Despite the paramount importance in metal(loid) detoxification by phytochelatin synthase (PCS) genes, no comprehensive analysis of their evolutionary patterns has been carried out in land plants in general and in crops in particular. A phylogenetic large-scale analysis of gene duplication in angiosperms was carried out followed by in vitro recombinant protein assays as well as complementation analysis (growth, thiol-peptides, elements) of Arabidopsis cad1-3 mutant with four representative PCS genes from two model crop species, Malus domestica and Medicago truncatula. We uncovered a so far undetected ancient tandem duplication (D duplication) spanning the whole core eudicotyledon radiation. Complementation with PCS genes from both D-subclades from M. domestica and M. truncatula displayed clear in vivo conservation of the differences between D1 and D2 paralogous proteins in plant growth, phytochelatin, and glutathione pools, as well as element contents under metal(loid) stress. In vitro recombinant PCS analysis identified analogous patterns of differentiation, showing a higher activity of D2 PCS genes, so far largely overlooked, compared to their paralogs from the D1 clade. This suggests that in many other crop species where the duplication is present, the D2 copy might play a significant role in metal(loid) detoxification. The retention of both PCS paralogs and of their functional features for such long divergence time suggests that PCS copy number could be constrained by functional specialization and/or gene dosage sensitivity. These results uncover the patterns of PCS evolution in plant genomes and of functional specialization of their paralogs in the genomes of two important model crops.

Arsenic is a widespread metalloid that even at low concentrations is highly toxic to most plant species. While the transcriptional responses associated to arsenic tolerance have been widely investigated in vascular plants, comparatively little is known in their sister lineage, the bryophytes. Most importantly, functional evidence of whether the same genes play major roles in arsenic tolerance responses in these two anciently diverged land plant lineages is currently largely missing. In this study, we identified by RNA-Seq a highly reliable set of differentially expressed genes (DEGs) responding to arsenite toxicity in the model bryophyte Marchantia polymorpha. We then explored the evolutionary level of functional conservation of seven upregulated DEGs by Agrobacterium-mediated transformation in the highly arsenic-sensitive cad1-3 mutant of Arabidopsis thaliana as a representative of tracheophytes, and characterized fresh weight, malondialdehyde production and total arsenic content in dry biomass of transgenic lines. While two of the tested M. polymorpha DEGs did not significantly enhance arsenic tolerance, the remaining five DEGs, when overexpressed in cad1-3, conferred maximal levels of tolerance, measured as biomass accumulation, between 56 % and 100 % of WT Col-0 plants. Among them, a putative 1-cys peroxiredoxin restored growth, protection from lipid peroxidation and capacity to accumulate arsenic to levels indistinguishable from those of WT. These results provide functional evidence for the considerable conservation of arsenic tolerance responses between M. polymorpha and A. thaliana, suggesting that M. polymorpha can be a valid model for the identification of evolutionarily deeply conserved genes for the genetic improvement of crops for arsenic tolerance.

Complex matrices such as soil have a range of measurable characteristics, and thus data to describe them can be considered multidimensional. These characteristics can be strongly influenced by factors that introduce confounding effects that hinder analyses. Traditional statistical approaches lack the flexibility and granularity required to adequately evaluate such matrices, particularly those with large dataset of varying data types (i.e. quantitative non-compositional, quantitative compositional). We present a statistical workflow designed to effectively analyse complex, multidimensional systems, even in the presence of confounding variables. The developed methodology involves exploratory analysis to identify the presence of confounding variables, followed by data decomposition (including strategies for both compositional and non-compositional quantitative data) to minimise the influence of these confounding factors such as sampling site/location. These data processing methods then allow for common patterns to be highlighted in the data, including the identification of biomarkers and determination of non-trivial associations between variables. We demonstrate the utility of this statistical workflow by jointly analysing the chemical composition and fungal biodiversity of New Zealand vineyard soils that have been managed with either organic low-input or conventional input approaches. By applying this pipeline, we were able to identify biomarkers that distinguish viticultural soil from both approaches and also unearth links and associations between the chemical and metagenomic profiles. While soil is an example of a system that can require this type of statistical methodology, there are a range of biological and ecological systems that are challenging to analyse due to the complex interplay of global and local effects. Utilising our developed pipeline will greatly enhance the way that these systems can be studied and the quality and impact of insight gained from their analysis.

The arsenic-specific ACR3 transporter plays pivotal roles in As detoxification in yeast and a group of ancient tracheophytes, the ferns. Despite putative ACR3 genes being present in the genomes of bryophytes, whether they have the same relevance also in this lineage is currently unknown. In this study, we characterized the MpACR3 gene from the bryophyte Marchantia polymorpha L. through a multiplicity of functional approaches ranging from phylogenetic reconstruction, expression analysis, loss- and gain-of-function as well as genetic complementation with an MpACR3 gene tagged with a fluorescent protein. Genetic complementation demonstrates that MpACR3 plays a pivotal role in As tolerance in M. polymorpha, with loss-of-function Mpacr3 mutants being hypersensitive and MpACR3 overexpressors more tolerant to As. Additionally, MpACR3 activity regulates intracellular As concentration, affects its speciation and controls the levels of intracellular oxidative stress. The MpACR3::3xCitrine appears to localize at the plasma membrane and possibly in other endomembrane systems. Taken together, these results demonstrate the pivotal function of ACR3 detoxification in both sister lineages of land plants, indicating that it was present in the common ancestor to all embryophytes. We propose that Mpacr3 mutants could be used in developing countries as low-cost and low-technology visual bioindicators to detect As pollution in water.

Origin authentication methods are pivotal in counteracting frauds and provide evidence for certification systems. For these reasons, geographical origin authentication methods are used to ensure product origin. This study focused on the origin authentication (i.e. at the producer level) of a typical mountain cheese origin using various approaches, including shotgun metagenomics, volatilome, near infrared spectroscopy, stable isotopes, and elemental analyses. DNA-based analysis revealed that viral communities achieved a higher classification accuracy rate (97.4 ± 2.6 %) than bacterial communities (96.1 ± 4.0 %). Non-starter lactic acid bacteria and phages specific to each origin were identified. Volatile organic compounds exhibited potential clusters according to cheese origin, with a classification accuracy rate of 90.0 ± 11.1 %. Near-infrared spectroscopy showed lower discriminative power for cheese authentication, yielding only a 76.0 ± 31.6 % classification accuracy rate. Model performances were influenced by specific regions of the infrared spectrum, possibly associated with fat content, lipid profile and protein characteristics. Furthermore, we analyzed the elemental composition of mountain Caciotta cheese and identified significant differences in elements related to dairy equipment, macronutrients, and rare earth elements among different origins. The combination of elements and isotopes showed a decrease in authentication performance (97.0 ± 3.1 %) compared to the original element models, which were found to achieve the best classification accuracy rate (99.0 ± 0.01 %). Overall, our findings emphasize the potential of multi-omics techniques in cheese origin authentication and highlight the complexity of factors influencing cheese composition and hence typicity.

Windstorms and salvage logging lead to huge soil disturbance in alpine spruce forests, potentially affecting soil-living arthropods. However, the impacts of forest loss and possible interactions with underlying ecological gradients on soil microarthropod communities remain little known, especially across different environmental conditions. Here we used DNA metabarcoding approach to study wind-induced disturbances on forest communities of springtails and soil mites. In particular, we aimed to test the effect of forest soil disturbance on the abundance, richness, species composition, and functional guilds of microarthropods. We sampled 29 pairs of windfall-forest sites across gradients of elevation, precipitation, aspect and slope, 2 years after a massive windstorm, named Vaia, which hit North-Eastern Italy in October 2018. Our results showed that wind-induced disturbances led to detrimental impacts on soil-living communities. Abundance of microarthropods decreased in windfalls, but with interacting effects with precipitation gradients. Operative Taxonomic Units (OTU) richness strongly decreased in post-disturbance sites, particularly affecting plant-feeder trophic guilds. Furthermore, species composition analyses revealed that communities occurring in post-disturbance sites were different to those in undisturbed forests (i.e., stands without wind damage). However, variables at different spatial scales played different roles depending on the considered taxon. Our study contributes to shed light on the impacts on important, but often neglected arthropod communities after windstorm in spruce forests. Effects of forest disturbance are often mediated by underlying large scale ecological gradients, such as precipitation and topography. Massive impacts of stronger and more frequent windstorms are expected to hit forests in the future; given the response we recorded, mediated by environmental features, forest managers need to take site-specific conservation measures.

In this study, hydrochar (HC), a carbon-rich product originated from hydrothermal conversion treatment (HTC), was obtained from wastes of the wine and dairy industries. The effect of mixing secondary char and compost was tested, before and after the aerobic mixing of compost (COM) and HC at increasing doses (from 15 to 75 Mg ha-1 DM), in an effort to lower the HC phytotoxicity due to potential phytotoxic compounds of secondary char. The results indicated that, after the aerobic stabilization, the mix HC/COM was able to double the plant growth in comparison to COM alone. The presence of easily degradable organic compounds probably led to poor stability of HC, increased microbial activity and, consequently, root anoxia when used at high doses. Chemical, spectroscopic and thermal investigation confirmed this hypothesis. In particular, HC shows a high content of dissolved organic matter, characterized by the presence of small molecules, which is negatively correlated with the growth index of lettuce. Furthermore, thermal characterization suggests a higher proportion of less complex and thermally stable molecular compounds in HC in comparison to COM. Therefore, co-composting of HC allows obtaining a useful amendment to support soil organic matter and fertility.

The European chestnut characterizes both the landscape and economy of mountainous Italian areas. In recent years, new canopy disorders have been reported: “chestnut yellows”, often ascribed to phytoplasma and/or nutrient deficiency, and “chestnut mosaic”, associated with a virus (ChMV). Therefore, research was carried out in four Italian regions to describe the two symptomatic frames and assess their etiology. Surveys were conducted on 101 chestnut trees (23 with mosaic, 38 with yellowing, and 40 without symptoms). The phytosanitary status was monitored, and the new canopy disorders were detected, distinguishing between yellowing and mosaic. Moreover, leaf samples were collected for molecular and nutrient analyses. No phytoplasma infection was recorded, while ChMV was detected in 91.3% of samples with mosaic symptoms, 31.6% of yellowing samples, and 30.0% of asymptomatic samples. Yellowing was associated with Mn deficiency. On the other hand, ChMV-infected and healthy leaves had similar mineral contents, showing that mosaic symptoms are induced by the virus. Both disorders negatively affected photosynthesis efficiency. These phytosanitary problems are present in Italian chestnut woods and cause local effects, and a relationship with other biotic and abiotic factors can be hypothesized. Considering the increase in new records, these symptoms represent an emerging issue whose impact and spread need to be further monitored.

Bacteria have a fundamental role in determining the fitness of grapevine, the composition of grapes and the features of wines but at present, little information is available. In this work, the bacteria colonizing the different portions of grapevine (bark, leaves and grapes) were explored in the vineyards of the Alpine region of Trentino, considering the impact of different environmental and agronomical variables. The vineyards included in the work were selected based on their different geographical positions (altitude) and grapevine training systems in order to explore the whole variability of the grapevine ecosystem. Moreover, the surface amount of copper was measured on grapes and leaves during the vegetative growth. Bacterial analysis, performed using plate counts and Illumina MiSeq, revealed an increase in the concentration of grape bacteria proportional to the progress of the ripening stage. Conversely, the peak of bacterial concentration onto leaf and bark samples occurred in August, probably due to the more favourable environmental conditions. In bark samples, the bacterial microbiota reached the 7 log CFU/cm(2), while 6 log UFC/g were measured in grape samples. A remarkable biodiversity was observed, with 13 phyla, 35 classes, 55 orders, 78 families and 95 genera of bacteria present. The presence of some taxa (Alphaproteobacteria, Desulfovibrionaceae, Clostriadiales, Oscillospira, Lachnospiraceae and Bacteroidales) was ubiquitous in all vineyards, but differences in terms of relative abundance were observed according to the vegetative stage, altitude of the vineyard and training system. Bacteria having oenological implication (Lactobacillus, Pediococcus and Oenococcus) were detected in grape samples collected in August, in low abundance. The data revealed a complex bacterial ecosystem inside the vineyard that, while maintaining common traits, evolves according to environmental and agronomical inputs. This study contributes to define the role of bacteria in the complex balance established in each vineyard between human actions and agricultural environment, known as terroir.

Recently new resistant and/or tolerant genotypes to the main cryptogams (downy and powdery mildew) were identified, to be used in enhancing sustainable viticulture. These cultivars (result of interspecific crossings between Vitis vinifera cultivars and other Vitis species of American and Asian origin) are experiencing a relevant expansion in Europe in viticultural regions characterized by high rainfall per year. In two sites of Trentino (Northeastern Italy) with different soil pH levels, and where these conditions are widespread, an important study was scheduled. During the period 2015-2019, fifteen resistant varieties were monitored in order to have information about agronomical and nutritional behavior. Yearly, at fruit set nutritional aspects through leaf analyses and leaf green color (SPAD indexes) were monitored. At pre-veraison, photosynthetically active biomass behavior (Normalized Difference Vegetation Index NDVI indexes) was controlled. Weather conditions of different years strongly changed the availability of nutrients in vines in relation to different genotypes and their color of vegetation and vigor. Collected results allow to suggest interventions aimed at a more suitable nutritional management for resistant varieties, in comparison of Vitis vinifera , above all for nitrogen (N), potassium (K), and zinc (Zn); in particular, for magnesium (Mg), early foliar treatments should be recommended, especially in years where rainfall is concentrated in the spring months.

In intensive fruit growing systems, the recovery and maintenance of soil fertility play a crucial role in both environmental protection and sustainable support to plant productivity. The circular economy approach adopted at the EU level strongly promotes the use of organic products instead of mineral fertilizers. This work focuses on two different soil improvers, compost from the organic fraction of municipal solid waste digestate (CO) and “matured” manure, produced after a fast and controlled aerobic treatment in an aerated pile (MM), which were applied in three apple orchards with different soil tillage. The soil improvers have been characterized for amendment and fertilizing properties. After the amendment, the soils were sampled twice a year (Spring and Autumn) for three years. Each sample has been characterized for texture, pH, cation exchange capacity, nutrients, soil organic matter, and micronutrients. The amendments obtained differed on C, N, P, and K contents, but had similar biological stability. The main effects on soils were the increasing of N and soil organic matter after compost application, while the use of matured manure mainly act on available P and exchangeable K. The treatments showed significant effects among fields with a linear increasing trend only for compost. Matured manure showed more effects in earlier times. The data collected aim to improve the knowledge about sustainable management of soil organic matter and organic nutrients in intensive fruit-growing agriculture by using local products.

The high demand for wine in Europe has increased the impact of viticulture on the environment. In line with European objectives, more sustainable agronomic practices have spread as an alternative to traditional management. This study aimed to compare, in a vineyard of Pinot blanc and Rhine Riesling in northeast Italy, the integrated agronomic practices (INT) with two types of organic management (ORG1—cattle manure and ORG2—green manure), in terms of production, grape quality, pest susceptibility, and soil nutrient availability. The results, after the fifth, sixth, and seventh year of testing, showed that organic management obtained a yield and vegetative features comparable to INT. Grape quality also did not show considerable overall differences between the theses in the must properties, despite the higher total sugar content and lower yeast available in ORG1. In the three-year period, the management of downy mildew, powdery mildew, and rot, as well as the soil fertilization, with the products available in organic farming proved to be comparable to the INT method. The application of cattle manure contributed by enriching the soil in K and P, while a balanced green manure mix has proven to be the best agronomic practice in terms of the release of mineral N during the phenological stages of greatest need of the vine. Organic management appears as an agronomic strategy able quantitatively and qualitatively support the vineyard system.

Alpine pastures have not yet been extensively studied with regard to the presence of alkaloids in herbaceous plants. In this work the alkaloid profiles were characterised from a selection of 62 herbs collected from alpine pastures in north-eastern Italy. High-performance liquid chromatography coupled to a hybrid quadrupole-orbitrap mass spectrometer was used to evaluate the presence of 41 different alkaloids and quantify them using a targeted approach. Provisionally, 118 alkaloids were identified, including both free and glycosylated forms, making use of a homemade database and a suspect screening approach. rucifoline, gramine, heliotrine, lycopsamine, seneciphylline, and veratramine were quantified with concentrations ranging from 6 to about 100 µg kg-1 in 6 plants. Herbaceous plants belonging to the most well-represented plant families (Poaceae, 9 species; Asteraceae, 7; Lamiaceae, 6) showed distinct and characteristic alkaloid profiles and were correctly reclassified with an average accuracy of 85% (Partial Least Squares - Discriminant Analysis).

The intensive exploitation of agricultural land has caused a depletion of soil organic carbon (SOC) and a decline in soil fertility, with a consequent decrease in the productivity of agroecosystems, also contributing to atmospheric GHG emissions. The ability of soil to storage organic carbon (OC) depends on its persistence and susceptibility to decomposition. SOC is generally partitioned into fractions differing in protection mechanisms and in turn decomposability. This study aimed to compare the OC pool repartition in soils managed with three different fertilization practices (mineral – CONV, manure – ORGM and green manure – ORG-GM) during a medium-term trial in vineyard, in order to understand the best solution in increasing C sequestration. The OC fractionation by acid hydrolysis allowed separating three fractions of OC: a labile, quickly mineralizable pool, a recalcitrant pool with a slower turnover and a stable pool protected by microbial attack. The results showed that the periodic application of organic matrices (manure and green manure), although did not increase total OC, enriched the soil along time with readily available OC, thus promoting the release of nutrients. Green manure has also been shown to promote the accumulation of stabilized OC, able to improve the structure of the soil and, therefore, its fertility.

Bryophytes are a large group of plants commonly used as bioindicators of metal and metalloid pollution. However, very little is known about the bryophyte genes responsible for metal(loid) detoxification, a knowledge that could provide novel tools for environmental monitoring applications.By genetic transformation and genome editing, in this study we obtained lines of the model bryophyte Marchantia polymorpha with de-regulated activity of the phytochelatin synthase (MpPCS) enzyme, responsible for the biosynthesis of metal(loid) chelators phytochelatins (PCn). Lack of PCn causes hypersensitivity to cadmium but has only subtle effects on sensitivity to excess of other highly toxic metals and the metalloid arsenic in M. polymorpha. Besides, our results indicate that MpPCS has a minor role in the maintenance of essential metals like zinc.As liverworts are sister to tracheophytes, these results suggest that the primary ancestral function of PCS genes in the common ancestor of all land plants may have been the detoxification of the non-essential cadmium ion. Hypersensitivity to cadmium further suggests that the Mppcs mutants could become useful bioindicators to specifically detect environmental contaminations of cadmium through direct visual assessment of plant growth and pigmentation.

Avocado (Persea americana Mill) is a fruit consumed worldwide due to its valuable organoleptic and health-promoting properties. To date, no information is available about the isotopic composition of this foodstuff and this study determines the stable isotope composition of five bio-elements (C, N, S, H and O) and the composition profile of 46 macro, micro and trace elements. The stable isotope and elemental profiles were determined in 131 avocados from different producing regions (Spain, Brazil, Chile, Colombia, Kenya, Mexico, Peru, South Africa) collected over three years to study the main geographical differences. A PLS-DA model was performed combining the stable isotopes with the elemental profile, making it possible to distinguish the Spanish from the non-Spanish avocados with a high prediction accuracy (98% correct classification). The results of this study highlight the potential of stable isotope ratios and elemental profiles for tracing the geographical origin of avocados.

The search for valid alternatives to cultivated varieties of Vitis vinifera has recently identified new resistant and/or tolerant genotypes to the main cryptogams (downy and powdery mildew), to be used in enhancing sustainable viticulture. These cultivars represent the result of interspecific crossings, obtained from some important European research centers in viticulture, using Vitis vinifera cultivars and other Vitis species of American and Asian origin. In two sites of northeastern Italy characterized by different soil pH levels, during the period 2015-2019, 15 resistant cultivars 'Aromera', 'Baron', 'Bronner', 'Cabernet Cantor', 'Cabernet Cortis', 'Cabernet Carbon', 'Cabino', 'Helios', 'Johanniter', 'Monarch', 'Muscaris', 'Prior', 'Regent', 'Solaris', and 'Souvignier gris' - were monitored in order to have information about agronomical and nutritional behaviour. Yearly, 15 plants for each cultivar/site were evaluated at fruit set, with regard to nutritional aspects through leaf analyses and leaf green colour (SPAD indexes), as well as for photosynthetically active biomass behaviour (normalized difference vegetation index - NDVI indexes) at pre-veraison. Weather conditions of different years strongly changed the absorption and availability of nutrients in plants and their behaviour for both indexes. 'Regent' and 'Johanniter' showed the highest nitrogen (N) uptake, while 'Solaris', 'Muscaris', and 'Monarch' had the lowest leaf contents. 'Baron' and 'Cabernet Carbon' showed the highest levels of vigor associated with greener leaves, while 'Cabernet Cortis' and 'Prior' had the lowest values of NDVI and SPAD. 'Cabernet Cortis' and 'Solaris' revealed potassium (K) deficiencies, while 'Bronner' and 'Souvignier gris' enhanced the availability of this cation, strongly depressing magnesium (Mg). 'Johanniter', 'Regent', and 'Monarch' had low Mg uptake too. 'Monarch', 'Aromera', and 'Bronner' showed low zinc (Zn) nutritional status in the leaves. Collected results allows us to suggest interventions aimed at a more suitable nutritional management for resistant cultivars, in confront of Vitis vinifera, above all for N, K, and Zn; in particular for Mg, early foliar treatments should be recommended, especially in years where rainfall is concentrated in the spring months.

The stable isotopes of five bio-elements (δ13C, δ15N, δ34S, δ2H, δ18O) and the elemental composition profile combined with chemometrics were used to differentiate the geographical origin of commercial samples of mango, aimed at contributing to the existence of a Spanish protected geographical indication (PGI). Eighty commercial mangoes from all over the world (Spain, Mexico, Peru, Brazil, Ivory Coast, Equatorial Guinea and Senegal), collected during two consecutive harvests, were analysed and assessed by means of univariate and multivariate data analyses, establishing the main variation sources linked to the origin of the mangoes. The data obtained from each analytical platform were assessed separately and in combination, resulting in three models whose discriminatory power (Spanish vs others) was compared. The model fitted for the stable isotope approach yielded a sensitivity of 100%, meaning that the Spanish mangoes were correctly classified in all the cases, against the 97.6% for multi-element analysis. The combined use of stable isotopes and elemental composition led to the most accurate geographical discrimination of Spanish mangoes, making it possible to select three stable isotope ratios (δ15N, δ18O, δ34S) and six elements (Cd, Se, Cs, As, Pb and Ba) as the main indicators of the geographic origin of mango. The results of this study revealed the feasibility of tracing the provenance of mango through either of the two analytical strategies or by combining them to obtain a more accurate classification.

Umbrian tobacco of the Virginia Bright variety is one of the most appreciated tobaccos in Europe, and one characterized by an excellent yield. In recent years, the Umbria region and local producers have invested in introducing novel practices (for production and processing) focused on environmental, social, and economic sustainability. Due to this, tobacco from Umbria is a leading commodity in the global tobacco industry, and it claims a high economic value. The aim of this study is then to assess if elemental and isotopic compositions can be used to protect the quality and geographical traceability of this particular tobacco. For the first time the characteristic value ranges of the stable isotope ratios of the bio-elements as a whole (δ2H, δ13C, δ15N, δ18O, and δ34S) and of the concentration of 56 macro- and micro-elements are now available, determined in Virginia Bright tobacco produced in two different areas of Italy (Umbria and Veneto), and from other worldwide geographical regions. The ranges of variability of elements and stable isotope ratios had slightly different results, according to the three geographical origins considered. In particular, Umbria samples presented significantly lower content of metals potentially dangerous for human health. The results of this first exploratory work highlight the possibility of characterizing tobacco from Umbria, and suggest widening the scope of the survey throughout Italy and foreign regions, in order to be used to describe the geographical origin of tobacco in general and verify the origin of the products on the market.