Analysis of Petrogenic Hydrocarbons in Plant Tissues: A Simple GC-MS-Based Protocol to Distinguish Biogenic Hydrocarbons from Diesel-Derived Compounds.

Diesel contamination of farming soils is of great concern because hydrocarbons are toxic to all forms of life and can potentially enter the food web through crops or plants used for remediation. Data on plant ability to uptake, translocate and accumulate diesel-derived compounds are controversial not only due to the probable diverse attitude of plant species but also because of the lack of a reliable method with which to distinguish petrogenic from biogenic compounds in plant tissues. The purpose of this study was to set up a GC-MS-based protocol enabling the determination of diesel-derived hydrocarbons in plants grown in contaminated soil for assessing human and ecological risks, predicting phytoremediation effectiveness and biomass disposal. To this end, two plant species, Vicia sativa L. and Secale cereale L., belonging to two diverse vascular plant families, were used as plant models. They were grown in soil spiked with increasing concentrations of diesel fuel, and the produced biomass was used to set up the hydrocarbon extraction and GC-MSD analysis. The developed protocol was also applied to the analysis of Typha latifolia L. plants, belonging to a different botanical family and grown in a long-time and highly contaminated natural soil. Results showed the possibility of distinguishing diesel-derived compounds from biogenic hydrocarbons in most terrestrial vascular plants, just considering the total diesel compounds in the n-alkanes carbon range C10-C26, where the interference of biogenic compounds is negligible. Diesel hydrocarbons quantification in plant tissues was strongly correlated (0.92 < r2 < 0.99) to the concentration of diesel in spiked soils, suggesting a general ability of the considered plant species to adsorb and translocate relatively low amounts of diesel hydrocarbons and the reliability of the developed protocol.

Does Caulerpa prolifera with Its Bacterial Coating Represent a Promising Association for Seawater Phytoremediation of Diesel Hydrocarbons?

Anthropic diesel-derived contamination of Mediterranean coastal waters is of great concern. Nature-based solutions such as phytoremediation are considered promising technologies to remove contaminants from marine environments. The aim of this work was to investigate the tolerance of the Mediterranean autochthonous seaweed Caulerpa prolifera (Forsskal) Lamouroux to diesel fuel and its hydrocarbon degradation potential. Changes in C. prolifera traits, including its associated bacterial community abundance and structure, were determined by fluorescence microscopy and next-generation sequencing techniques. Thalli of C. prolifera artificially exposed to increasing concentration of diesel fuel for 30 days and thalli collected from three natural sites with different levels of seawater diesel-derived hydrocarbons were analysed. Gas chromatography was applied to determine the seaweed hydrocarbon degradation potential. Overall, in controlled conditions the lower concentration of diesel (0.01%) did not affect C. prolifera survival and growth, whereas the higher concentration (1%) resulted in high mortality and blade damages. Similarly, only natural thalli, collected at the most polluted marine site (750 mg L-1), were damaged. A higher abundance of epiphytic bacteria, with a higher relative abundance of Vibrio bacteria, was positively correlated to the health status of the seaweed as well as to its diesel-degradation ability. In conclusion, C. prolifera tolerated and degraded moderate concentrations of seawater diesel-derived compounds, especially changing the abundance and community structure of its bacterial coating. The protection and exploitation of this autochthonous natural seaweed-bacteria symbiosis represents a useful strategy to mitigate the hydrocarbon contamination in moderate polluted Mediterranean costal environments.

High Phenotypic Plasticity in a Prominent Plant Invader along Altitudinal and Temperature Gradients.

Studies on plant growth and trait variation along environmental gradients can provide important information for identifying drivers of plant invasions and for deriving management strategies. We used seeds of the annual plant invader Ambrosia artemisiifolia L. (common ragweed) collected from an agricultural site in Northern Italy (226 m. a.s.l; Mean Annual Air Temperature: 12.9 °C; precipitations: 930 mm) to determine variation in growth trajectories and plant traits when grown along a 1000-m altitudinal gradient in Northern Italy, and under different temperature conditions in the growth chamber (from 14/18 °C to 26/30 °C, night/day), using a non-liner modeling approach. Under field conditions, traits related to plant height (maximum height, stem height, number of internodes) followed a three-parameter logistic curve. In contrast, leaf traits (lateral spread, number of leaves, leaf length and width) followed non-monotonic double-Richards curves that captured the decline patterns evident in the data. Plants grew faster, reaching a higher maximum plant height, and produced more biomass when grown at intermediate elevations. Under laboratory conditions, plants exhibited the same general growth trajectory of field conditions. However, leaf width did not show the recession after the maximum value shown by plants grown in the field, although the growth trajectories of some individuals, particularly those grown at 18 °C, showed a decline at late times. In addition, the plants grown at lower temperatures exhibited the highest value of biomass and preserved reproductive performances (e.g., amount of male inflorescence, pollen weight). From our findings, common ragweed exhibits a high phenotypic plasticity of vegetative and reproductive traits in response to different altitudes and temperature conditions. Under climate warming, this plasticity may facilitate the shift of the species towards higher elevation, but also the in situ resistance and (pre)adaptation of populations currently abundant at low elevations in the invasive European range. Such results may be also relevant for projecting the species management such as the impact by possible biocontrol agents.

Allergenicity at component level of sub-pollen particles from different sources obtained by osmolar shock: A molecular approach to thunderstorm-related asthma outbreaks.

BACKGROUND: The so-called "thunderstorm asthma" (TA) is an uncommon but dramatic outbreak of asthma attacks occurring during a thunderstorm in the pollen and moulds season. Mechanisms which make the pollen able to enter the deeper airways and provoke severe asthma symptoms are still unclear. OBJECTIVE: To test the hypothesis that sub-pollen particles (SPPs) originated from the rupture by an osmotic shock of pollen associated with TA contain allergens. METHODS: After hydration, SPPs released from pollen grains of grass, pellitory, olive, cypress, ragweed and birch were isolated and determined by microscopy. Allergens were determined by in vitro ELISA inhibition tests indirectly using the sera from 10 polyreactive patients. An inhibition <50% was considered as negative, 50%-75% moderate and > 75% complete. RESULTS: The inhibition experiments showed that the SPPs from birch and cypress were unable to inhibit serum IgE reactivity to Bet v 1 and Cup a 1, respectively. Ragweed SPPs inhibited ragweed pollen extract and Amb a 1 by 75.8 ± 0.11% and 81.2 ± 0.15%, respectively. Olive and pellitory SPPs retained almost the whole IgE-binding capability in all cases tested. Grass SPPs inhibited 32 ± 0.06% of Lolium perenne Lol p 1 and 65% of Phleum pratense extracts, but results were highly variable for individual allergens (97.5%-0.03% for Phl p 2, 45.3 ± 0.12% for Phl p 5, 24.7 ± 0.22% for Phl p 6, and 38.3 ± 0.2% for Phl p 1). CONCLUSIONS: Inhibition experiments confirm the hypothesis that SSPs obtained after the osmotic shock of pollen involved in TA, namely grass, pellitory and olive tree pollen, contain allergens and therefore they can induce severe asthma attacks during thunderstorms.

Seeding and Overseeding Native Hayseed Support Plant and Soil Arthropod Communities in Agriculture Areas.

Using native seed mixtures to create or recover grassland habitats in rotation to crops or in strips surrounding fields is considered a cost-effective practice to enhance ecosystem resilience and agro-biodiversity. The aim of this research was to assess the effects of native hayseed mixtures on plant and microarthropod communities in an agricultural area of Northern Italy. Three different experimental treatments were set up. The first was a control (C) (i.e., non-seeded plots left to spontaneous vegetation succession after ploughing no deeper than 15 cm). The second, hayseed seeded (Hs) after ploughing no deeper than 15 cm. The third experimental treatment was hayseed overseeded (Ov) on the resident plant community after only a superficial harrowing. Ov plots exhibited the preeminent positive effects on the total productivity and quality of the grassland in terms of total vegetation cover, cover and richness of typical grassland species (i.e., Molinio-Arrhenatheretea species), and cover of legumes, grasses and perennial species. Moreover, Ov sites exhibited the highest abundance of microarthropod taxa and soil biological quality (QBS-ar) but only in spring, when the disturbance of ploughing negatively affected Hs and C plots. On the other hand, Hs sites showed a great reduction of invasive alien (i.e., Ambrosia artemisiifolia and Artemisia verlotiorum) and segetal weed species (i.e., Capsella bursa-pastoris and Spergula arvensis) in terms of cover. This study provides valuable indication on using hayseed mixtures to create grassland habitats as reservoir of native flora and soil biodiversity in agriculture areas.

The interactive effect of herbivory, nutrient enrichment and mucilage on shallow rocky macroalgal communities.

This paper focuses on the interactive short and long-term effect of three different stressors on a macroalgal assemblage. Three stressors are considered: herbivory, nutrients and mucilage. The experiment was conducted in Tavolara Punta Coda Cavallo Marine Protected Area (Mediterranean Sea) during a bloom of the benthic mucilage-producing microalga Chrysophaeum taylorii (Pelagophyceae); this microalga is recently spreading in the Mediterranean Sea. On a rocky substratum, 36 plots 20 × 20 cm in size were prepared. Factorial combinations of three experimental treatments were applied in triplicate, including three grazing levels crossed with two nutrient enrichment and two mucilage removal treatments. Significant differences were observed among treatments 8 weeks later, at the end of summer. In particular, dark filamentous algae were more abundant in all enriched plots, especially where mucilage and macroalgae had been removed; a higher percent cover of crustose coralline algae was instead observed where nutrients had been increased and no grazing pressure acted. Furthermore, the abundance of Dictyota spp. and Laurencia spp. was significantly higher in enriched mucilage-free plots where the grazing pressure was null or low. However, the effects of the treatments on the overall assemblage of the macroalgal community were not long persistent (36 weeks later). These results illustrate the capacity of a shallow-water macroalgal community to quickly recover from the simultaneous impacts of herbivory, nutrient enrichment, and mucilage.

Ambrosia artemisiifolia L. temperature-responsive traits influencing the prevalence and severity of pollinosis: a study in controlled conditions.

BACKGROUND: Ambrosia artemisiifolia L. is one of the most important sources of allergenic pollen in many regions of the world. Its health impact increased over the last decades and is expected to further increase in consequence of climate change. However little information is available on the specific role played by temperature on allergy rising. The aim of this work was to evaluate the effect of temperature on A. artemisiifolia growth, flowering and pollen allergenicity, the major plant functional traits influencing the prevalence and severity of pollinosis. RESULTS: Plants were grown in controlled conditions at three thermal regimes: "Low" (LT: 18-14 °C light-dark), "Intermediate" (IT: 24-20 °C light-dark) and "High" (HT: 30-26 °C light-dark). During plant development, plant vegetative and reproductive morpho-functional traits were measured and, at the end of plant life-cycle, mature pollen was collected and analyzed for its allergenic properties by slot blot, 1D- and 2D-western blot (by using a pool of sera from ragweed-allergic patients) and liquid chromatography-tandem mass spectrometry. A. artemisiifolia showed a great development plasticity leading to a broad temperature tolerance. Shoot architecture, growth rate, number of male inflorescence and pollen allergenicity were temperature-responsive traits. Pollen allergenicity increased in parallel with temperature and differences were related to allergen synthesis and Amb a 1-IgE-binding. Flavonoids whose concentration in pollen decreased with the increase of temperature, were recognized as the cause of the negligible Amb a 1-IgE binding in LT pollen. CONCLUSIONS: Results show that temperature governs plant development and pollen allergenicity influencing the temporal and spatial magnitude of subject exposure to allergens.

Effect of Soil pH on the Growth, Reproductive Investment and Pollen Allergenicity of Ambrosia artemisiifolia L.

Despite the importance of soil reaction for alien plant establishment, few and incomplete studies have included this key factor so far. In this study, we investigated the effects of soil pH on the germination, growth (plant height, width, dry weight, etc.) and reproductive investment (inflorescence size and n° of flowers) of Ambrosia artemisiifolia (common ragweed), an allergenic species that is highly invasive and alien in Europe, through a replicated experiment in controlled conditions. In addition, we determined if soil pH has an effect on the total pollen allergenicity of the species. After preliminary germination tests on agar at different pH (from pH4 to pH8), plants were grown in natural soils with pH values of 5 (acid), 6 (sub-acid) and 7 (neutral) obtained by modifying a natural soil by liming methods (calcium hydroxide solution). Results showed that plants grown at pH7 were shorter and developed leaves at a slower rate than those grown at pH5 and pH6; plants grown at pH7 did not produce flowers and pollen. We also observed that, at pH5 and pH6, larger plants (as assessed by the dry weight of the aerial biomass) had both larger and more numerous inflorescences and emitted pollen earlier. Finally, the IgE-binding signal was higher in pollen samples collected from plants grown at pH5 (Integrated Optical Density, IOD, range: 1.12-1.25) than in those grown at pH6 (IOD range: 0.86 -1.03). Although we acknowledge the limitations of only testing the effects of pH in controlled conditions, this study suggests that soil pH greatly affects the growth and development of A. artemisiifolia and indicates that it may have a role in limiting the distribution and hazardousness of this plant. Future field tests should therefore assess the effectiveness of liming in the management and control of ragweed and other alien species.

Role of commercial harbours and recreational marinas in the spread of non-indigenous fouling species.

The role of commercial harbours as sink and source habitats for non-indigenous species (NIS) and the role of recreational boating for their secondary spread were investigated by analysing the fouling community of five Italian harbours and five marinas in the western Mediterranean Sea. It was first hypothesised that NIS assemblages in the recreational marinas were subsets of those occurring in commercial harbours. However, the data did not consistently support this hypothesis: the NIS pools of some marinas significantly diverged from harbours even belonging to the same coastal stretches, including NIS occurring only in marinas. This study confirms harbours as hotspots for marine NIS, but also reveals that numbers of NIS in some marinas is higher than expected, suggesting that recreational vessels effectively facilitate NIS spread. It is recommended that this vector of NIS introduction is taken into account in the future planning of sustainable development of maritime tourism in Europe.

The invasive microalga Chrysophaeum taylorii: Interactive stressors regulate cell density and mucilage production.

The benthic mucilage producing microalga Chrysophaeum taylorii Lewis and Bryan (Pelagophyceae) has recently received attention for its rapid spread in the Mediterranean Sea, where its blooms have remarkable detrimental effects. So far no information on C. taylorii response to multiple stressors, especially in terms of mucilage hyperproduction, is available in the literature yet, and a manipulative field experiment in this topic was designed in Tavolara Punta Coda Cavallo Marine Protected Area. The aim of the study was to test the effects of nutrient enrichment (addition of nutrients), mechanical disturbance (partial and total benthic organisms removal) and hydrodynamics (increased water turbulence) on C. taylorii cell density and mucilage abundance. To the purpose, the three above mentioned stressors were simulated and the three treatments were assigned to 20 × 20 cm plots following a full-factorial design (n = 3). Interactive effects of the three stressors affected significantly both benthic C. taylorii cell density and mucilage cover although differently. Mechanical disturbance and high hydrodynamics produced consistent effects on cell density and mucilage production (i.e. the former factor enhancing and the latter decreasing). Nutrient enrichment on the contrary led to contrasting effects, promoting cell abundance and inhibiting mucilage production. Therefore, important mucilage blooms are expected in oligotrophic sheltered coastal locations where barren areas are present.

Consumer depletion alters seagrass resistance to an invasive macroalga.

Few field studies have investigated how changes at one trophic level can affect the invasibility of other trophic levels. We examined the hypothesis that the spread of an introduced alga in disturbed seagrass beds with degraded canopies depends on the depletion of large consumers. We mimicked the degradation of seagrass canopies by clipping shoot density and reducing leaf length, simulating natural and anthropogenic stressors such as fish overgrazing and water quality. Caulerpa racemosa was transplanted into each plot and large consumers were excluded from half of them using cages. Potential cage artifacts were assessed by measuring irradiance, scouring by leaf movement, water flow, and sedimentation. Algal invasion of the seagrass bed differed based on the size of consumers. The alga had higher cover and size under the cages, where the seagrass was characterized by reduced shoot density and canopy height. Furthermore, canopy height had a significant effect depending on canopy density. The alteration of seagrass canopies increased the spread of C. racemosa only when large consumers were absent. Our results suggest that protecting declining habitats and/or restoring fish populations will limit the expansion of C. racemosa. Because MPAs also enhance the abundance and size of fish consuming seagrass they can indirectly promote algal invasion. The effects of MPAs on invasive species are context dependent and require balancing opposing forces, such as the conservation of seagrass canopy structure and the protection of fish grazing the seagrass.