cd did not cause serious disturbance of growth and physiological parameters in the studied poplar clones. Cd concentrations in plant tissues reflected external concentrations. In treated plants, root contents increased from 38.57 to 511.51 ppm, leaf contents from 0.91 to 7.50, while stem contents ranged from 1.37 to 9.50 ppm.