Integration and holistic analysis of multiple multidimensional soil data sets.

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.

Elucidation of arsenic detoxification mechanism in Marchantia polymorpha: The role of ACR3.

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.

Discriminative power of DNA-based, volatilome, near infrared spectroscopy, elements and stable isotopes methods for the origin authentication of typical Italian mountain cheese using sPLS-DA modeling.

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.

Impact of forest disturbance on microarthropod communities depends on underlying ecological gradients and species traits.

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.

Evaluating the potential of hydrochar as a soil amendment.

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.

Alkaloid profiling of Italian alpine herbs using high resolution mass spectrometry (Orbitrap-MS).

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).

Spatial and Seasonal Structure of Bacterial Communities Within Alpine Vineyards: Trentino as a Case Study.

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/cm2, 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.

Matured Manure and Compost from the Organic Fraction of Solid Waste Digestate Application in Intensive Apple Orchards.

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.

Elemental and Isotopic Characterization of Tobacco from Umbria.

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.

Microbiome of vineyard soils is shaped by geography and management.

BACKGROUND: Despite their importance as a reservoir of biodiversity, the factors shaping soil microbial communities and the extent by which these are impacted by cultivation are still poorly understood. Using 16S rRNA gene and ITS sequencing, we characterized the soil microbiota of vineyards and of neighboring permanent grassland soils in the Italian province of Trentino, and correlated their structure and composition to location, chemical properties of the soil, and land management. RESULTS: Bacterial communities had a core of conserved taxa accounting for more than 60% of the reads of each sample, that was influenced both by geography and cultivation. The core fungal microbiota was much smaller and dominated by geography alone. Cultivation altered the structure and composition of the soil microbiota both for bacteria and fungi, with site-specific effects on their diversity. The diversity of bacterial and fungal communities was generally inversely correlated across locations. We identified several taxa that were impacted by the chemical properties and texture of the soil. CONCLUSIONS: Our results highlight the different responses of bacterial and fungal communities to environmental factors and highlight the need to characterize both components of the soil microbiota to fully understand the factors that drive their variability.

Isotopic and elemental profiles of Mediterranean buffalo milk and cheese and authentication of Mozzarella di Bufala Campana PDO: An initial exploratory study.

Mozzarella di Bufala Campana (MBC) is a PDO cheese produced from whole buffalo milk in specific regions of southern Italy. Due to the high price and the limited amount of buffalo milk, MBC is potentially subject to mislabelling. Stable isotope ratio analysis combined with elemental analysis is one powerful technique for detecting the authenticity of PDO cheeses. Here, the elemental and isotopic profiles of authentic samples of buffalo milk and the corresponding MBC samples collected in the reference area in winter and summer are presented in an initial exploratory study. By merging MBC-PDO samples with non-PDO samples of buffalo mozzarella produced both inside and outside the reference area, a model was developed to classify product categories for this cheese. Despite the differences occurring during processing, along with differences in the season and production area, the model was effective in distinguishing PDO and non-PDO mozzarella, particularly when non-PDO cheeses were made outside the MBC reference area.

Characterisation and geographical traceability of Italian goji berries.

This work characterises and distinguishes Italian and Asian goji berries, combining three analytical approaches to determine 5 light stable isotope ratios, 57 mineral elements and 14 carotenoids. The study analyses stable isotope ratios and provides full HPLC-DAD-MS characterisation of the carotenoid fraction in goji berries for the first time. The isotopic composition of Italian berries was characterised by δ13C values ranging between -28.2‰ and -25.7‰, δ15N between 2.3 and 7.3‰, δ34S between 2.6 and 7.9‰, and D/H between -112 and -49‰. Italian goji berries were shown to have a particular composition and interesting nutritional characteristics, specifically a higher total carotenoid content (355 mg/100 g dw mean content vs 198 mg/100 g dw) and higher content of some micronutrients: K, B, Cu, Mo, Se, and Zn. On the basis of δ34S, Y, U, Cu, Rb, zeaxanthin palmitate and total carotenoid content, discriminant analysis correctly classified all samples according to their geographical origin.

Botanical origin characterisation of tannins using infrared spectroscopy.

Different approaches to analysing the botanical origin of tannins have been proposed in the last fifteen years, but are generally time consuming and require the use of advanced instrumentation. This study aims to suggest an effective, easy, rapid and cheap method based on the acquisition of FT-IR spectra of 3g/L hydroalcoholic tannin solutions, overcoming possible disadvantages due to sample or particle size inhomogeneity. 114 commercial powder tannins from 7 different botanical sources (oak, chestnut, gall, quebracho, tea, grape skin and grape seed) were collected and the FT-IR spectra were acquired in the region 926-5011cm-1. Partial Least Squares regression, Discriminant Analysis and Artificial Neural Networks were applied to FT-IR spectra to investigate the possibility of differentiating the 7 botanical origins. The best results were obtained using Discriminant Analysis, with 95% correct re-classification, and 97% grouping of grape skin and seed in a single source.

The impact of different barrel sanitation approaches on the spoilage microflora and phenols composition of wine.

Careful control of spoilage microflora inside wine containers is a key issue during winemaking. To date, attention has been paid to the development of an effective protocol for the eradication of spoilage agents, especially Brettanomyces, from barrels. Few studies have taken into account the modifications caused by sanitation treatments in wine and wood barrels. In the present study the effects of two sanitation treatments (ozone and sodium hydroxide) on barrel spoilage microflora and the composition of the wine stored inside them were evaluated. The phenols of wine (38 compounds) were characterised using a UHPLC-MS during the first 3 months of wine ageing, to see possible alterations in composition due to the chemical exchange from wood to wine in presence of sanitising agents. With the same scope, a panel of 13 judges carried out sensorial analysis of wines. The results showed that the tested treatments had little effect on the organoleptic characteristics of wines, but underline the different performance of the sanitation treatments in terms of eradicating microorganisms.

δ(15) N from soil to wine in bulk samples and proline.

The feasibility of using δ(15) N as an additional isotopic marker able to link wine to its area of origin was investigated. The whole production chain (soil-leaves-grape-wine) was considered. Moreover, the research included evaluation of the effect of the fermentation process, the use of different types of yeast and white and red vinification, the addition of nitrogen adjuvants and ultrasound lysis simulating wine ageing. The δ(15) N of grapes and wine was measured in bulk samples and compounds, specifically in proline, for the first time. Despite isotopic fractionation from soil to wine, the δ(15) N values of leaves, grapes, wine and particularly must and wine proline conserved the variability of δ(15) N in the growing soil. Fermentation and ultrasound treatment did not affect the δ(15) N values of grape must, which was therefore conserved in wine. The addition of inorganic or organic adjuvants was able to influence the δ(15) N of bulk wine, depending on the amount and the difference between the δ(15) N of must and that of the adjuvant. The δ(15) N of wine proline was not influenced by adjuvant addition and is therefore the best marker for tracing the geographical origin of wine. Copyright © 2016 John Wiley & Sons, Ltd.

Verifying the botanical authenticity of commercial tannins through sugars and simple phenols profiles.

Commercial tannins from several botanical sources and with different chemical and technological characteristics are used in the food and winemaking industries. Different ways to check their botanical authenticity have been studied in the last few years, through investigation of different analytical parameters. This work proposes a new, effective approach based on the quantification of 6 carbohydrates, 7 polyalcohols, and 55 phenols. 87 tannins from 12 different botanical sources were analysed following a very simple sample preparation procedure. Using Forward Stepwise Discriminant Analysis, 3 statistical models were created based on sugars content, phenols concentration and combination of the two classes of compounds for the 8 most abundant categories (i.e. oak, grape seed, grape skin, gall, chestnut, quebracho, tea and acacia). The last approach provided good results in attributing tannins to the correct botanical origin. Validation, repeated 3 times on subsets of 10% of samples, confirmed the reliability of this model.

Validation of methods for H, C, N and S stable isotopes and elemental analysis of cheese: results of an international collaborative study.

RATIONALE: PDO cheeses, such as Parmigiano Reggiano and Grana Padano, which cost more than double generic similar cheeses, must be protected against mislabelling. The aim of this study was to validate the methods for the isotopic and elemental analysis of cheese, in order to support official recognition of their use in authenticity assessment. METHODS: An international collaborative study based on blind duplicates of seven hard cheeses was performed according to the IUPAC protocol and ISO Standards 5725/2004 and 13528/2005. The H, C, N and S stable isotope ratios of defatted cheese determined using Isotope Ratio Mass Spectrometry (IRMS) and the content of Li, Na, Mn, Fe, Cu, Se, Rb, Sr, Mo, Ba, Re, Bi, U in cheese after acid microwave digestion using Inductively Coupled Plasma Mass Spectrometry or Optical Emission Spectrometry (ICP-MS or -OES) were measured in 13 different laboratories. RESULTS: The average standard deviations of repeatability (sr) and reproducibility (sR) were 0.1 and 0.2 ‰ for δ(13)C values, 0.1 and 0.3 ‰ for δ(15)N values, 2 and 3 ‰ for δ(2)H values, and 0.4 and 0.6 ‰ for δ(34)S values, thus comparable with results of official methods and the literature for other food matrices. For elemental data, the average RSDr and RSDR values ranged between 2 and 11% and between 9 and 28%, respectively, consistent with methods reported by the FDA and in the literature for cheese. CONCLUSIONS: The validation data obtained here can be submitted to the standardisation agencies to obtain official recognition for the methods, which is fundamental when they are used in commercial disputes and legal debates.

Silicification of wood adopted for barrel production using pure silicon alkoxides in gas phase to avoid microbial colonisation.

The paper presents a new approach, covering wood with silica-based material in order to protect it from spoilage due to microbial colonisation and avoiding the loss of the natural features of the wood. Wood specimens derived from wine barrels were treated with methyltriethoxysilane in gas phase, leading to the deposition of a silica nanofilm on the surface. (29)Si and (13)C solid state Nuclear Magnetic Resonance and Scanning Electron Microscope-Energy Dispersive X-ray analysis observations showed the formation of a silica polymeric film on the wood samples, directly bonding with the wood constituents. Inductively Coupled Plasma-Mass Spectroscopy quantification of Si showed a direct correlation between the treatment time and silica deposition on the surface of the wood. The silica-coated wood counteracted colonisation by the main wine spoilage microorganisms, without altering the migration from wood to wine of 21 simple phenols measured using a HPLC-Electrochemical Coulometric Detection.

Botanical traceability of commercial tannins using the mineral profile and stable isotopes.

Commercial tannins are natural polyphenolic compounds extracted from different plant tissues such as gall, the wood of different species and fruit. In the food industry they are mainly used as flavourings and food ingredients, whereas in winemaking they are classified as clarification agents for wine protein stabilisation, although colour stabilisation, metal removal, unpleasant thiol removal and rheological correction are also well-known and desired effects. Due to their particular technical properties and very different costs, the possibility of correct identification of the real botanical origin of tannins can be considered a primary target in oenology research and in fulfilling the technical and economic requirements of the wine industry. For some categories of tannins encouraging results have already been achieved by considering sugar or polyphenolic composition. For the first time this work verifies the possibility of determining the botanical origin of tannins on the basis of the mineral element profile and analysis of the (13) C/(12) C isotopic ratio. One hundred two commercial tannins originating from 10 different botanical sources (grapes, oak, gall, chestnut, fruit trees, quebracho, tea, acacia, officinal plants and tara) were analysed to determine 57 elements and the (13) C/(12) C isotopic ratio, using inductively coupled plasma mass spectrometry and isotope-ratio mass spectrometry, respectively. Forward stepwise discriminant analysis provided good discrimination between the 8 most abundant groups, with 100% correct re-classification. The model was then validated five times on subsets of 10% of the overall samples, randomly extracted, achieving satisfactory results. With a similar approach it was also possible to distinguish toasted and untoasted oak tannins as well as tannins from grape skin and grape seeds.

Use of Fourier transform infrared spectroscopy to create models forecasting the tartaric stability of wines.

Tartaric instability of wines still represents a serious problem in terms of the commercial value of bottled wines, particularly whites, leading consumers to be suspicious as regards the effective healthiness or wholesomeness of products. The study, carried out on 536 Italian wines, investigated the potential of using Fourier Transform Infrared Spectroscopy, distinguishing between white and red/rosé wines, to create models predicting the instability of wines, assessed in comparison to two of the most widespread methods of reference: the "mini-contact test" (10 min, 0 °C, KHT) and the "cooling test" (5 days, -4 °C). The models proposed, constructed using 80% of the samples and based on Partial Least Squares-Regression and Artificial Neural Networks, were shown to work well in terms of correct classification (from 89% to 97%) of the external validation subset (20%). As regards the more problematical question of technical management of wines before bottling, in the worst cases only 4-6% of unstable samples were erroneously classified as stable.