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The increasing use of graphene-related materials (GRMs) in everyday-life products raises concerns for their possible release into the environment and consequent impact on organisms. GRMs have widely varying effects on plants and, according to recent evidences, graphene oxide (GO) has the potential to interfere with the sexual reproduction owing to its acidic properties and production residues. Here, stigmas of the model plant Cucurbita pepo (summer squash) were subjected to simulated dry depositions of GO and GO purified from production residues (PGO). Stigmas were then hand-pollinated and GRM deposition was checked by ESEM and confocal microscopy. Analysis of stigma integrity, pH homeostasis and pollen-stigma interactions did not reveal negative effects. Fruit and seed production were not affected, but GO depositions of 22.1 ± 7.2 ng mm-2 affected the normal development of seeds, decreasing seed dimensions, seed germination and germination speed. The elemental analysis revealed that GO has significant quantities of production residues, such as strong acids and oxidants, while PGO has only traces, which justifies the differences observed in the effects caused by the two materials. Our results show that GO depositions of up to 11.1 ± 3.6 ng mm-2, which fall within the variation range of total dry particulate matter depositions reported in the literature, are safe for reproduction of C. pepo. This is the first "safety" limit ever recorded for depositions of "out-of-the-box" GO concerning the reproduction of a seed plant. If confirmed for wind-pollinated species, it might be considered for policymaking of GRMs emissions in the air.

In bioaccumulation studies, sample devitalization through acid washing or oven drying is commonly applied to enhance the element accumulation efficiency of moss sample. Such aspect, however, has never been considered in biomonitoring surveys using lichens. In this study, the trace element accumulation performance of living (L) and dead (D) samples of the lichen Pseudevernia furfuracea was compared by a side-by-side transplanting at 40 sites in a large, mixed land use area of NE Italy for 8 weeks. Devitalization was achieved without any physico-chemical treatments, by storing lichen samples in a dark cool room for 18 months. Health status of lichens was assessed before and after the sample exposure by chlorophyll fluorescence emission. Although elemental analysis of the two exposed sample sets revealed a similar trace element pollution scenario, the content of 13 out of the 24 selected elements was higher in D samples. By expressing results as exposed-to-unexposed (EU) ratio, D samples show a higher bioaccumulation signal in 80% of transplant sites for Al, Ca, Fe, Hg, Pb and Ti. Overall, the health status of lichen samples might lead to interpretational discrepancies when EU ratio is classified according to the recently proposed bioaccumulation scale.

Kranner, Ilse/0000-0003-4959-9109; Holzinger, Andreas/0000-0002-7745-3978, PubMed: 33223597, Evidence is emerging that phytohormones represent key inter-kingdom signalling compounds supporting chemical communication between plants, fungi and bacteria. The roles of phytohormones for the lichen symbiosis are poorly understood, particularly in the process of lichenization, i.e. the key events which lead free-living microalgae and fungi to recognize each other, make physical contact and start developing a lichen thallus. Here, we studied cellular and extracellularly released phytohormones in three lichen mycobionts, Cladonia grayi, Xanthoria parietina and Tephromela atra, grown on solid medium, and the effects of indole-3-acetic acid (IAA) on their respective photobionts, Asterochloris glomerata, Trebouxia decolorans, Trebouxia sp. Using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) we found that mycobionts produced IAA, salicylic acid (SA) and jasmonic acid (JA). IAA represented the most abundant phytohormone produced and released by all mycobionts, whereas SA was released by X. parietina and T. atra, and JA was released by C. grayi only. With a half-life of 5.2 days, IAA degraded exponentially in solid BBM in dim light. When IAA was exogenously offered to the mycobionts' compatible photobionts at "physiological" concentrations (as released by their respective mycobionts and accumulated in the medium over seven days), the photobionts' water contents increased up to 4.4%. Treatment with IAA had no effects on the maximum quantum yield of photosystem II, dry mass, and the contents of photosynthetic pigments and alpha-tocopherol of the photobionts. The data presented may be useful for designing studies aimed at elucidating the roles of phytohormones in lichens., Austrian Science Fund (FWF)Austrian Science Fund (FWF); FWFAustrian Science Fund (FWF) [P32092-B32, I1951-B16], Open access funding provided by Austrian Science Fund (FWF).; We also thank the FWF for financial support (grants P32092-B32 to IK and I1951-B16 to AH).

Phytohormones are pivotal signaling compounds in higher plants, in which they exert their roles intracellularly, but are also released for cell-to-cell communication. In unicellular organisms, extracellularly released phytohormones can be involved in chemical crosstalk with other organisms. However, compared to higher plants, hardly any knowledge is available on the roles of phytohormones in green algae. Here, we studied phytohormone composition and extracellular release in aero-terrestrial Trebouxiophyceae. We investigated (a) which phytohormones are produced and if they are released extracellularly, and if extracellular phytohormone levels are (b) affected by environmental stimuli, and (c) differ between lichen-forming and non-lichen-forming species. Three free-living microalgae (Apatococcus lobatus, Chloroidium ellipsoideum, and Myrmecia bisecta) and three lichen-forming microalgae (Asterochloris glomerata, Trebouxia decolorans, and Trebouxia sp.) were studied. Algae were grown on solid media and the following cellular phytohormones were identified by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS): indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellin A4 (GA4 ), and zeatin (ZT). Furthermore, IAA, IBA, ABA, jasmonic acid (JA), gibberellin A3 (GA3 ), and GA4 were found to be released extracellularly. IAA and ABA were released by all six species, and IAA was the most concentrated. Phytohormone release was affected by light and water availability, especially IAA in A. glomerata, Trebouxia sp., and C. ellipsoideum. No clear patterns were observed between lichen-forming and non-lichen-forming species. The results are envisaged to contribute valuable baseline information for further studies into the roles of phytohormones in microalgae.

Products containing graphene-related materials (GRMs) are becoming increasingly common, allowing GRM nanoparticles (NPs) to enter the environment during their life cycle. Thanks to their lightness and bidimensional geometry, GRM NPs can be easily dispersed in the air and travel very long distances. The flowers of wind-pollinated plants may be exposed to airborne GRMs, being apt to intercept pollen from the air and, inevitably, other airborne particles. Here, stigmas of four wind-pollinated plants (Corylus avellana, common hazel; Juglans regia, walnut; Quercus ilex, holm oak; Zea mays, maize) were exposed to airborne graphene oxide (GO) and GO purified from production residues (PGO) at a concentration of 3.7 ng m-3. Subsequently, the stigmas were pollinated and the adhesion of GOs and their effects on stigma integrity and pollen-stigma interaction were examined. The effect of GO NPs in presence of liquid water on the stigma of C. avellana was also investigated. GOs NPs were intercepted by all species, but their effect varied among them. GO reduced pollen adhesion in J. regia and Q. ilex, whereas pollen germination was unaffected in all four species. The presence of a film of water neither completely removed GO NPs from the stigma, nor it enhanced the toxic effect of GO acidity. PGO never affected pollen-stigma interaction, indicating that the phytotoxic substances used for the production of GO, still in traces in commercial GO, are the main cause of GO toxicity. These results reconfirm the need to verify GRMs effects also on key biological processes beside single model organisms.

Fungal–algal relationships—both across evolutionary and ecological scales—are finely modulated by the presence of the symbionts in the environments and by the degree of selectivity and specificity that either symbiont develop reciprocally. In lichens, the green algal genus Trebouxia Puymaly is one of the most frequently recovered chlorobionts. Trebouxia species-level lineages have been recognized on the basis of their morphological and phylogenetic diversity, while their ecological preferences and distribution are still only partially unknown. We selected two cosmopolitan species complexes of lichen-forming fungi as reference models, i.e., Rhizoplaca melanophthalma and Tephromela atra, to investigate the diversity of their associated Trebouxia spp. in montane habitats across their distributional range worldwide. The greatest diversity of Trebouxia species-level lineages was recovered in the altitudinal range 1,000–2,500 m a.s.l. A total of 10 distinct Trebouxia species-level lineages were found to associate with either mycobiont, for which new photobionts are reported. One previously unrecognized Trebouxia species-level lineage was identified and is here provisionally named Trebouxia “A52.” Analyses of cell morphology and ultrastructure were performed on axenically isolated strains to fully characterize the new Trebouxia “A52” and three other previously recognized lineages, i.e., Trebouxia “A02,” T. vagua “A04,” and T. vagua “A10,” which were successfully isolated in culture during this study. The species-level diversity of Trebouxia associating with the two lichen-forming fungi in extreme habitats helps elucidate the evolutionary pathways that this lichen photobiont genus traversed to occupy varied climatic and vegetative regimes.

Canopy-forming brown algae support highly productive ecosystems whose decline has been attributed to the interplay of several anthropogenic disturbances. Climate change could have disruptive effects on the biology of these species, but the role of temperature in the development of early life stages is poorly understood. The aim of this study was to assess the response of Ericaria giacconei, a winter-reproducing Southern–Mediterranean endemic species, to thermal stress by testing five temperatures (12, 15, 18, 24, and 28°C) on adults and early stages. Chlorophyll a fluorescence of adult plants was measured at 0, 24, 72, and 120 h on nine fronds in each of the three aquaria per treatment. To assess egg release, zygote settlement, and embryo growth rate, approximately 1,200 receptacles were cultured on six Petri dishes per temperature treatment, and 10 random subsections of 2 ×2 mm were examined in three Petri dishes at 0, 20, 44, and 92 h after fertilization. Adult plants showed a plastic physiological response, and thermal stress had no significant effect on PSII efficiency. Embryos fully developed only at 12 and 15°C. Mortality increased at 18 and 24°C, and no zygotes survived at 28°C. In a scenario of further increasing temperatures, the effects of warming could affect the recruitment of E. giacconei and increase its vulnerability to further stresses. These effects on the survival of early stages, which are the bottleneck for the long-term survival of the species, should be taken into account in conservation and restoration measures to maintain canopy-forming macroalgal populations and associated biodiversity and ecosystem services.

In this study, the effects of ozone (O3) on the physiology of the lichen Pseudevernia furfuracea var. furfuracea previously subjected to field stressing conditions were assessed. Samples collected in a pristine site were exposed for 6 weeks at 3 sites characterized by different pollution, e.g. elemental and PAH depositions (site RU, close to wood-burning house chimneys; site UI, close to cast-ironworks; site CK, in a semi-natural context). Afterwards, samples were transferred to controlled fumigation chambers, where they were either O3-treated for 2 weeks (250 ppb O3 for 5 h day−1, O3+ samples) or not (0 ppb O3, O3− samples). Three physiological markers (Fv/Fm, maximum quantum yield of primary photochemistry; MDA, malondialdehyde content; potassium leakage) as well as elemental and PAH concentrations were measured in matched sets of sample replicates at each experimental step. Data were explored by multivariate techniques and the effects of field exposure and fumigation were tested by generalized linear models (GLM). Detrimental effects on MDA and Fv/Fm were observed limited to samples exposed in RU and UI sites. Physiological parameters in O3-treated samples showed heterogeneous variation patterns with respect to field-exposed ones. A recovery of Fv/Fm was observed in RU- and UI-exposed samples, whereas a significant increase of MDA was highlighted limited to CK O3+ and CK O3− samples, possibly related to a “chamber effect”. Overall, the impairment caused by ozonation was limited, proving the strong O3-tolerance of our test species. Interestingly, the content of the most abundant 4-ring PAHs in RU O3+ samples, which underwent the highest field enrichment of PAHs, was significantly lower than that of matched RU O3− samples. This suggested a possible role of ozone in degrading PAHs at thallus level, with interesting interpretative repercussions in the context of transplant-based surveys aimed at evaluating PAH depositions when O3 ground levels are high.

In biomonitoring of airborne elements with native lichens, two allopatric species could be used jointly only when bioaccumulation mechanisms are the same, and differences negligible. The present study aims at (i) investigating the bioaccumulation performance for 12 elements in one-year-old lobes of Flavoparmelia caperata and Xanthoria parietina (i.e. the two lichen native biomonitors most used in the peri-Mediterranean regions) collected side-by-side on the same trees at 17 sampling sites. Both species were characterized morphologically and by X-ray diffraction analysis to evaluate specific thallus area and oxalate content, respectively. Results show that Ca and chalcophile elements (Cd, Cu, Pb and Zn) were more concentrated in F. caperata, lithophile and siderophile elements (Cr, Fe, and Ti) in X. parietina, whereas Al, Li, Mn, and Ni were accumulated in similar amounts. The outstanding content of Ca-oxalate deposits in F. caperata underlies the higher content of Ca, Cd and Pb observed in this species. On the contrary, the micro-anatomy of the upper cortex of X. parietina, which is more porous than in F. caperata, allows a higher transfer of litophile and siderophile elements. The adoption of interspecific calibration factors for biomonitoring purposes is thoroughly discussed, and the choice of using a single native species made by the most recent guidelines for biomonitoring airborne elements with lichens is fully supported.

In the Mediterranean Sea, marine forests constituted by Cystoseira sensu lato (s.l.) species (Fucales, Phaeophyceae) support highly productive ecosystems, whose decline was ascribed to the interplay of several anthropogenic impacts. Recently, this decline has also been attributed to the increase of seawater temperatures and thermal anomalies, which can alter the reproductive phenology, germling growth and population viability of Cystoseira s.l. species. To manage the conservation of these communities is fundamental to evaluate how current and future foreseen warming scenarios will affect the thermal physiology of different life stages of these species. Moreover, a recognized strategy of management is the identification and protection of contemporary climatic refugia, which may safeguard the persistence of these habitat-forming species. The present research focused on Cystoseira hyblaea Giaccone, which is endemic to the Sicily Channel (Central Mediterranean Sea) and has a narrow distributional range, being only reported in Punta D’Aliga, where it was no longer found, in Cap Bon (Northern Tunisian coast) and in Portopalo di Capo Passero (Sicily, Southern Italy). This species lives in the intertidal and upper sublittoral zone on both semi-exposed and exposed rocky coasts, and its maximum vegetative and reproductive development occurs during winter. Considering the ongoing warming scenario, C. hyblaea could be severely threaten being an intertidal species and due to its cold affinity, the winter reproductive phenology and the restricted distributional range. Therefore, the aim of this study was to assess the thermal tolerance of both embryos and adults of C. hyblaea, in order to evaluate which stage might be the most vulnerable to climate change, and to foresee the possible fate and argue about the future conservation status of this species under a predicted global warming. To perform this research, egg release, zygote settlement, embryo development and adult photosynthetic efficiency were studied, on both early life stages and adults, under five temperature treatments (12, 15, 18, 24, 28 °C) in controlled mesocosms. In particular, ca. 1200 receptacles were cultivated on 6 Petri dishes per temperature treatment. At 0, 20, 44, 92 h after fertilization, 10 random subareas of 2x2 mm were examined in 3 Petri. Chlorophyll a fluorescence of adults was measured at 0, 24, 72, 120 h on 9 fronds in each of the 3 aquaria per treatment. Through the trial, it was observed that the receptacles’ release efficiency did not vary significantly among temperatures. However, the settlement efficiency of C. hyblaea increased from 12 to 18 °C and decreased at 28 °C. Indeed, the extremely low settlement efficiency was at 28 °C, due to the fact that zygotes had undergone lysis because of thermal stress. Regarding the early life stages, embryos showed a complete development only at 12 and 15 °C, while yet at 18 °C mortality increased sharply, being total at 28 °C. In particular, the highest developmental rate was observed at 15 °C (highest percentage of embryos with rhizoids already at 20 h AF), suggesting that this temperature constitutes the thermal optimum for reproduction and development of early developmental stages. On the contrary, adults of C. hyblaea showed a more plastic physiological response and thermal stress did not significantly affect photosynthetic efficiency. The present study demonstrated that adults of C. hyblaea resulted not substantially affected by heat with an expected ability to acclimatise also to elevated temperatures. Contrarily, the early developmental stages resulted strictly stenotherm, requiring a very narrow range of low temperatures for an effective recruitment and development (12-15 °C). Therefore, the recruitment could represent the most vulnerable process to the impacts of climate change in the population dynamics of C. hyblaea. Moreover, the results confirm the cold affinity of this species and suggest that the localities, in which C. hyblaea is confined, are acting as potential climatic refugia. If failure in the recruitment process is reiterated for several years, it will lead to an impoverishment of C. hyblaea populations, ultimately hindering their long-term viability in its current range. Given the fragmented and restricted range and increased frequency of extreme climatic events, C. hyblaea may undergo local extinction. Considering this perspective, the areas in which C. hyblaea manages to survive should receive a higher level of protection. Moreover, this species should be included in the IUCN Red List of Species (IUCN, 2021), being classified as a “Critically endangered” or “Rare”.

Lichens and their isolated symbionts are potentially valuable resources for biotechnological approaches. Especially mycobiont cultures that produce secondary lichen products are receiving increasing attention, but lichen mycobionts are notoriously slow-growing organisms. Sufficient biomass production often represents a limiting factor for scientific and biotechnological investigations, requiring improvement of existing culturing techniques as well as methods for non-invasive assessment of growth. Here, the effects of pH and the supplement of growth media with either D-glucose or three different sugar alcohols that commonly occur in lichens, D-arabitol, D-mannitol and ribitol, on the growth of the axenically cultured mycobiont isolated from the lichen Xanthoria parietina were tested. Either D-glucose or different sugar alcohols were offered to the fungus at different concentrations, and cumulative growth and growth rates were assessed using two-dimensional image analysis over a period of 8 weeks. The mycobiont grew at a pH range from 4.0 to 7.0, whereas no growth was observed at higher pH values. Varying the carbon source in Lilly-Barnett medium (LBM) by replacing 1% D-glucose used in the originally described LBM by either 1%, 2% or 3% of D-mannitol, or 3% of D-glucose increased fungal biomass production by up to 26%, with an exponential growth phase between 2 and 6 weeks after inoculation. In summary, we present protocols for enhanced culture conditions and non-invasive assessment of growth of axenically cultured lichen mycobionts using image analysis, which may be useful for scientific and biotechnological approaches requiring cultured lichen mycobionts. Supplementary Information The online version contains supplementary material available at 10.1007/s11557-021-01707-7.

Atmospheric dispersion model (ADM) simulations are increasingly used as management tools in air pollution monitoring programs, even in the absence of proper validation. Biomonitors can provide important information for ADM validation, but an open question is their temporal frame of application, particularly when native organisms are used. In this study, we tested two alternative ADM simulating the total suspended particulate (TSP) released by a coal power station, against the element content of two native lichens collected at 40 sites, integrated by soil samples. The ADM simulations differed by the time references: the 6-month period preceding lichen sampling, approximately corresponding to the estimated age of the samples (Mod. A), and the whole year 2005, representative of the local average conditions and used in the plant authorization processes (Mod. B). A generalized regression model analysis clearly showed that the Cr, Pb and V content of lichen samples was spatially associated to the outcomes of Mod. A, but not with Mod. B. Interestingly, the Cr content of lichen samples consistently correlated to TSP concentration predicted by Mod. A along two transects placed downwind from the coal power station. This result was corroborated by an air particulate matter sampling which pointed out that air Cr concentrations increased during the operative period of the source. Overall, our results suggest that lichen bioaccumulation data can proficiently be used to validate ADM simulations if the exposure time of the biological samples is consistent with the temporal domain of the ADM simulations.

Environmental DNA (eDNA) metabarcoding and metagenomics analyses can improve taxonomic resolution in biodiversity studies. Only recently, these techniques have been applied in aerobiology, to target bacteria, fungi and plants in airborne samples. Here, we present a nine-month aerobiological study applying eDNA metabarcoding in which we analyzed simultaneously airborne diversity and variation of fungi and plants across five locations in North and Central Italy. We correlated species composition with the ecological characteristics of the sites and the seasons. The most abundant taxa among all sites and seasons were the fungal genera Cladosporium, Alternaria, and Epicoccum and the plant genera Brassica, Corylus, Cupressus and Linum, the latter being much more variable among sites. PERMANOVA and indicator species analyses showed that the plant diversity from air samples is significantly correlated with seasons, while that of fungi varied according to the interaction between seasons and sites. The results consolidate the performance of a new eDNA metabarcoding pipeline for the simultaneous amplification and analysis of airborne plant and fungal particles. They also highlight the promising complementarity of this approach with more traditional biomonitoring frameworks and routine reports of air quality provided by environmental agencies.

Lichens can withstand extreme desiccation to water contents of ≤ 0.1 g H2O g–1 DW, and in the desiccated state are among the most extremotolerant organisms known. Desiccation-tolerant life-forms such as seeds, mosses and lichens survive ‘vitrification’, that is the transition of their cytoplasm to a ‘glassy’ state, which causes metabolism to cease. However, our understanding of the mechanisms of desiccation tolerance is hindered by poor knowledge of what reactions occur in the desiccated state. Using Flavoparmelia caperata as a model lichen, we determined at what water contents vitrification occurred upon desiccation. Molecular mobility was assessed by dynamic mechanical thermal analysis, and the de- and re-epoxidation of the xanthophyll cycle pigments (measured by HPLC) was used as a proxy to assess enzyme activity. At 20 °C vitrification occurred between 0.12–0.08 g H2O g−1 DW and enzymes were active in a ‘rubbery’ state (0.17 g H2O g−1 DW) but not in a glassy state (0.03 g H2O g−1 DW). Therefore, desiccated tissues may appear to be ‘dry’ in the conventional sense, but subtle differences in water content will have substantial consequences on the types of (bio)chemical reactions that can occur, with downstream effects on longevity in the desiccated state.

Phytohormones are pivotal signaling compounds in higher plants, in which they exert their roles intracellularly, but are also released for cell‐to‐cell communication. In unicellular organisms, extracellularly released phytohormones can be involved in chemical crosstalk with other organisms. However, compared to higher plants, hardly any knowledge is available on the roles of phytohormones in green algae. Here, we studied phytohormone composition and extracellular release in aero‐terrestrial Trebouxiophyceae. We investigated (a) which phytohormones are produced and if they are released extracellularly, and if extracellular phytohormone levels are (b) affected by environmental stimuli, and (c) differ between lichen‐forming and non‐lichen‐forming species. Three free‐living microalgae (Apatococcus lobatus, Chloroidium ellipsoideum, and Myrmecia bisecta) and three lichen‐forming microalgae (Asterochloris glomerata, Trebouxia decolorans, and Trebouxia sp.) were studied. Algae were grown on solid media and the following cellular phytohormones were identified by ultra‐high‐performance liquid chromatography coupled with tandem mass spectrometry (UHPLC‐MS/MS): indole‐3‐acetic acid (IAA), indole‐3‐butyric acid (IBA), abscisic acid (ABA), gibberellin A4 (GA4), and zeatin (ZT). Furthermore, IAA, IBA, ABA, jasmonic acid (JA), gibberellin A3 (GA3), and GA4 were found to be released extracellularly. IAA and ABA were released by all six species, and IAA was the most concentrated. Phytohormone release was affected by light and water availability, especially IAA in A. glomerata, Trebouxia sp., and C. ellipsoideum. No clear patterns were observed between lichen‐forming and non‐lichen‐forming species. The results are envisaged to contribute valuable baseline information for further studies into the roles of phytohormones in microalgae.

The metabolic regulations of evergreen Mediterranean Quercus ilex trees growing at extra-urban and urban sites with different percentages of surrounding impervious pavements and distance to major traffic roads were investigated at the peak of the summer drought season. Plants from extra-urban site experienced lower water stress due to a fine regulation of reactive oxygen species (ROS, concomitant rise of anion superoxide and hydrogen peroxide content, +55 and +44%), abscisic acid and water soluble carbohydrates (+111 and +9%). The level of ROS was kept under the injury threshold by both enzymatic and metabolic antioxidants. Plants from the sub-urban site followed a different strategy, with a lower production of ROS and the concomitant reduction in phenols and carotenoids. Despite these differences, indications for stress-induced injury were not observed in any of the sites. Our work shows new relations between urban stress conditions and metabolic regulations of trees growing under such harsh environments.

The protocols commonly applied in surveys with lichens as biomonitors of airborne trace elements require analyses of samples derived from thalli or parts of thalli grown in the last year before sampling, under the postulation that samples of the same size are of the same age. Unfortunately, the influence of ecological site-specific factors on lichen growth is still largely ignored, so that samples of the same size collected in environmentally and climatically diverse sites might actually differ in age. This work aims at quantifying the influence of climatic conditions on the radial growth rates (RaGRs) of Xanthoria parietina, one of the most popular lichen biomonitors. RaGR was monitored in seven populations distributed along an altitudinal transect of 30 km in the Classical Karst (NE Italy), from 20 to 500 m above sea level. For c. 17 months, lobe growth was measured seasonally with a digital calliper, and site-specific climatic variables were monitored by means of thermo-hygrometric sensors and implemented by meteorological data. Finally, the lobe growth of X. parietina was modelled as a function of 18 environmental variables. Results revealed that thalli of relatively dry sites had significantly lower seasonal RaGR with respect to moister ones. Considering that cumulative precipitations were equally distributed along the transect, it was concluded that RaGR of X. parietina is affected negatively by high air temperatures and positively by high relative humidity. The importance of RaGR variation in lichen bioaccumulation studies is critically discussed.

The environmental biodegradability profile of graphene related materials (GRMs) is important to know in order to predict whether these materials will accumulate in soil or will be transformed by primary decomposers. In this study, few-layer graphene (FLG) was exposed to living and devitalized axenic cultures of two white-rot basidiomycetes (Bjerkandera adusta and Phanerochaete chrysosporium) and one soil saprotrophic ascomycete (Morchella esculenta) with or without lignin, for a period of four months. Over this time, the increase of fungal biomass and presence of H2O2 and oxidizing enzymes [laccase/peroxidase and lignin peroxidase (LiP)] in growth media was assessed by gravimetric and spectrophotometric measurements, respectively. Raman spectroscopy and transmission electron microscopy (TEM) were used to compare the structure of FLG before and after incubation. All of the test fungi decreased pH in growth media and released H2O2 and laccase/peroxidase, but only basidiomycetes released LiP. Independent of growth media composition all fungi were found to be capable to oxidize FLG to a graphene oxide-like material, including M. esculenta, which released only laccase/peroxidase, i.e. the most common enzymes among primary decomposers. These findings suggest that FLG involuntarily released into terrestrial environments would likely be oxidized by soil microflora.

Heat shock treatments are an innovative and eco-friendly method to devitalize biodeteriogens of outdoor stone monuments. They consist in short (up to 6 h), mild (40–60 °C) thermal treatments applied to artificially wet surfaces, and are effective against lichens and bryophytes. Epilithic green algae, a polyphyletic and diversified group of photoautotrophs, are among the most important colonizers of stone monuments, forming conspicuous biofilms which cause chemical, physical and aesthetical damage to the substratum. Like lichens and bryophytes, they are able to face the extreme conditions of a stone surface but their resistance mechanisms are only partially known. The present study aims to test the applicability of heat shock treatments to six morphologically and phylogenetically distant green microalgae. Their survival mechanisms have been investigated in relation to photosynthesis and content of selected polyols and non-reducing sugars. Chlorophyll a fluorescence measurements and observations at the epifluorescence microscope demonstrate that the thermal treatments cause negative effects on all the species, although they do not necessarily kill the whole populations, as observed in all the lichens and bryophytes tested so far. The survival capability is discussed in relation to the production of extracellular polymeric substances and non-reducing sugars.

International audience; Graphene related materials (GRMs) are currently being used in products and devices of everyday life and this strongly increases the possibility of their ultimate release into the environment as waste items. GRMs have several effects on plants, and graphene oxide (GO) in particular, can affect pollen germination and tube growth due to its acidic properties. Despite the socio-economic importance of sexual reproduction in seed plants, the effect of GRMs on this process is still largely unknown. Here, Corylus avellana L. (common Hazel) pollen was germinated in-vitro with and without 1−100 μg mL−1 few-layer graphene (FLG), GO and reduced GO (rGO) to identify GRMs effects alternative to the acidification damage caused by GO. At 100 μg mL−1 both FLG and GO decreased pollen germination, however only GO negatively affected pollen tube growth. Furthermore, GO adsorbed about 10 % of the initial Ca2+ from germination media accounting for a further decrease in germination of 13 % at the pH created by GO. In addition, both FLG and GO altered the normal tip-focused reactive oxygen species (ROS) distribution along the pollen tube. The results provided here help to understand GRMs effect on the sexual reproduction of seed plants and to address future in-vivo studies.

The effects of two graphene-based materials (GBMs), few-layers graphene (FLG) and graphene oxide (GO), were studied in the aeroterrestrial green microalga

The interpretation of lichen bioaccumulation data is of paramount importance in environmental forensics and decision-making processes. By implementing basic ideas underlying previous interpretative scales, new dimensionless, species-independent &ldquo, bioaccumulation scales&rdquo, for native and transplanted lichens are proposed. Methodologically consistent element concentration datasets were populated with data from biomonitoring studies relying on native and transplanted lichens. The scale for native lichens was built up by analyzing the distribution of ratios between element concentration data and species-specific background concentration references (B ratios), herein provided for Flavoparmelia caperata and Xanthoria parietina (foliose lichens). The scale for transplants was built up by analyzing the distribution of ratios between element concentration in exposed and unexposed samples (EU ratio) of Evernia prunastri and Pseudevernia furfuracea (fruticose lichens). Both scales consist of five percentile-based classes, namely, &ldquo, Absence of&rdquo, &ldquo, Low&rdquo, Moderate&rdquo, High&rdquo, and &ldquo, Severe&rdquo, bioaccumulation. A comparative analysis of extant interpretative tools showed that previous ones for native lichens suffered from the obsolescence of source data, whereas the previous expert-assessed scale for transplants failed in describing noticeable element concentration variations. The new scales, based on the concept that pollution can be quantified by dimensionless ratios between experimental and benchmark values, overcome most critical points affecting the previous scales.

In bioaccumulation studies, the interpretation of pollutant contents in the target biomonitor has to be performed by assessing a deviation from an unaltered reference condition. A common strategy consists in the comparison with background element content (BEC) values, often built up by uncritically merging methodologically heterogeneous data. In this respect, the acid digestion of samples was identified as a major step affecting BEC data. Here, the analytical outcomes of two acid mixtures were compared on a set of matched paired samples of the lichen Pseudevernia furfuracea, a widely used biomonitor for which BEC values based on partial digestion were previously provided. The standard reference material BCR 482 (P. furfuracea) was used to validate analytical procedures consisting of either a HF total mineralization or an aqua regia partial one, both associated to ICP-MS multi-element analysis. In particular, the performance of the procedures was evaluated by comparing analytical results of field samples with the accuracy obtained on BCR aliquots (measured-to-expected percentage ratio). The total digestion showed a better performance for Al, As, Ba, Ca, Cd, Cu, Fe, Mn, Ni, Se, Sn, and Zn, whereas the opposite was found for Cr, Co, P, and S. Moreover, new BEC values were provided for P. furfuracea using a consolidated statistical approach, after a total sample digestion with hydrofluoric acid. The multivariate investigation of the background variability of 43 elements in 57 remote Italian sites led to the identification of geographically homogeneous areas for which BEC values are provided for use as reference in biomonitoring applications.

Melanised and non-melanised thalli of the devitalized lichen biomonitor Pseudevernia furfuracea have higher binding site concentration than mosses, adsorbing 30% more Zn2+, regardless of melanin presence. Despite the broad use of lichens as biomonitors of airborne trace elements, the surface chemistry and metal adsorption parameters of these organisms are still poorly known. The current investigation is aimed at (i) quantifying the acid-base surface properties and the first-order physical-chemical parameters of Cu2+ and Zn2+ adsorption of devitalized Pseudevernia furfuracea, a lichen commonly used in biomonitoring of airborne trace elements, and (ii) comparing the results with those available for moss biomonitors. Equilibrium constants and metal-binding site concentrations were calculated with a thermodynamic model by taking into account the presence/absence of ancillary extracellular cell wall compounds, namely melanin and acetone-soluble lichen substances. An acid–base titration experiment performed in the pH range of 3–10 showed that melanised and non-melanised P. furfuracea samples have lower pHPZC (3.53–3.99) and higher metal-binding site concentrations (0.96–1.20 mmol g−1) compared to that of the mosses investigated so far at the same experimental conditions. Melanin biosynthesis increased the content of carboxyl and phosphoryl groups and reduces that of amine/polyphenols. Cu2+ and Zn2+ adsorption was unaffected by the degree of melanisation while the removal of extracellular lichen substances slightly decreased Zn2+ adsorption. Although Cu2+ and Zn2+ adsorption parameters related to P. furfuracea surfaces were 3 times lower than in the mosses, lichen samples adsorbed the same amount of Cu2+ and 30% more Zn2+. The present study contributes in understanding the role of ancillary cell wall compounds in Cu2+ and Zn2+ adsorption in a model lichen. It also provides a first comparison between the surface physico-chemical characteristics of lichens and mosses frequently used as biomonitors of trace elements.

Green roofs are gaining momentum in the arid and semi-arid regions due to their multiple benefits as compared with conventional roofs. One of the most critical steps in green roof installation is the selection of drought and heat tolerant species that can thrive under extreme microclimate conditions. We monitored the water status, growth and survival of 11 drought-adapted shrub species grown on shallow green roof modules (10 and 13cm deep substrate) and analyzed traits enabling plants to cope with drought (symplastic and apoplastic resistance) and heat stress (root membrane stability). The physiological traits conferring efficiency/safety to the water transport system under severe drought influenced plant water status and represent good predictors of both plant water use and growth rates over green roofs. Moreover, our data suggest that high substrate temperature represents a stress factor affecting plant survival to a larger extent than drought per se. In fact, the major cause influencing seedling survival on shallow substrates was the species-specific root resistance to heat, a single and easy measurable trait that should be integrated into the methodological framework for screening and selection of suitable shrub species for roof greening in the Mediterranean.

Products containing graphene-related materials (GRMs) are becoming quite common, raising concerns for environmental safety. GRMs have varying effects on plants, but their impact on the sexual reproduction process is largely unknown. In this study, the effects of few-layer graphene (FLG) and a similarly layered phyllosilicate, muscovite mica (MICA), were tested in vivo on the reproductive structures, i.e., pollen and stigma, of Cucurbita pepo L. ssp. pepo &lsquo, greyzini&rsquo, (summer squash, zucchini). Pollen was exposed to FLG or MICA, after careful physical-chemical characterization, at concentrations of 0.5 and 2 mg of nanomaterial (NM) per g of pollen for up to six hours. Following this, pollen viability was tested. Stigmas were exposed to FLG or MICA for three hours and then analyzed by environmental scanning electron microscopy to verify possible alterations to their surface. Stigmas were then hand-pollinated to verify the effects of the two NMs on pollen adhesion and in vivo pollen germination. FLG and MICA altered neither pollen viability nor the stigmatic surface. However, both NMs equivalently decreased pollen adhesion and in vivo germination compared with untreated stigmas. These effects deserve further attention as they could impact on production of fruits and seeds. Importantly, it was shown that FLG is as safe as a naturally occurring nanomaterial.

Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.

Drought tolerance was greater in the whole lichen than in its isolated photobiont. Cell turgor state has an influence on the functionality of photosynthetic process in lichens. Irreversible thermodynamics is widely used to describe the water relations of vascular plants. However, poikilohydrous organisms like lichens and aeroterrestrial microalgae have seldom been studied using this approach. Water relations of lichens are generally addressed without separate analysis of the mycobiont and photobiont, and only few studies have correlated changes in photosynthetic efficiency of dehydrating lichens to accurate measurements of their water potential components. We measured water potential isotherms and chlorophyll a fluorescence in the lichen Flavoparmelia caperata harvested in different seasons, as well as in its isolated photobiont, the green alga Trebouxia gelatinosa, either exposed to water stress cycles or fully hydrated. No significant seasonal trends were observed in lichen water relations parameters. Turgor loss point and osmotic potential of the whole thallus were significantly lower than those measured in the photobiont, while differences between the water stressed photobiont and controls were not significant. Dehydration-induced drop of F v/F m was correlated with turgor loss, revealing that the photosynthetic activity of lichens partly depends on their turgor level. We provided one of the first quantitative evidences of the influence that turgor status could exert on the functionality of photosynthetic processes in lichens.

The production of graphene based materials (GBMs) is steadily increasing but the effects of the possible release of GBMs in the environment are far from being understood. Graphene oxide (GO) is among the most active GBMs and it causes widely varying effects on the vegetative body of seed plants. However, nothing is known yet about its potential effects on the reproductive process. This study addresses the effects of GO on pollen germination and pollen tube elongation in the model species Nicotiana tabacum and in the non-model species Corylus avellana. In vitro germination experiments were conducted without or with GO (control and treated samples, respectively) at concentrations of 25, 50 and 100 μg mL−1. Pollen germination and tube elongation were affected at GO concentrations ≥50 μg mL−1, decreasing by 20% and 19% in N. tabacum and by 68% and 58% in C. avellana, respectively. GO did not affect the viability of N. tabacum pollen, but doubled the frequency of bent tubes. Microscopy observations of pollen tubes exposed to a cell-permeant, dual-excitation ratiometric pH indicator revealed that GO affected the intracellular pH homeostasis. Further germination experiments on C. avellana conducted by inverting the pH conditions of the control and treated (100 μg GO mL−1) samples demonstrated that the main factor influencing the pollen performance is the acidic properties of GO. This might affect the reproductive process of numerous seed plants thus being relevant from an environmental point of view.

Considerata l’elevata eterogeneità dell’inquinamento urbano e l’aumento stagionale delle concentrazioni di O3 troposferico, la valutazione degli effetti combinati di diversi inquinanti sulla fisiologia dei licheni assume carattere centrale nell’ottica di un consolidamento metodologico del biomonitoraggio di inquinanti aerodiffusi. In questo lavoro si valutano gli effetti combinati di IPA, elementi e O3 sulla fisiologia di Pseudevernia furfuracea, un biomonitor di amplissimo utilizzo. Essendo inoltre l’ozonazione una tecnica consolidata per la rimozione di IPA da matrici contaminate, a fortiori si ipotizza che l’esposizione dei talli a concentrazioni simil-estive di O3 possa comportare un decremento degli IPA accumulati. I talli sono stati esposti per 6 settimane in siti caratterizzati da diverse fonti emissive e grado di inquinamento. A valle dell’esposizione i campioni sono stati incubati per 2 settimane in camere di fumigazione con o senza O3. Il contenuto di 18 IPA (GC/MS) e 25 elementi (ICP-MS), nonché il contenuto di malondialdeide (MDA), il rilascio di ioni K+ e il parametro Fv/Fm sono stati misurati prima e dopo l’esposizione e la fumigazione. L’effetto dei trattamenti sui parametri fisiologici e sul contenuto di IPA sono stati testati tramite modelli lineari generalizzati (GLM). Sia l’esposizione che la fumigazione hanno avuto effetti statisticamente significativi sul contenuto di MDA ma non su altri marker fisiologici. In particolare Fv/Fm ha subito decrementi significativi rispetto alla condizione pre-esposizione solo a seguito dell’esposizione nei siti più inquinati, così come il rilascio di ioni K+, mentre il contenuto di potassio e fosforo è risultato significativamente inferiore nei talli fumigati rispetto a quelli non fumigati. La concentrazione di IPA a 4 anelli (la classe di IPA più abbondante) è inoltre risultata sistematicamente inferiore nei talli fumigati, confermando un effetto di degradazione di tali composti da parte dell’O3.

The relation between water status and expression profiles of desiccation -related genes has been studied in the desiccation tolerant (DT) aeroterrestrial green microalga Trebouxia gelatinosa, a common lichen photobiont. Algal colonies were desiccated in controlled conditions and during desiccation water content (WC) and water potential (Ψ) were measured to find the turgor loss point (Ψtlp). Quantitative real-time PCR was performed to measure the expression of ten genes related to photosynthesis, antioxidant defense, expansins, heat shock proteins (HSPs), and desiccation related proteins in algal colonies collected during desiccation when still at full turgor (WC > 6 g H2O g−1 dry weight), immediately before and after Ψtlp (−4 MPa; WC∼1 g H2O g−1 dry weight) and before and after complete desiccation (WC < 0.01 g H2O g−1 dry weight), quantifying the HSP70 protein levels by immunodetection. Our analysis showed that the expression of eight out of ten genes changed immediately before and after Ψtlp. Interestingly, the expression of five out of ten genes changed also before complete desiccation, i.e. between 0.2 and 0.01 g H2O g−1 dry weight. However, the HSP70 protein levels were not affected by changes in water status. The study provides new evidences of the link between the loss of turgor and the expression of genes related to the desiccation tolerance of T. gelatinosa, suggesting the former as a signal triggering inducible mechanisms.

Il tempio di Atena o Athenaion (Paestum, Salerno), monumento tra i più famosi del paesaggio archeologico campano, è rimasto per lunghissimo tempo esposto agli eventi meteorici, privo dell’originaria copertura di malta di finitura. Nel tempo tutte le superfici litiche sono state colonizzate da organismi, in prevalenza licheni, i quali esercitano un’azione biodeteriogena sul substrato e possono provocare un considerevole danno estetico. Per questo motivo nell’ultimo decennio le superfici lapidee del tempio sono state soggette a numerosi interventi di restauro. Attualmente la situazione più rilevante è quella relativa al pavimento della cella del tempio il quale presenta una copertura lichenica a mosaico prevalentemente dominata da due specie, Bagliettoa parmigera (J.Steiner), con tallo emi-endolitico di colore bianco cenere, e Verrucaria nigrescens Pers., con tallo epilitico d colore da marrone a nero. La copertura raggiunge in alcuni tratti anche valori prossimi al 100% e il danno estetico e meccanico apportato da questa comunità è stimabile come molto alto. Per questi motivi, in questo studio si è ritenuto opportuno applicare metodi di restauro classici e verificare nuovi protocolli di trattamento per migliorare e rendere più duratura l’eradicazione delle patine biologiche. A tal proposito, il protocollo standard che fan uso di sostanze biocide, quali Biotin R, T e New Des 50, è stato affiancato da blandi trattamenti termici (40-60 °C) applicati su talli di V. nigrescens. L’efficacia dei trattamenti è stata verificata attraverso misure di parametri di fluorescenza clorofilliana e misure di integrità di membrana mediante microscopia confocale. Tra le sostanze biocide il Biotin R è risultato il meno efficace mentre il New Des 50 quello con l’effetto più forte sia sui parametri di fluorescenza che sull’integrità delle membrane cellulari. L’applicazione di trattamenti termici ha avuto un effetto analogo, molto maggiore su talli lichenici idratati. La combinazione dei biocidi con i trattamenti termici si è rivelata molto più efficace dei trattamenti singoli suggerendo la possibilità in futuro di ridurre le quantità di biocidi utilizzati e quindi ridurre l’impatto di queste sostanze sulla salute degli operatori e dell’ambiente.

In biomonitoring, the knowledge of background element content (BEC) values is an essential pre-requisite for the correct assessment of pollution levels. Here, we estimated the BEC values of a highly performing biomonitor, the epiphytic lichen Pseudevernia furfuracea, by means of a careful review of literature data, integrated by an extensive field survey. Methodologically homogeneous element content datasets, reflecting different exposure conditions across European and extra-European countries, were compiled and comparatively analysed. Element content in samples collected in remote areas was compared to that of potentially enriched samples, testing differences between medians for 25 elements. This analysis confirmed that the former samples were substantially unaffected by anthropogenic contributions, and their metrics were therefore proposed as a first overview at supra-national background level. We also showed that bioaccumulation studies suffer a huge methodological variability. Limited to original field data, we investigated the background variability of 43 elements in 62 remote Italian sites, characterized in GIS environment for anthropization, land use, climate and lithology at different scale resolution. The relationships between selected environmental descriptors and BEC were tested using Principal Component Regression (PCR) modelling. Elemental composition resulted significantly dependent on land use, climate and lithology. In the case of lithogenic elements, regression models correctly reproduced the lichen content throughout the country at randomly selected sites. Further descriptors should be identified only for As, Co, and V. Through a multivariate approach we also identified three geographically homogeneous macro-regions for which specific BECs were provided for use as reference in biomonitoring applications.

In this contribution, new data concerning bryophytes, fungi, and lichens of the Italian flora are presented. It includes new records and confirmations for the bryophyte genera Diplophyllum and Ptychostomum, the fungal genera Arrhenia, Gymnosporangium, and Sporidesmium and the lichen genera Arthonia, Coenogonium, Flavoplaca, Gyalolechia, Parmotrema, Peltigera, Pterygiopsis, Squamarina, Tornabea, and Waynea.

A comparative study of isolated vs. lichenized Trebouxia sp. showed that lichenization does not influence the survival capability of the alga to the photo-oxidative stress derived from prolonged desiccation. Coccoid algae in the Trebouxia genus are the most common photobionts of chlorolichens but are only sporadically found in soil or bark outside of a lichen. They all appear to be desiccation tolerant, i.e. they can survive drying to water contents of below 10 %. However, little is known about their longevity in the dry state and to which extent lichenization can influence it. Here, we studied the longevity in the dry state of the lichenized alga (LT) Trebouxia sp. in the lichen Parmotrema perlatum, in comparison with axenically grown cultures (CT) isolated from the same lichen. We report on chlorophyll fluorescence emission and reactive oxygen species (ROS) production before desiccation, after 15–45 days in the dry state under different combinations of light and air humidity and after recovery for 1 or 3 days in fully hydrated conditions. Both the CT and the LT were able to withstand desiccation under high light (120 µmol photons m−2 s−1 for 14 h per day), but upon recovery after 45 days in the dry state the performance of the CT was better than that of the LT. By contrast, the quenching of excess light energy was more efficient in the LT, at high relative humidities especially. ROS production in the LT was influenced mostly by light exposure, whereas the CT showed an oxidative burst independent of the light conditions. Although lichenization provides benefits that are essential for the survival of the photobiont in high-light habitats, Trebouxia sp. can withstand protracted periods of photo-oxidative stress even outside of a lichen thallus.

Lichens belonging to Parmeliaceae are highly diversified, but most of them share an extremely conserved morpho-chemical trait: the lower cortex is heavily melanized. The adaptive value of this character is still uncertain. Melanins are ubiquitous compounds found in most organisms since they fulfil several biological functions including defense against UV radiation, oxidizing agents, microbial stress, and metal complexation. This work aims to establish whether melanization can affect the elemental content of lichen thalli. The relative abundance of macro- (Ca, K and S) and micro- (Fe, Mn and Zn) nutrients in melanized and non-melanized pseudotissues of nine species was first evaluated by a non-destructive micro-X-ray fluorescence elemental analysis on either the upper and lower cortex, and on the internal medulla, which was artificially exposed to the mechanical removal of the lower cortex. Afterwards, the total concentration of the same elements was measured in composite samples by inductively coupled plasma atomic emission spectroscopy after acidic digestion. In order to verify whether Fe and Zn are chemically bound to the melanized pseudotissues, a sequential elution experiment was performed on two species: the two-side heavily melanized Melanelixia glabratula and the one-side lightly melanized Punctelia subrudecta. The content of Fe and Zn was higher in the melanized species than in the non-melanized ones. Species deprived of their melanized lower cortex showed a sharp decrease in Fe but not in Zn, suggesting that the melanized lower cortex is involved in Fe complexation, whereas Zn is homogeneously distributed throughout the thallus.