Investigating Metabolic Plant Response toward Deoxynivalenol Accumulation in Four Winter Cereals.

Deoxynivalenol (DON) is a phytotoxic agent supporting the spread of fungal diseases in cereals worldwide, i.e., fusarium head blight. It is known that DON accumulation may elicit changes in plant secondary metabolites in response to pathogen attack. This study maps the changes in selected secondary metabolite classes upon DON contamination occurring in fifteen Triticum spp. genotypes, among them emmer, spelt, and soft wheat, and 2 tritordeum varieties, cultivated in two different sites and over two harvest years. The main phenolic classes (i.e., alkylresorcinols, soluble, and cell-wall bound phenolic acids) were targeted analyzed, while changes in the lipidome signature were collected through untargeted HRMS experiments. The results, obtained across multiple Triticum species and in open fields, confirmed the modulation of first-line biological pathways already described in previous studies involving single cereal species or a limited germplasm, thus reinforcing the involvement of nonspecific chemical defenses in the plant response to pathogen attack.

Mycotoxin occurrence in kernels and straws of wheat, barley, and tritordeum.

Thirty-two varieties of common and durum wheat, hordeum, barley, and tritordeum collected over two harvesting years (2020 and 2021) were investigated for the presence of multiple Fusarium-related mycotoxins in asymptomatic plants. DON, 3-AcDON, 15-AcDON, T-2, HT-2, and ZEN together with the emerging mycotoxin ENN B and the major modified form of DON, namely DON3Glc, were quantified by means of UHPLC-MS/MS. Overall, DON and ENN B were the most frequently detected mycotoxins, albeit large inter-year variability was observed and related to different climate and weather conditions. Straws had higher mycotoxin contents than kernels and regarding DON occurrence tritordeum was found to be the most contaminated group on average for both harvesting years, while barley was the less contaminated one. Emerging mycotoxin ENN B showed comparable contents in kernels compared to straw, with a ratio close to 1 for tritordeum and barley. Regarding the occurrence of the other evaluated mycotoxins, T-2 and HT-2 toxins have been spotted in a few tritordeum samples, while ZEN has been frequently found only in straw from the harvesting year 2020. The data collected confirms the occurrence of multiple Fusarium mycotoxins in straws also from asymptomatic plants, highlighting concerns related to feed safety and animal health. The susceptibility of Tritordeum, hereby reported for the first time, suggests that careful measures in terms of monitoring, breeding, and cultural choices should be applied when dealing with his emerging crop.

Erucin Exerts Cardioprotective Effects on Ischemia/Reperfusion Injury through the Modulation of mitoKATP Channels.

Modulation of mitochondrial K channels represents a pharmacological strategy to promote cardioprotective effects. Isothiocyanates emerge as molecules capable of releasing hydrogen sulfide (H2S), an endogenous pleiotropic gasotransmitter responsible for anti-ischemic cardioprotective effects also through the involvement of mitoK channels. Erucin (ERU) is a natural isothiocyanate resulting from the enzymatic hydrolysis of glucosinolates (GSLs) present in Eruca sativa Mill. seeds, an edible plant of the Brassicaceae family. In this experimental work, the specific involvement of mitoKATP channels in the cardioprotective effect induced by ERU was evaluated in detail. An in vivo preclinical model of acute myocardial infarction was reproduced in rats to evaluate the cardioprotective effect of ERU. Diazoxide was used as a reference compound for the modulation of potassium fluxes and 5-hydroxydecanoic acid (5HD) as a selective blocker of KATP channels. Specific investigations on isolated cardiac mitochondria were carried out to evaluate the involvement of mitoKATP channels. The results obtained showed ERU cardioprotective effects against ischemia/reperfusion (I/R) damage through the involvement of mitoKATP channels and the consequent depolarizing effect, which in turn reduced calcium entry and preserved mitochondrial integrity.

Untargeted Metabolomics of Meat Digests: Its Potential to Differentiate Pork Depending on the Feeding Regimen.

Meat quality seems to be influenced by the dietary regimes applied for animal feeding. Several research studies are aimed at improving meat quality, preserving it from oxidative processes, by the incorporation of antioxidant components in animal feeding. The main part of these studies evaluates meat quality, determining different parameters directly on meat, while few research studies take into account what may happen after meat ingestion. To address this topic, in this study, an in vitro gastrointestinal digestion protocol was applied to two different pork muscles, longissimus dorsi and rectus femoris, obtained from pigs fed with different diets. In detail, two groups of 12 animals each were subjected to either a conventional diet or a supplemented diet with extruded linseeds as a source of omega-3 fatty acids and plant extracts as a source of phenolics antioxidant compounds. The digested meat was subjected to an untargeted metabolomics approach. Several metabolites deriving from lipid and protein digestion were detected. Our untargeted approach allowed for discriminating the two different meat cuts, based on their metabolomic profiles. Nonetheless, multivariate statistics allowed clearly discriminating between samples obtained from different animal diets. In particular, the inclusion of linseeds and polyphenols in the animal diet led to a decrease in metabolites generated from oxidative degradation reactions, in comparison to the conventional diet group. In the latter, fatty acyls, fatty aldehydes and oxylipins, as well as cholesterol and vitamin D3 precursors and derivatives, could be highlighted.

Occurrence of Ergot Alkaloids in Major and Minor Cereals from Northern Italy: A Three Harvesting Years Scenario.

Ergot alkaloids (EAs), mycotoxins produced mainly by fungi of the Claviceps genus, have been frequently reported in rye, while their increasingly frequent occurrence in other cereals is likely related to weather conditions, with the incidence of ergot sclerotia in winter grains being related to heavy rainfall and moist soils at critical periods. However, compared to other regulated mycotoxins, data about the prevalence and occurrence of EAs in major and minor cereals harvested in the Mediterranean growing areas are still scant. In this regard, the current study reported the occurrence of EAs in 18 genotypes of winter cereals harvested over 3 years from an experimental field located in North Italy which were analyzed by HPLC-MS/MS. Results indicate a widespread occurrence of all the major EAs in all the considered cereal crops, especially under supportive meteorological conditions. EA contamination was dependent on the harvest year (p < 0.0001) which was particularly high in 2020 for all the considered species. The results also demonstrated a large co-occurrence of EAs with 98 cereal samples out of 162 contaminated with at least one of the 12 EAs (60% positive samples) in the range LOD: 15,389 µg/kg (median value: 2.32 µg/kg), expressed as the sum of the EAs. Rye was confirmed to be the crop more susceptible to the fungal infection (EAs content up to 4,302 µg/kg). To the best of our knowledge, we have reported the accumulation of EAs in tritordeum (LOD: 15,389 µg/kg) and in emmer (LOD: 1.9 µg/kg) for the first time.

Hazelnut (Corylus avellana L.) shells as a potential source of dietary fibre: impact of hydrothermal treatment temperature on fibre structure and degradation compounds.

BACKGROUND: Hemicellulose extraction from lignocellulosic biomasses has gained interest over the years, and hydrothermal treatment is one of the most common methods employed for this purpose. This work aimed to deeply study hazelnut (Corylus avellana L.) shells as a new source of dietary fibre, evaluating the effect of hydrothermal treatment temperatures on the type and structure of fibre extracted, but also on the formation of side-products derived from lignocellulose degradation. RESULTS: Different process temperatures led to diverse polysaccharides in the hydrothermal extract. Pectin was identified for the first time in hazelnut shells when experimenting with extraction at 125 °C, whereas at 150 °C a heterogeneous mixture of pectin, xylan, and xylo-oligosaccharides was present. The highest yield in terms of total fibre was gained at 150 and 175 °C, and then decreased again at 200 °C. Finally, more than 500 compounds from different chemical classes were putatively identified and they appeared to be present in the extracted fibre with a different distribution and relative amount, depending on the heat treatment severity. A generally high content of phenols, phenyls, oligosaccharides, dehydro-sugars, and furans was observed. CONCLUSIONS: Modulation of the hydrothermal treatment temperature allows fibre extracts with very different compositions, and therefore different potential end uses, to be obtained from hazelnut shells. A sequential temperature-based fractionation approach, as a function of the severity of the extraction parameters, can also be considered. Nevertheless, the study of the side-compounds formed from lignocellulosic matrix degradation, as a function of the applied temperature, needs to be fully addressed for a safe introduction of the fibre extract within the food chain. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Implementing the Use of Collision Cross Section Database for Phycotoxin Screening Analysis.

The increased consumption of blue-green algae (BGA)-based dietary supplements has raised concern about their food safety, especially about cyanotoxin presence. The hyphenation of liquid chromatography with ion mobility mass spectrometry represents a relevant tool to screen several compounds in a large variety of food matrices. In this work, ultrahigh-performance liquid chromatography coupled to traveling wave ion mobility spectrometry/quadrupole time-of-flight mass spectrometry (UHPLC-TWIMS-QTOF) was employed to establish the first comprehensive TWIMS-derived collision cross section database (TWCCSN2) for phycotoxins. The database included 20 cyanotoxins and 1 marine toxin. Accurate m/z, retention times, and TWCCSN2 values were obtained for 81 adducts in positive and negative electrospray (ESI+/ESI-) modes. Reproducibility and robustness of the TWCCSN2 measurements were determined to be independent of the matrix. A screening was carried out on 19 commercial BGA dietary supplements of different composition. Cyanotoxins were confidently identified in five samples based on retention time, m/z, and TWCCSN2.

Perenniality, more than genotypes, shapes biological and chemical rhizosphere composition of perennial wheat lines.

Perennial grains provide various ecosystem services compared to the annual counterparts thanks to their extensive root system and permanent soil cover. However, little is known about the evolution and diversification of perennial grains rhizosphere and its ecological functions over time. In this study, a suite of -OMICSs - metagenomics, enzymomics, metabolomics and lipidomics - was used to compare the rhizosphere environment of four perennial wheat lines at the first and fourth year of growth in comparison with an annual durum wheat cultivar and the parental species Thinopyrum intermedium. We hypothesized that wheat perenniality has a greater role in shaping the rhizobiome composition, biomass, diversity, and activity than plant genotypes because perenniality affects the quality and quantity of C input - mainly root exudates - hence modulating the plant-microbes crosstalk. In support of this hypothesis, the continuous supply of sugars in the rhizosphere along the years created a favorable environment for microbial growth which is reflected in a higher microbial biomass and enzymatic activity. Moreover, modification in the rhizosphere metabolome and lipidome over the years led to changes in the microbial community composition favoring the coexistence of more diverse microbial taxa, increasing plant tolerance to biotic and abiotic stresses. Despite the dominance of the perenniality effect, our data underlined that the OK72 line rhizobiome distinguished from the others by the increase in abundance of Pseudomonas spp., most of which are known as potential beneficial microorganisms, identifying this line as a suitable candidate for the study and selection of new perennial wheat lines.

A multiplatform metabolomics/reactomics approach as a powerful strategy to identify reaction compounds generated during hemicellulose hydrothermal extraction from agro-food biomasses.

Hydrothermal treatment is commonly used for hemicelluloses extraction from lignocellulosic materials. In this study, we thoroughly investigated with a novel approach the metabolomics of degradation compounds formed when hazelnut shells are subjected to this type of treatment. Three different complementary techniques were combined, namely GC-MS, 1H NMR, and UHPLC-IM-Q-TOF-MS. Organic acids, modified sugars and aromatic compounds, likely to be the most abundant chemical classes, were detected and quantified by NMR, whereas GC- and LC-MS-based techniques allowed to detect many molecules with low and higher Mw, respectively. Furans, polyols, N-heterocyclic compounds, aldehydes, ketones, and esters appeared, among others. Ion mobility-based LC-MS method was innovatively used for this purpose and could allow soon to create potentially useful datasets for building specific databases relating to the formation of these compounds in different process conditions and employing different matrices. This could be a very intelligent approach especially in a risk assessment perspective.

Anthocyanin Content and Fusarium Mycotoxins in Pigmented Wheat (Triticum aestivum L. spp. aestivum): An Open Field Evaluation.

Twelve Triticum aestivum L. spp. aestivum varieties with pigmented grain, namely one red, six purple, three blue, and two black, were grown in open fields over two consecutive years and screened to investigate their risk to the accumulation of multiple Fusarium-related mycotoxins. Deoxynivalenol (DON) and its modified forms DON3Glc, 3Ac-DON, 15Ac-DON, and T-2, HT-2, ZEN, and Enniatin B were quantified by means of UHPLC-MS/MS, along with 14 different cyanidin, petunidin, delphinidin, pelargonidin, peonidin, and malvidin glycosides. A significant strong influence effect of the harvesting year (p = 0.0002) was noticed for DON content, which was more than doubled between harvesting years growing seasons (mean of 3746 µg kg-1 vs. 1463 µg kg-1). In addition, a striking influence of varieties with different grain colour on DON content (p < 0.0001) emerged in combination with the harvesting year (year×colour, p = 0.0091), with blue grains being more contaminated (mean of 5352 µg kg-1) and red grain being less contaminated (mean of 715 µg kg-1). The trend was maintained between the two harvesting years despite the highly variable absolute mycotoxin content. Varieties accumulating anthocyanins in the pericarp (purple coloration) had significantly lower DON content compared to those in which aleurone was involved (blue coloration).

Metabolite Profiling and Bioactivities of Leaves, Stems, and Flowers of Rumex usambarensis (Dammer) Dammer, a Traditional African Medicinal Plant.

The comprehensive identification of secondary metabolites represents a fundamental step for the assessment of bioactivities and pharmacological properties of traditional herbal drugs. Rumex usambarensis (Dammer) Dammer has been described as a multipurpose remedy in different African traditional pharmacopoeias, but its phytochemical profile has not been properly investigated. Herein we report a high throughput metabolomic screening, based on ultra-high performance liquid chromatography-travelling wave ion mobility spectrometry quadrupole time-of-flight (UHPLC-TWINS-QTOF), which was performed for the first time on different R. usambarensis plant parts. By applying high-resolution mass spectrometry-based metabolomics and chemometric analysis, a complete discrimination of different aerial parts was obtained, with the annotation of 153 significant metabolites in leaves, stems, and flowers, suggesting an easy authentication and discrimination route. Phytochemical data were correlated to antimicrobial and antioxidant properties. Flavonoids, benzopyranes, chromones, and xanthones derivatives, along with a richer phytocomplex, might be responsible for the stronger bioactivities obtained from flowers.

The B1080/B1192 molecular marker identifies hemp plants with functional THCA synthase and total THC content above legal limit.

In Cannabis sativa L. the presence of delta 9-tetrahydrocannabinolic acid (THCA) above legal limit is a challenging issue that still restricts the industrial exploitation of this promising crop. In recent years, the interest of entrepreneurs and growers who see hemp as a dynamic and profitable crop was joined by the growing knowledge on C. sativa genetics and genomics, accelerated by the application of high throughput tools. Despite the renewed interest in the species, much remains to be clarified, especially about the long-standing problem of THCA in hemp inflorescences, which could even result in the seizure of the whole harvest. Although several hypotheses have been formulated on the accumulation of this metabolite in industrial varieties, none is conclusive yet. In this work, individuals of a population of the hemp cultivar 'FINOLA' obtained from commercial seeds were investigated for total THC level and examined at molecular level. A marker linked to THCA synthase was found at a high incidence in both male and female plants, suggesting a considerable genetic variability within the seed batch. Full-length sequences encoding for putatively functional THCA synthases were isolated for the first time from the genome of both female and male plants of an industrial hemp variety and, using transcriptional analysis, the THCA synthase expression was quantified in mature inflorescences of individuals identified by the marker. Biochemical analyses finally demonstrated for these plants a 100% association between the predicted and actual chemotype.

Phylogeny and mycotoxin profiles of pathogenic Alternaria and Curvularia species isolated from date palm in southern Tunisia.

Date palm (Phoenix dactylifera L.), is a widely cultivated crop across North Africa, with about 300 thousand tons of fruits produced per year, in Tunisia. A wide range of fungal pathogens has been associated with leaf spots of date palm, Alternaria species being the most frequently reported. Symptomatic leaves of Deglet Nour variety were randomly collected in six localities in Tunisia. We used a polyphasic approach to identify 45 Alternaria and five Curvularia strains isolated from date palm, confirming their pathogenicity. Sequencing of allergen Alt-a1, glyceraldehyde-3-phosphate dehydrogenase (gpd) and calmodulin genes allowed us to group 35 strains in Alternaria Section, and 10 strains in Ulocladioides section. Based on sequencing analyses of Internal Transcribed Spacer, gpd and elongation factor genomic regions, all Curvularia strains were identified as Curvularia spicifera. All Alternaria and Curvularia species tested on date palm plantlets proved to be pathogenic, fulfilling Koch's postulates. Although no significant differences were observed among the species, the highest mean disease severity index was observed in A. arborescens, while the lowest corresponded to C. spicifera. The capability of these strains to produce mycotoxins in vitro was evaluated. None of the A. consortialis strains produced any known Alternaria mycotoxin, whereas more than 80% of the strains included in Alternaria section Alternaria produced variable amounts of multiple mycotoxins such as alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid and tentoxin. Curvularia spicifera strains produced detectable traces of fumonisins B. This work reports a first comprehensive multidisciplinary study of mycotoxigenic Alternaria species and C. spicifera associated with leaf spot disease on date palm.

Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples.

This work evaluates the potential of ion mobility spectrometry (IMS) to improve the analytical performance of current liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determination of ergot alkaloids (EAs) in cereal samples. Collision cross section (CCS) values for EA epimers are reported for the first time to contribute to their unambiguous identification. Additionally, CCS values have been inter-laboratory cross-validated and compared with CCS values predicted by machine-learning models. Slight differences were observed in terms of CCS values for ergotamine, ergosine and ergocristine and their corresponding epimers (from 3.3 to 4%), being sufficient to achieve a satisfactory peak-to-peak resolution for their unequivocal identification. A LC-travelling wave ion mobility (TWIM)-MS method has been developed for the analysis of EAs in barley and wheat samples. Signal-to-noise ratio (S/N) was improved between 2.5 and 4-fold compared to the analog LC-TOF-MS method. The quality of the extracted ion chromatograms was also improved by using IMS.

Assessing food authenticity through protein and metabolic markers.

This chapter aims to address an issue of ancient origins, but more and more topical in a globalized world in which consumers and stakeholders are increasingly aware: the authenticity of food. Foods are systems that can also be very complex, and verifying the correspondence between what is declared and the actual characteristics of the product is often a challenging issue. The complexity of the question we want to answer (is the food authentic?) means that the answer is equally articulated and makes use of many different analytical techniques. This chapter will consider the chemical analyses of foods aimed at guaranteeing their authenticity and will focus on frontier methods that have been developed in recent years to address the need to respond to ever-increasing guarantees of authenticity. Targeted and non-targeted approaches will be considered for verifying the authenticity of foods, through the study of different classes of constituents (proteins, metabolites, lipids, flavors). The numerous approaches available (proteomics, metabolomics, lipidomics) and the related analytical techniques (LC-MS, GC-MS, NMR) are first described from a more general point of view, after which their specific application for the purposes of authentication of food is addressed.

An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance.

KEY MESSAGE: pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes. Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be upregulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences, respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results does not confirmed pKFDV02 as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

A molecular insight into the lipid changes of pig Longissimus thoracis muscle following dietary supplementation with functional ingredients.

In this work, the Longissimus thoracis pig skeletal muscle was used as a model to investigate the impact of two different diets, supplemented with n-3 polyunsaturated fatty acids from extruded linseed (L) and polyphenols from grape skin and oregano extracts (L+P), on the lipidomic profile of meat. A standard diet for growing-finishing pigs (CTRL) was used as a control. Changes in lipids profile were investigated through an untargeted lipidomics and transcriptomics combined investigation. The lipidomics identified 1507 compounds, with 195 compounds fitting with the MS/MS spectra of LipidBlast database. When compared with the CTRL group, the L+P diet significantly increased 15 glycerophospholipids and 8 sphingolipids, while the L diet determined a marked up-accumulation of glycerolipids. According to the correlations outlined between discriminant lipids and genes, the L diet may act preventing adipogenesis and the related inflammation processes, while the L+P diet promoted the expression of genes involved in lipids' biosynthesis and adipogenic extracellular matrix formation and functioning.

Evolutionary Wheat Populations in High-Quality Breadmaking as a Tool to Preserve Agri-Food Biodiversity.

Plant biodiversity preservation is one of the most important priorities of today's agriculture. Wheat (Triticum spp. L.) is widely cultivated worldwide, mostly under a conventional and monovarietal farming method, leading to progressive biodiversity erosion. On the contrary, the evolutionary population (EP) cultivation technique is characterized by mixing and sowing together as many wheat genotypes as possible to allow the crop to genetically adapt over the years in relation to specific pedoclimatic conditions. The objective of this study was to assess the nutritional, chemical and sensory qualities of three different breads obtained using different organic EP flours, produced following a traditional sourdough process and compared to a commercial wheat cultivar bread. Technological parameters, B-complex vitamins, microelements, dietary fibre and phenolic acids were determined in raw materials and final products. Flours obtained by EPs showed similar characteristics to the commercial wheat cultivar flour. However, significant differences on grain technological quality were found. The breads were comparable with respect to chemical and nutritional qualities. Overall, the sensory panellists rated the tasted breads positively assigning the highest score to those produced with EPs flours (6.75-7.02) as compared to commercial wheat cultivar-produced bread (cv. Bologna, 6.36).

Mass Spectrometry Imaging Disclosed Spatial Distribution of Defense-Related Metabolites in Triticum spp.

Fusarium Head Blight is the most common fungal disease that strongly affects Triticum spp., reducing crop yield and leading to the accumulation of toxic metabolites. Several studies have investigated the plant metabolic response to counteract mycotoxins accumulation. However, information on the precise location where the defense mechanism is taking place is scarce. Therefore, this study aimed to investigate the specific tissue distribution of defense metabolites in two Triticum species and use this information to postulate on the metabolites' functional role, unlocking the "location-to-function" paradigm. To address this challenge, transversal cross-sections were obtained from the middle of the grains. They were analyzed using an atmospheric-pressure (AP) SMALDI MSI source (AP-SMALDI5 AF, TransMIT GmbH, Giessen, Germany) coupled to a Q Exactive HF (Thermo Fisher Scientific GmbH, Bremen, Germany) orbital trapping mass spectrometer. Our result revealed the capability of (AP)-SMALDI MSI instrumentation to finely investigate the spatial distribution of wheat defense metabolites, such as hydroxycinnamic acid amides, oxylipins, linoleic and α-linoleic acids, galactolipids, and glycerolipids.