Phytomonitoring and Phytoremediation of Environmental Pollutants.

Since 1973, with the activation of the Environmental Action Program (EAP), the European Union has produced a substantial body of legislative packages aimed at improving the environmental quality; as a result, air, water, and soil pollution have significantly been reduced, as stated by the European Commission in the EAP 2020 [...].

Seed Priming by Low-Dose Radiation Improves Growth of Lactuca sativa and Valerianella locusta.

Valerian salad and lettuce are edible species that are easy to grow rapidly, and have traits useful for commercial purposes. The consumption of these species is increasing worldwide for their nutritional properties. Seed germination and seedling development are critical stages in the life cycle of plants. Seed priming, including the use of high-energy radiation, is a set of techniques based on the idea that low stress levels stimulate plant responses, thereby improving seed germination and plant growth. In this study, we evaluated in hydroponic culture (i) the germination performance; (ii) morphological traits; and (iii) antioxidant and phenol contents at different endpoints in Lactuca sativa and Valerianella locusta that were developed from seeds exposed to X-rays (1 Gy and 10 Gy doses). Under radiation, biomass production increased in both species, especially in lettuce, where also a reduction in the mean germination time occurred. Radiation increased the level of phenols during the first growth weeks, under both doses for lettuce, and only 1 Gy was required for valerian salad. The species-specific responses observed in this research suggest that the use of radiations in seed priming needs to be customized to the species.

Optimizing Moss and Lichen Transplants as Biomonitors of Airborne Anthropogenic Microfibers.

Anthropogenic microfibers (mfs) are synthetic particles composed of cellulose (cotton, rayon, acetate, etc.) or petrochemical-based polymers (i.e., microplastics-MPs) that are less than 5 mm in length. The accumulation of mfs, including MPs, in the moss Hypnum cupressiforme and the lichen Pseudevernia furfuracea was compared in a transplant experiment lasting 6 weeks. We also tested the effects of the bag used for transplants on the accumulation of mfs. Anthropogenic particles trapped by both biomonitors were mostly filamentous (99% mfs), and their number was overall higher in the moss (mean ± s.d. 102 ± 24) than in the lichen (mean ± s.d. 87 ± 17), at parity of sample weight. On average, mfs found in lichen were significantly longer than those found in moss bags, suggesting that lichens are less efficient at retaining smaller mfs. Exposure without the net yielded a higher mfs number accumulation in both species, indicating that "naked" transplants provide greater sensitivity. The calculation of daily fluxes evidenced a loss of mfs in the lichen, suggesting the presence of more stable bonds between moss and mfs. Raman microspectroscopy carried out on about 100 debris confirms the anthropogenic nature of mfs, of which 20% were MPs. Overall results indicate that moss is preferable to lichen in the biomonitoring of airborne mfs especially when exposed naked.

Evaluation of morpho-physiological responses and genotoxicity in Eruca sativa (Mill.) grown in hydroponics from seeds exposed to X-rays.

Due to its potential applications in cultivated plants, ionizing radiation (IR) and its effect on organisms is increasingly studied. Here we measured the effects of ionizing radiation on Eruca sativa by analyzing plants from irradiated seeds (1 and 10 Gy) grown in hydroponics. We measured several morpho-physiological traits and genotoxicity. Radiation stress induced a noticeable variability of the morpho-physiological traits highlighting decreased plant vigor. Shoot length and leaf number were significantly higher in 1 Gy-treated samples, whereas root length was significantly higher in 10 Gy treated plants. Stomata number significantly increased with IR dose, whereas both pigment and Rubisco content decreased under radiation stress. Phenol content significantly increased in 1 Gy treated samples, otherwise from total antioxidants, which were not different from control. Most results could find a feasible explanation in a hormesis-like pattern and in a decreased plant vigor under radiation stress. IR induced genotoxic damage, evaluated by ISSR markers, in 15 day old leaves; specifically, a severe decrease in the genome template stability was observed. However, a partial recovery occurred after 2 weeks, especially under the lowest dose (i.e., 1 Gy), suggesting that DNA damage detection and repair mechanisms are active. Pigment content and genotoxic damage may serve as proxies for evaluating plant responses to IR stress, since they show univocal dose-dependent trends. The use of more checkpoints for analyses and more doses over a wider range, as well as the focus on different metabolites, could help elucidate plant response in terms of morpho-physiological changes.

Biomonitoring of Airborne Microplastic Deposition in Semi-Natural and Rural Sites Using the Moss Hypnum cupressiforme.

We show that the native moss Hypnum cupressiforme can be used as a biomonitor of atmospheric microplastics (MPs). The moss was collected in seven semi-natural and rural sites in Campania (southern Italy) and was analyzed for the presence of MPs, according to standard protocols. Moss samples from all sites accumulated MPs, with fibers representing the largest fraction of plastic debris. Higher numbers of MPs and longer fibers were recorded in moss samples from sites closer to urbanized areas, likely as the results of a continuous flux from sources. The MP size class distribution showed that small size classes characterized sites having a lower level of MP deposition and a high altitude above sea level.

First Report of Seedling Damping-Off of industrial Hemp (Cannabis sativa L.) caused by seed-transmitted Alternaria rosae in Italy.

In the years 2020-2021 as part of the activity of the Campania region hemp fiber project, variety comparison trials were carried out on 7 hemp varieties among those relevant for bast fiber production. During the trials, in particular on the cv. Fibrante, a consistent problem was noted: a noticeable germination failure (80-90%) occurred during the emergence of seedlings. Therefore, experiments were conducted to ascertain the possible presence of seed-borne pathogens. Tests were carried out on 100 seeds that were surface disinfected with 2% sodium hypochlorite solution for 3 min, rinsed in sterile distilled water three times and dried on sterile filter paper. The seeds were plated on potato dextrose agar (PDA Oxoid™) amended with 100 mg L-1 of streptomycin sulphate, kept at 24°C in the dark and observed daily. Growing colonies were subcultured on PDA for 10 days and, subsequently, twenty purified fungal isolates were obtained by single spore isolation. Colonies of these isolates on PDA were initially grayish-white and then turned dark olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these isolates produced light brown and solitary conidiophore with septum. Conidia were obclavate or pyriform, brown, with 1-3 transverse septa and 0-3 longitudinal septa, and measured 12.5 to 28.5 × 5 to 15 µm (n=50). The morphological characteristics observed under the light microscope were consistent with that of Alternaria spp. (Simmons 2007). In order to characterize the representative isolate, total DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and 3 genes were PCR-amplified: the ITS spacer using the primer pair ITS1-ITS4 (White et al., 1990), the transcription elongation factor 1- using the primer pair EF1-983F/ EF1-2218R (Rehner and Buckley., 2005) and the RNA polymerase II second largest subunit (RPB2) using the primer pair RPB2-5F2/fRPB2-7cR (Sung et al 2007; Liu et al 1999). The size-expected amplicons were purified and sequenced at the BMR Genomics (Padova, Italy) and the resulting sequences were deposited in GenBank under the accession numbers ON556507, ON601003, ON601004. BLAST-n analysis revealed 98 to 99% nucleotide identity with some representative isolates of Alternaria rosae E.G. Simmons & C.F. Hill (KU375630.1, XM_046169884.1, XM_046168987.1). To fulfill Koch's postulates, 100 hemp-certified seed were disinfected as mentioned above, left to germinate on the water-agar to discard potentially infected seeds and finally sowed in sterile peat-soil mix (1:1 v/v). The inoculum consisted of 10 mL of 105 conidial suspension obtained by the representative isolate (Ar_H1). Negative control seeds were inoculated with sterile water. After 5-7 days 100% of inoculated seedlings showed weak germinative vigor with yellowing of the epicotyls and dark areas on the root. The tissue narrowed and turned necrotic with abundant white mycelium covering the entire seedling. Small pieces of necrotic roots were plated on PDA and the same Alternaria-like colonies grew in 10 days. DNA sequencing confirmed the presence of A. rosae. Alternaria spp. are fungi that produce a wide range of toxic metabolites, harmful to food safety in the food uses of the seed. This finding further highlights that the quality of the hemp seed must be considered as a priority aspect in the entire hemp supply chain. To the best of our knowledge, this is the first report of A. rosae as seed-borne fungus on hemp.

Metals Induce Genotoxicity in Three Cardoon Cultivars: Relation to Metal Uptake and Distribution in Extra- and Intracellular Fractions.

Heavy metal-polluted soil represents an important stress condition for plants. Several studies demonstrated that growth inhibition under metal stress and metal-induced damages, including genotoxicity, is particularly pronounced at the early stages of seedling growth. Moreover, it is reported that heavy metals enter the cytoplasm to exert their detrimental effect, including DNA damage. In this work, we estimated (i) metal-induced genotoxicity by ISSR molecular markers and (ii) the distribution of the metal fractions between symplast and apoplast by EDTA washing, in three cultivars of Cynara cardunculus var. altilis (L.) DC (Sardo, Siciliano, and Spagnolo), grown in hydroponics for 15 days with Cd or Pb: In line with the literature, in all cultivars, the genotoxic damage induced by Pb was more severe compared to Cd. However, a cultivar-specific response was evidenced since Spagnolo showed, under metal stress, a significantly higher genome template stability compared to the other examined cultivars. The lower genotoxicity observed in Spagnolo could depend on the lower intracellular metal concentration measured in this cultivar by chemical analysis. Accordingly, light microscopy highlighted that Spagnolo developed smaller and more numerous epidermal cells under metal stress; these cells would provide a larger wall surface offering a wider metal sequestration compartment in the apoplast.

Potential Use of Cardunculus Biomass on Pleurotus eryngii Production: Heteroglycans Content and Nutritional Properties (Preliminary Results).

The new perspective of using waste biomass to cultivate mushrooms as a source of protein for human nutrition, in line with the circular economy principles, is receiving increasing attention in the scientific community and represents great wealth in terms of environmental sustainability. Pleurotus eryngii is a mushroom also known as cardunculus mushroom due to its ability to grow on this plant. This study explores the potential intrinsic properties of cardunculus (for example, the presence of inulin in the roots) as raw material for the growth of cardunculus mushrooms, and the influence on heteroglycan content and nutrition parameters of the fruiting bodies. Both mycelium and fruiting bodies were used to determine the heteroglycan content in the presence of inulin or cardunculus roots rich in inulin. To produce heteroglycans from P. eryngii in greater quantities and shorter times without having to wait for the formation of the fruiting bodies, the mycelium could be used. The results showed that the presence of cardunculus biomass positively influences the heteroglycan content of P. eryngii. In terms of nutritional parameters, higher contents of polyphenols, flavonoids, anthocyanins, and antioxidant activity were detected in P. eryngii grown on the cardunculus stem and root substrate. In conclusion, recycling cardunculus biomass to generate growth blocks for edible mushrooms is a winning choice due to the opportunity to use this biomass waste, which is gaining more and more attention due to the increase in cultivated areas and the use of fruiting bodies of P. eryngii as a functional food and source of molecules with potential biological activities.

Mobile Biomonitoring of Atmospheric Pollution: A New Perspective for the Moss-Bag Approach.

In this work the potential of moving moss-bags, fixed to bicycles, to intercept particulate matter (PM) and linked metal(loid)s was tested for the first time. Seven volunteers carried three moss-bags for fifty days while commuting by bicycle in the urban area of Antwerp, Belgium. Moreover, one bike, equipped with mobile PM samplers, travelled along four routes: urban, industrial, green route and the total path, carrying three moss-bags at each route. The saturation isothermal remanent magnetization (SIRM) signal and chemical composition (assessed by HR-ICP-MS) of the moss samples indicated that the industrial route was the most polluted. Element fluxes (i.e., the ratio between element daily uptake and the specific leaf area) could discriminate among land uses; particularly, they were significantly higher in the industrial route for Ag, As, Cd and Pb; significantly lowest in the green route for As and Pb; and comparable for all accumulated elements along most urban routes. A comparison with a previous experiment carried out in the same study area using similar moss-bags at static exposure points, showed that the element fluxes were significantly higher in the mobile system. Finally, PM2.5 and PM10 masses measured along the four routes were consistent with element fluxes.

Multi-elemental profile and enviromagnetic analysis of moss transplants exposed indoors and outdoors in Italy and Belgium.

Air pollution represents one of the major concerns worldwide, fueled by the increasing urbanization and related PM production worsening air quality in open air as well as in confined environments. In the present work, exposure to atmospheric metal pollution was investigated in 20 paired indoor (I)-outdoor (O) sites located in two urban areas of Italy and Belgium, by chemical (ICP-MS) and magnetic (saturation isothermal remanent magnetization, SIRM) analyses of Hypnum cupressiforme moss exposed in bags. After 12 weeks, the elemental profiles of the moss material exposed in the two countries largely overlapped, except for some elements which specifically accumulated in Belgium (Ag, As, Cd, Mo, Pb and Sb) and in Italy (Ca, Mg, Co, Cr, Sr, Ti and U). Element concentrations were higher in moss exposed outdoors, with the Italian sites mostly showing a terrigenous footprint, and the Belgian sites mostly affected by elements of environmental concern (e.g., As, Pb, Sb). The Indoor/Outdoor ratios (mostly lower than 0.75) indicated indoor pollution as strongly affected by outdoor pollution, although specific elements could be of indoor origin or magnified in indoor environments (e.g., Al, Ag, Cd and Co). In line with the chemical analysis, the SIRM signal was significantly higher in outdoor than indoor moss material. A positive, significant correlation was observed between SIRM and several accumulated elements indicating SIRM analysis as a powerful tool to predict the level of metal pollution. Moss bags were confirmed as a useful and versatile tool to highlight metal contamination even in confined environments, an essential prerogative in the perspective of the evaluation of the total exposure risk for humans to these pollutants.

Facing metal stress by multiple strategies: morphophysiological responses of cardoon (Cynara cardunculus L.) grown in hydroponics.

The contamination of environments by heavy metals has become an urgent issue causing undesirable accumulations and severe damages to agricultural crops, especially cadmium and lead which are among the most widespread and dangerous metal pollutants worldwide. The selection of proper species is a crucial step in many plant-based restoration approaches; therefore, the aim of the present work was to check for early morphophysiological responsive traits in three cultivars of Cynara cardunculus (Sardo, Siciliano, and Spagnolo), helping to select the best performing cultivar for phytoremediation. For all three tested cultivars, our results indicate that cardoon displays some morphophysiological traits to face Cd and Pb pollution, particularly at the root morphology level, element uptake ability, and photosynthetic pigment content. Other traits show instead a cultivar-specific behavior; in fact, stomata plasticity, photosynthetic pattern, and antioxidant power provide different responses, but only Spagnolo cv. achieves a successful strategy attaining a real resilience to metal stress. The capacity of Spagnolo plants to modify leaf structural and physiological traits under heavy metal contamination to maintain high photosynthetic efficiency should be considered an elective trait for its use in contaminated environments.

Exploring the phytoremediation potential of Cynara cardunculus: a trial on an industrial soil highly contaminated by heavy metals.

Metal uptake capacity and growth patterns of three cardoon cultivars (Sardo, Siciliano, and Spagnolo) were investigated for phytoremediation in a metal-contaminated soil. Metal concentrations after one and two growth rounds were measured in soil and plants by ICP-MS. Potentially and promptly available metal fractions were estimated by EDTA and ammonium nitrate. Plant weight, water and nitrogen content, and life span were considered as growth parameters. After the first growth cycle, a significant accumulation of all elements was observed in roots, whereas only Cd, Cu, Fe, Pb, and Sb were significantly translocated to the shoots. After the second growth cycle, a further significant increase, especially in roots, was observed in all cultivars and for all elements considered. High percentages of the potentially available fraction were found for As, Cd, Cu, Pb, and Sb. Metal concentrations in plants were far higher than the promptly available fraction, indicating an increase of metal bioavailability during culture rounds. A severe decrease of weight and life span was observed in Sardo and Siciliano grown on polluted soil, along with an increase in N content. The cultivar Spagnolo, harmonizing a high ability of metal uptake and tolerance, proved the most efficient cultivar in metal phytoremediation.

Overall plant responses to Cd and Pb metal stress in maize: Growth pattern, ultrastructure, and photosynthetic activity.

This study provides a full description of the responses of the crop energy plant Zea mays to stress induced by Cd and Pb, in view of a possible extensive use in phytoattenuation of metal-polluted soils. In this perspective, (i) the uptake capability in root and shoot, (ii) the changes in growth pattern and cytological traits, and (iii) the photosynthetic efficiency based on photochemistry and the level of key proteins were investigated in hydroponic cultures. Both metals were uptaken by maize, with a translocation factor higher for Cd than Pb, but only Cd-treated plants showed a reduced growth compared to control (i.e., a lower leaf number and a reduced plant height), with a biomass loss up to 40%, at the highest concentration of metal (10-3 M). The observation of cytological traits highlighted ultrastructural damages in the chloroplasts of Cd-treated plants. A decline of Rubisco and D1 was observed in plants under Cd stress, while a relevant increase of the same proteins was found in Pb-treated plants, along with an increase of chlorophyll content. Fluorescent emission measurements indicated that both metals induced an increase of NPQ, but only Cd at the highest concentration determined a significant decline of Fv/Fm. These results indicate a different response of Z. mays to individual metals, with Pb triggering a compensative response and Cd inducing severe morpho-physiological alterations at all investigated levels. Therefore, Z. mays could be successfully exploited in phytoattenuation of Pb-polluted soil, but only at very low concentrations of Cd to avoid severe plant damages and biomass loss.

Genotoxic effect of Pb and Cd on in vitro cultures of Sphagnum palustre: An evaluation by ISSR markers.

In the present work, the genotoxic effect of cadmium and lead supplied in a laboratory trial, was investigated for the first time in the moss Sphagnum palustre, by ISSR molecular markers. A total of 169 reproducible bands were obtained with 12 primers, ten of which gave polymorphisms (i.e., appearance/disappearance of bands), indicating a clear genotoxic effect induced by the metals. Both metals induced a decrease of the genome template stability in a dose dependent manner. At concentration >10-5 Cd also induced a general toxic effect in S. palustre, leading to chlorophyll degradation and moss death. Moreover, we followed the fate of supplied heavy metals into the moss tissue by SEM-EDX to see if they entered the cells. SEM-EDX observations on moss cultures treated with equimolar concentrations of the two metals showed that most Pb precipitated in form of particles on moss surface, while Cd did not aggregate in particles and was not found on moss surface. In light of these findings, we concluded that probably Pb induced a genotoxic effect at lower intracellular concentrations than Cd.

Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma.

The chromatin-modifying enzyme lysine-specific demethylase 1, KDM1A/LSD1 is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells and its overexpression correlated with aggressive disease, poor differentiation and infaust outcome. Here, we show that LSD1 physically binds MYCN both in vitro and in vivo and that such an interaction requires the MYCN BoxIII. We found that LSD1 co-localizes with MYCN on promoter regions of CDKN1A/p21 and Clusterin (CLU) suppressor genes and cooperates with MYCN to repress the expression of these genes. KDM1A needs to engage with MYCN in order to associate with the CDKN1A and CLU promoters. The expression of CLU and CDKN1A can be restored in MYCN-amplified cells by pharmacological inhibition of LSD1 activity or knockdown of its expression. Combined pharmacological inhibition of MYCN and LSD1 through the use of small molecule inhibitors synergistically reduces MYCN-amplified Neuroblastoma cell viability in vitro. These findings demonstrate that LSD1 is a critical co-factor of the MYCN repressive function, and suggest that combination of LSD1 and MYCN inhibitors may have strong therapeutic relevance to counteract MYCN-driven oncogenesis.