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.

Influence of Agronomic Practices on the Antioxidant Compounds of Pigmented Wheat (Triticum aestivum spp. aestivum L.) and Tritordeum (× Tritordeum martinii A. Pujadas, nothosp. nov.) Genotypes.

Twelve pigmented wheat genotypes, one tritordeum, and one common wheat were grown in three field experiments under varying nitrogen (N) fertilization rates to investigate the contributions of genotype, environment, and fertilization on the levels of phenolic acids, anthocyanins, carotenoids and antioxidant capacity of the grains. Soluble phenolic acids increased significantly (+16%) in the environment with high soil N content, while bound phenolic acids and anthocyanins decreased (-16 and -57%). N fertilization affected the agronomic and qualitative traits but had limited effects on some bioactive compounds (bound phenolic acids and anthocyanins). The greatest differences appeared among the color groups and within the same color types, with the black group showing the most anthocyanins and phenolic acids (34.4 and 1207 mg·kg-1) and the highest antioxidant capacity. Some of the cultivars could be promising for the development of innovative supply chains and the production of functional foods, as they showed good yield and quality performances, and good antioxidant features.

The cultivation of rye in marginal Alpine environments: a comparison of the agronomic, technological, health and sanitary traits of local landraces and commercial cultivars.

Rye is a secondary crop that is characterized by a higher tolerance to climatically less favorable conditions than other cereal species. For this reason, rye was historically used as a fundamental raw material for bread production and as a supply of straw in northern parts of Europe as well as in mountain environments, such as Alpine valleys, where locally adapted landraces have continued to be cultivated over the years. In this study, rye landraces collected in different valleys in the Northwest Italian Alps have been selected as the most genetically isolated within their geographical contexts and cultivated in two different marginal Alpine environments. The traits concerning their agronomy, mycotoxin contamination, bioactive content, as well as their technological and baking quality were assessed to characterize and compare rye landraces with commercial wheat and rye cultivars. Rye cultivars showed the same grain yield level as wheat in both environments. Only the genotype selected from the Maira Valley was characterized by tall and thin culms and a proneness to lodging, thereby resulting in a lower yield capacity. Among the rye cultivars, the hybrid one presented the highest yield potential, but also the highest susceptibility to the occurrence of ergot sclerotia. However, the rye cultivars, especially the landraces, were characterized by higher concentrations of minerals, soluble fibers, and soluble phenolic acids, and thus both their flours and breads had superior antioxidant properties. A 40% substitution of refined wheat flour with whole-grain rye flour led to a higher dough water absorption and a lower stability, thereby resulting in lower loaf volumes and darker products. Agronomically and qualitatively speaking, the rye landraces diverged significantly from the conventional rye cultivars, thus reflecting their genetic distinctiveness. The landrace from the Maira Valley shared a high content in phenolic acids and good antioxidant properties with the one from the Susa Valley and, when combined with wheat flour, turned out to be the most suitable for bread making. Overall, the results have highlighted the suitability of reintroducing historic rye supply chains, based on the cultivation of local landraces in marginal environments and the production of value-added bakery goods.

Effect of enrichment of rice snacks with pulse seed coats on phenolic compound content, product features and consumer hedonic response.

This study investigated the impact of seed coats from peas (PC) and chickpeas (CC) (at 15 % and 30 % levels) on rice-based co-extruded snacks. Using PC and CC reduced the content of soluble (29 %) and cell-wall bound phenolic acids (21 %), but it enhanced the amount and the profile of flavonoids of rice-based snacks (up to 16 times with PC), resulting in significantly higher antioxidant activity (134 %). Snacks with 15 % CC showed a higher section area (about 335 versus 191 mm2) and a lower average pore radius (20.1 versus 23.9 mm) than PC-snacks; however, such features did not affect either texture or porosity. At 30 % level, PC resulted in a more porous structure (porosity: 73.1 versus 66.7 %) with smaller pores (17.2 versus 27.3 mm) and high firmness (55.9 versus 40.1 N). Consumers' acceptability evaluation revealed that samples containing pulse seed coat were comparable and preferred to the control (i.e., 100 % polished rice).

Pulse type and extrusion conditions affect phenolic profile and physical properties of extruded products.

The suitability of chickpea, red lentil and green pea for the production of extruded products (i.e., snacks and pasta) was studied. Besides starch and technological properties, the impact of processing on some bioactive compounds was evaluated. The best results were obtained for lentil: the snacks showed the lowest porosity (21 %), the highest average pore radius (18.8 µm), and high expansion (section area: 310 mm2; inner area: 114 mm2), while pasta exhibited low cooking loss (5.7 g 100 g-1) and high firmness (924 N). Extrusion-cooking reduced the soluble phenolic acid content (-45 %) and flavonoids (-41 %), but increased the cell-wall bound phenolic acids and antioxidant activity. The different pulses did not lead to a marked difference in the antioxidant activity of the extruded products, although the lentil maintained the highest flavonoid content after both processes.

The Effect of Foliar Fungicide and Insecticide Application on the Contamination of Fumonisins, Moniliformin and Deoxynivalenol in Maize Used for Food Purposes.

The fungal ear rot of maize cultivated in temperate areas is mainly due to the Fusarium species. The use of insecticides against European Corn Borer (ECB) reduces the severity of fungal ear rot as well as the fumonisin (FB) and moniliformin (MON) levels in maize kernels at harvest, which in turn results in a lowering of their effect on deoxynivalenol (DON) control. However, the direct fungicidal control of ear rot has rarely been implemented for maize, and the first studies reported conflicting results on the reduction of mycotoxins. In the present experiment, field trials were carried out in North Italy over three growing seasons to study the effect of fungicide application timings on maize to control mycotoxins, considering the interaction of the application with the insecticide treatment, according to a full factorial split plot design. The mycotoxin content was determined through LC-MS/MS analysis. The field trials showed a significant reduction in ECB severity (75%), fungal ear rot severity (68%), Fusarium Liseola section infection (46%), FBs (75%) and MON (79%) as a result of the insecticide application for all the years, while the DON content increased by 60%. On the other hand, a fungicide application alone or applied in plots protected by an insecticide was never effective for the fungal symptoms, infection or mycotoxin content. The results confirm that a correct insecticide application to control ECB damage is the most effective agrochemical solution for the control of fungal ear rot, FBs and MON.

5-n-Alkylresorcinol Profiles in Different Cultivars of Einkorn, Emmer, Spelt, Common Wheat, and Tritordeum.

5-n-Alkylresorcinols (AR) are bioactive compounds found in the edible parts of many cereals. Here, saturated and unsaturated homologues, including the oxidized forms 5-(2'-oxo) AR and their plant metabolites, were profiled by ultrahigh-performance liquid chromatography-ion mobility separation-high-resolution mass spectrometry in 18 cultivars of einkorn, emmer, spelt, common wheat, and tritordeum, cultivated in two consecutive years under uniform agronomic conditions. The average content of AR ranged between 672.5 ± 129.8 and 1408.9 ± 528.0 mg/kg, exceeding 2380 mg/kg in some samples and highlighting a superior content in tritordeum and in modern cultivars with respect to old wheat genotypes. By evaluating the effect of environmental and agronomic factors on the different variables, the harvest year resulted to be always significant, while location and variety influenced AR abundance only for some homologues. Furthermore, the spatial distribution of AR was investigated by mass spectrometry imaging using transversal cross sections of wheat kernels. Our results show that AR homologues are mainly localized in the testa and in the outer pericarp of wheat kernels.

Influence of H2O2-Induced Oxidative Stress on In Vitro Growth and Moniliformin and Fumonisins Accumulation by Fusarium proliferatum and Fusarium subglutinans.

Fusarium proliferatum and Fusarium subglutinans are common pathogens of maize which are known to produce mycotoxins, including moniliformin (MON) and fumonisins (FBs). Fungal secondary metabolism and response to oxidative stress are interlaced, where hydrogen peroxide (H2O2) plays a pivotal role in the modulation of mycotoxin production. The objective of this study is to examine the effect of H2O2-induced oxidative stress on fungal growth, as well as MON and FBs production, in different isolates of these fungi. When these isolates were cultured in the presence of 1, 2, 5, and 10 mM H2O2, the fungal biomass of F. subglutinans isolates showed a strong sensitivity to increasing oxidative conditions (27-58% reduction), whereas F. proliferatum isolates were not affected or even slightly improved (45% increase). H2O2 treatment at the lower concentration of 1 mM caused an almost total disappearance of MON and a strong reduction of FBs content in the two fungal species and isolates tested. The catalase activity, surveyed due to its crucial role as an H2O2 scavenger, showed no significant changes at 1 mM H2O2 treatment, thus indicating a lack of correlation with MON and FB changes. H2O2 treatment was also able to reduce MON and FB content in certified maize material, and the same behavior was observed in the presence and absence of these fungi, highlighting a direct effect of H2O2 on the stability of these mycotoxins. Taken together, these data provide insights into the role of H2O2 which, when increased under stress conditions, could affect the vegetative response and mycotoxin production (and degradation) of these fungi.

High-amylose corn in gluten-free pasta: Strategies to deliver nutritional benefits ensuring the overall quality.

High-amylose corn alone or in combination (25% and 50%) with conventional corn was used to produce gluten-free pasta. Flour pre-gelatinization in a tank (process A) or on a conveyor belt (process B) were tested. Resistant starch (RS), soluble (SPAs) and cell-wall bound phenolic acids (CWBPAs) and antioxidant capacity were significantly higher in high-amylose corn pasta. Cooked pasta from process B showed a higher SPA concentration, likely due to the lower cooking loss. The structure of pasta prepared with process B was more homogeneous, whereas it was more compact in the case of process A, as shown by a lower starch susceptibility to α-amylase hydrolysis, higher beginning of gelatinization temperature and lower water absorption. 25% HA represents a good compromise between high RS (4.2%) and good cooking behavior. At higher HA levels, process B is more suitable to obtain pasta with a better cooking quality.

Fate of regulated, masked, emerging mycotoxins and secondary fungal metabolites during different large-scale maize dry-milling processes.

The use of maize in the food chain could be mainly limited due to its contamination by mycotoxins. As scarce information is available, the current study is aimed at collecting new data on the co-occurrence and the fate of the most frequent masked, modified and emerging mycotoxins and other second fungal metabolites in maize food products and by-products. Three maize lots, obtained in different growing seasons, were processed using two different degermination processes, a dry-degermination system or a tempering-degermination one, in order to compare the interaction between mycotoxins and the dry-milling management system. Whole grain before and after cleaning, and all the products and the by-products were sampled twice for each lot and were subjected to a multi-mycotoxin LC-MS/MS analysis. More than 30 mycotoxins and other fungal metabolites, including masked or modified forms, co-occurred in all the maize milling fractions. Grain cleaning reduced all the detected fungal metabolites by 1.2-2 times, compared to the grain before cleaning. Animal feed flour showed the highest content of almost all the mycotoxins and fungal metabolites, with a consequent negative impact on animal health. Considering that for all the mycotoxins and fungal metabolites an inverse relationship with particle size was observed, flaking grits represented the healthiest maize products with the least contamination level, while the abatement was always lower for maize flour. Furthermore, the metabolites were variably redistributed in the maize fractions. The total aflatoxins, kojic acid, deoxynivalenol and its modified form, culmorin, and its associated forms, butenolide, fusaproliferin, fusaric acid, fusarinolic acid and, in some cases, zearalenone and its modified forms, and fusarin C were found to be concentrated significantly in the germ. Some of them also had a greater permanence in the maize food fractions and a weaker decontamination, both of which point to a higher risk of exposure for the end consumers. The co-occurrence of a such a high number of mycotoxins and fungal metabolites and their different fates during the dry-milling process have never been described before.

The effect of the amylose content and milling fractions on the physico-chemical features of co-extruded snacks from corn.

The suitability of corn fractions (break meal: 250-500 µm; flour: < 150 µm) from hybrids with different amylose contents (conventional: 18%; high-amylose: 42%; waxy: 2%) and their blends, to produce co-extruded snacks was assessed. Corn flour exhibited a higher content in total soluble phenolic acids (+34%) than break meal. The high-amylose hybrid maintained a higher antioxidant capacity and phenolic acid content (+52% for soluble and + 54% for cell-wall bound phenolic acids), even after extrusion, than the conventional one. Because of its gelatinization properties (high pasting and peak temperatures; low maximum viscosity), the high-amylose hybrid produced co-extruded snacks characterized by low section areas and large inner areas. The blends led to snacks whose features (sections and inner areas, porosity and hardness) did not follow a linear trend with the amylose content, suggesting the need for further studies to better understand the starch interactions that take place among the various hybrids.

Fumonisin Distribution in Maize Dry-Milling Products and By-Products: Impact of Two Industrial Degermination Systems.

In temperate areas, the main limitation to the use of maize in the food chain is its contamination by B-series fumonisins (FBs) during cultivation. Since the content of this group of mycotoxins may be distributed unevenly after milling, the aim of this study was to compare the distribution of FBs in maize fractions derived from two industrial dry-milling processes, that is, a dry-degermination (DD) system and a tempering-degermination (TD) system. Grain cleaning reduces FBs by about 42%. The germ of the two degermination processes showed a similar FB content of kernel after cleaning. Conversely, an animal feed flour resulted in a FB content that was two times higher than whole grain before cleaning. A significant FB reduction was observed in the milling fractions in both processes, with a higher reduction in the TD system than in the DD one. The average decontamination respect to uncleaned kernels in the DD process was 50%, 83% and 87%, for maize flour, break meal and pearl meal, respectively, while it was 78%, 88% and 94% in the TD process for small, medium and flaking grits, respectively. Among the milling fractions, the flaking grits with the highest particle size resulted in the highest FB reduction.

Influence of Agricultural Management on Phytochemicals of Colored Corn Genotypes ( Zea mays L.). Part 1: Nitrogen Fertilization.

In this study, the influence of nitrogen (N) fertilization (170 versus 300 kg of N/ha) on the content of bioactive compounds of whole-meal flour of 10 different colored corn genotypes was investigated. Considerable differences in antioxidant capacity and phytochemical concentrations were observed among genotypes. Higher N fertilization rates significantly ( p < 0.05) increased the content of both total cell-wall-bound phenolics and xanthophylls (lutein and zeaxanthin). Nevertheless, the main phenolic acids (ferulic, p-coumaric, and sinapic acids) as well as the antioxidant capacity and content of ß-cryptoxanthin, ß-carotene, and total anthocyanins did not show significant differences as far as the N fertilization rate is concerned. For corn cultivation, the application of high N fertilization rates, generally carried out to obtain higher grain yields, could positively influence the content of some bioactives particularly in years characterized by high rainfall levels responsible for N leaching from the soil.

Role of the European corn borer (Ostrinia nubilalis) on contamination of maize with 13 Fusarium mycotoxins.

The European corn borer (ECB) plays an important role in promoting Fusarium verticillioides infections and in the consequent fumonisin contamination in maize grain in temperate areas. The objective of this study was to evaluate whether the ECB feeding activity could also affect the occurrence of emerging mycotoxins in maize kernels. During the 2008-10 period, natural infestation of the insect was compared, in field research, with the protection of infestation, which was obtained by using an entomological net. The ears collected in the protected plots were free from ECB attack, while those subject to natural insect attacks showed a damage severity that varied from 10% to 25%. The maize samples were analysed by means of an LC-MS/MS-based multi-mycotoxin method, which led to the detection of various metabolites: fumonisins (FUMs), fusaproliferin (FUS), moniliformin (MON), bikaverin (BIK), beauvericin (BEA), fusaric acid (FA), equisetin (EQU), deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-G), zearalenone (ZEA), culmorin (CULM), aurofusarin (AUR) and butenolide (BUT). The occurrence of mycotoxins produced by Fusarium spp. of Liseola section was affected significantly by the ECB feeding activity. The presence of ECB injuries increased the FUMs from 995 to 4694 µg kg(-1), FUS from 17 to 1089 µg kg(-1), MON from 22 to 673 µg kg(-1), BIK from 58 to 377 µg kg(-1), BEA from 6 to 177 µg kg(-1), and FA from 21 to 379 µg kg(-1). EQU, produced by F. equiseti section Gibbosum, was also increased by the ECB activity, by 1-30 µg kg(-1) on average. Instead, the content of mycotoxins produced by Fusarium spp. of Discolor and Roseum sections was not significantly affected by ECB activity. As for FUMs, the application of a strategy that can reduce ECB damage could also be the most effective solution to minimise the other mycotoxins produced by Fusarium spp. of Liseola section.

Development of a rapid LC-DAD/FLD method for the simultaneous determination of auxins and abscisic acid in plant extracts.

Plant hormones play a crucial role in controlling plant growth and development. These groups of naturally occurring substances trigger physiological processes at very low concentrations, which mandate sensitive techniques for their quantitation. This paper describes a method to quantify endogenous (±)-2-cis-4-trans-abscisic acid, indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid. The method combines high-performance liquid chromatography (HPLC) with diode array and fluorescence detection in a single run. Hybrid tea rose 'Monferrato' matrices (leaves, petals, roots, seeds, androecium, gynoecium, and pollen) were used as references. Rose samples were separated and suspended in extracting methanol, after which (±)-2-cis-4-trans-abscisic acid and auxins were extracted by solvent extraction. Sample solutions were added first to cation solid phase extraction (SPE) cartridges and the eluates to anion SPE cartridges. The acidic hormones were bound to the last column and eluted with 5% phosphoric acid in methanol. Experimental results showed that this approach can be successfully applied to real samples and that sample preparation and total time for routine analysis can be greatly reduced.

Moniliformin analysis in maize samples from North-West Italy using multifunctional clean-up columns and the LC-MS/MS detection method.

A fast clean-up method has been developed to purify maize extracts and to detect moniliformin (MON) in maize samples from North-West Italy over a four-year period (2008-2011). The method is based on the use of MycoSep® 240 Mon clean-up columns (Romer Labs®). Samples were extracted using acetonitrile/water (84:16, v/v), and the extracts were purified with previously described clean-up columns. The liquid chromatography-tandem mass spectrography (LC-MS/MS) analysis has been carried out by means of hydrophilic interaction chromatography (HILIC), combined with negative electrospray mass spectrometry. The method has a recovery of 76-91% (relative standard deviation, RSD%: 6-14%), a limit of detection (LOD) of 1 µg kg⁻¹ and a limit of quantification (LOQ) of 4 µg kg⁻¹. Naturally contaminated maize (108 samples) was analysed for MON content. The average percentages of positive samples was 93% with the following ranges (µg kg⁻¹): 33-2606 (2008);

Determination of maize kernel hardness: comparison of different laboratory tests to predict dry-milling performance.

BACKGROUND: Numerous foods are produced from maize, and grain hardness has been described to have an impact on grain end-use value, and in particular for dry-milling performance. RESULTS: Thirty-three samples of commercial hybrids have been analysed for test weight (TW), thousand-kernel weight (TKW), hard:soft endosperm ratio (H/S), milling time (MT) and total milling energy (TME) through the Stenvert hardness test, coarse:fine material ratio (C/F), break force (HF) and break energy (HWF) through the puncture test, floating test (FLT), kernel dimensions and sphericity (S), protein (PC), starch (SC), lipid (LC), ash (AC) content and amylose:amylopectin ratio (AS/AP).Total grit yield (TGY) has been obtained through a micromilling procedure and used to compare the efficiency of the tests to predict the dry-milling performance. TW, H/S, MT, TME, C/F, FLT, S, PC, SC and AS/AP were significantly correlated with each other. TW has been confirmed to be a simple estimator of grain hardness. Among the hardness tests, C/F was shown to be the best descriptor of maize milling ability, followed by FLT. A good correlation with TGY has also been observed with H/S, MT, TME and PC, while SC, S and AS/AP seem to play a minor role. The puncture test (HF and HWF) did not offer good indications on the impact of hardness on kernel grinding properties. CONCLUSION: This study can be considered as a contribution towards determining kernel properties which influence maize hardness measurement in relation to the end-use processing performance.

Management of fumonisin contamination in maize kernels through the timing of insecticide application against the European corn borer Ostrinia nubilalis Hübner.

The European corn borer (ECB), Ostrinia nubilalis, is the principal pest of maize in Central and South Europe. It is known to promote Fusarium verticillioides infection in maize grain, a recognized producer of fumonisins. Field experiments were performed in 2006 and 2007 in two sites in NW Italy to determine the effects of the timing of insecticide application on ECB damage, fungal ear rot and fumonisin contamination under natural conditions. Different insecticide application timings were compared, from maize flowering to approximately 15 days after the flight peak of adult ECB. At harvest, the ears were rated for incidence and severity of ECB damage, fungal ear rot symptoms and fumonisin (FB(1) + FB(2)) contamination. In all years/sites, treatments applied at the beginning of consistent ECB flight activity were most effective in controlling insect damage on ears. Fungal ear rot and fumonisin contamination were significantly affected by ECB control. The efficacy of the best timing of insecticide application in controlling fumonisin contamination was, on average, 93% compared to the untreated control. Contamination levels of these mycotoxins increased with either an earlier or later treatment. Furthermore, an earlier insecticide application showed lower fumonisin contamination than a treatment applied after the adult flight peak. Production of maize kernels and maize-based foods that do not exceed the maximum international and EU permitted levels for fumonisins could be enhanced by appropriate insecticide treatment against second generation ECB. The optimum time for insecticide application is between the beginning of consistent adult flight activity and the flight peak.